Lotte B. Nielsen

Circulating Levels of MicroRNA from Children with Newly Diagnosed Type 1 Diabetes and Healthy Controls: Evidence That miR-25 Associates to Residual Beta-Cell Function and Glycaemic Control during Disease Progression

This study aims to identify key miRNAs in circulation, which predict ongoing beta-cell destruction and regeneration in children with newly diagnosed Type 1 Diabetes (T1D). We compared expression level of sera miRNAs from new onset T1D children and age-matched healthy controls and related the miRNAs expression levels to beta-cell function and glycaemic control. Global miRNA sequencing analyses were performed on sera pools from two T1D cohorts (n = 275 and 129, resp.) and one control group (n = 151). We identified twelve upregulated human miRNAs in T1D patients (miR-152, miR-30a-5p, miR-181a, miR-24, miR-148a, miR-210, miR-27a, miR-29a, miR-26a, miR-27b, miR-25, miR-200a); several of these miRNAs were linked to apoptosis and beta-cell networks. Furthermore, we identified miR-25 as negatively associated with residual beta-cell function (est.: −0.12, P = 0.0037), and positively associated with glycaemic control (HbA1c) (est.: 0.11, P = 0.0035) 3 months after onset. In conclusion this study demonstrates that miR-25 might be a “tissue-specific” miRNA for glycaemic control 3 months after diagnosis in new onset T1D children and therefore supports the role of circulating miRNAs as predictive biomarkers for tissue physiopathology and potential intervention targets.

Co-localisation of the Kir6.2/SUR1 channel complex with glucagon-like peptide-1 and glucose-dependent insulinotrophic polypeptide expression in human ileal cells and implications for glycaemic control in new onset type 1 diabetes

OBJECTIVE The ATP-dependent K+-channel (K(ATP)) is critical for glucose sensing and normal glucagon and insulin secretion from pancreatic endocrine alpha- and beta-cells. Gastrointestinal endocrine L- and K-cells are also glucose-sensing cells secreting glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotrophic polypeptide (GIP) respectively. The aims of this study were to 1) investigate the expression and co-localisation of the K(ATP) channel subunits, Kir6.2 and SUR1, in human L- and K-cells and 2) investigate if a common hyperactive variant of the Kir6.2 subunit, Glu23Lys, exerts a functional impact on glucose-sensing tissues in vivo that may affect the overall glycaemic control in children with new-onset type 1 diabetes. DESIGN AND METHODS Western blot and immunohistochemical analyses were performed for expression and co-localisation studies. Meal-stimulated C-peptide test was carried out in 257 children at 1, 6 and 12 months after diagnosis. Genotyping for the Glu23Lys variant was by PCR-restriction fragment length polymorphism. RESULTS Kir6.2 and SUR1 co-localise with GLP-1 in L-cells and with GIP in K-cells in human ileum tissue. Children with type 1 diabetes carrying the hyperactive Glu23Lys variant had higher HbA1C at diagnosis (coefficient = 0.61%, P = 0.02) and 1 month after initial insulin therapy (coefficient = 0.30%, P = 0.05), but later disappeared. However, when adjusting HbA1C for the given dose of exogenous insulin, the dose-adjusted HbA1C remained higher throughout the 12 month study period (coefficient = 0.42%, P = 0.03). CONCLUSIONS Kir6.2 and SUR1 co-localise in the gastrointestinal endocrine L- and K-cells. The hyperactive Glu23Lys variant of the K(ATP) channel subunit Kir6.2 may cause defective glucose sensing in several tissues and impaired glycaemic control in children with type 1 diabetes.

CTSH regulates β-cell function and disease progression in newly diagnosed type 1 diabetes patients

Significance In type 1 diabetes (T1D), the insulin-producing pancreatic β-cells are destroyed by the immune system. Both genetic and environmental factors contribute to T1D risk. Candidate genes for T1D identified by genome-wide association studies have been proposed to act at both the immune system and the β-cell levels. This study shows that the risk variant rs3825932 in the candidate gene cathepsin H (CTSH) predicts β-cell function in both model systems and human T1D. Collectively, our data indicate that higher CTSH expression in β-cells may protect against immune-mediated damage and preserve β-cell function, thereby representing a possible therapeutic target. Our study reinforces the concept that candidate genes for T1D may affect disease progression by modulating survival and function of the β-cells. Over 40 susceptibility loci have been identified for type 1 diabetes (T1D). Little is known about how these variants modify disease risk and progression. Here, we combined in vitro and in vivo experiments with clinical studies to determine how genetic variation of the candidate gene cathepsin H (CTSH) affects disease mechanisms and progression in T1D. The T allele of rs3825932 was associated with lower CTSH expression in human lymphoblastoid cell lines and pancreatic tissue. Proinflammatory cytokines decreased the expression of CTSH in human islets and primary rat β-cells, and overexpression of CTSH protected insulin-secreting cells against cytokine-induced apoptosis. Mechanistic studies indicated that CTSH exerts its antiapoptotic effects through decreased JNK and p38 signaling and reduced expression of the proapoptotic factors Bim, DP5, and c-Myc. CTSH overexpression also up-regulated Ins2 expression and increased insulin secretion. Additionally, islets from Ctsh−/− mice contained less insulin than islets from WT mice. Importantly, the TT genotype was associated with higher daily insulin dose and faster disease progression in newly diagnosed T1D patients, indicating agreement between the experimental and clinical data. In line with these observations, healthy human subjects carrying the T allele have lower β-cell function, which was evaluated by glucose tolerance testing. The data provide strong evidence that CTSH is an important regulator of β-cell function during progression of T1D and reinforce the concept that candidate genes for T1D may affect disease progression by modulating survival and function of pancreatic β-cells, the target cells of the autoimmune assault.

Relationship between ZnT8Ab, the SLC30A8 gene and disease progression in children with newly diagnosed type 1 diabetes

Autoantibodies against the newly established autoantigen in type 1 diabetes, zinc transporter 8, ZnT8, are presented as two types, ZnT8RAb and ZnT8WAb. The rs13266634 variant of the SLC30A8 gene has recently been found to determine the type of ZnT8Ab. The aim of this study was to explore the impact of this genetic variant and the ZnT8Ab on the residual beta-cell function during disease progression the first year after disease diagnosis in children with newly diagnosed type 1 diabetes. This cohort consists of 257 children aged < 16 years, all patients were newly diagnosed with type 1 diabetes. A Boost-test was carried out at 1, 6, and 12 months to characterize the residual beta-cell function. Carriers of the CC and CT genotype groups of the rs13266634 SNP of the SLC30A8 gene had higher stimulated C-peptide levels the first year after onset compared with those of the TT genotype group (29%, p = 0.034). CC genotype carriers were highly associated with the presence of ZnT8RAb subtype during disease progression (compared with TT, p < 0.0001). On the other hand, the TT genotype was associated with the presence of ZnT8WAb subtype during disease progression (compared with CC, p < 0.0001). The C allele of the SLC30A8 gene is associated with preserved beta-cell function in type 1 diabetes patients. The genetic determination of the rs13266634 variant on the ZnT8Ab specificity is sustained the first 12 months after the diagnosis of type 1 diabetes in a pediatric cohort.

The PTPN22 C1858T gene variant is associated with proinsulin in new-onset type 1 diabetes.

BackgroundThe protein tyrosine phosphatase nonreceptor type 2 (PTPN22) has been established as a type 1 diabetes susceptibility gene. A recent study found the C1858T variant of this gene to be associated with lower residual fasting C-peptide levels and poorer glycemic control in patients with type 1 diabetes. We investigated the association of the C1858T variant with residual beta-cell function (as assessed by stimulated C-peptide, proinsulin and insulin dose-adjusted HbA1c), glycemic control, daily insulin requirements, diabetic ketoacidosis (DKA) and diabetes-related autoantibodies (IA-2A, GADA, ICA, ZnT8Ab) in children during the first year after diagnosis of type 1 diabetes.MethodsThe C1858T variant was genotyped in an international cohort of children (n = 257 patients) with newly diagnosed type 1 diabetes during 12 months after onset. We investigated the association of this variant with liquid-meal stimulated beta-cell function (proinsulin and C-peptide) and antibody status 1, 6 and 12 months after onset. In addition HbA1c and daily insulin requirements were determined 1, 3, 6, 9 and 12 months after diagnosis. DKA was defined at disease onset.ResultsA repeated measurement model of all time points showed the stimulated proinsulin level is significantly higher (22%, p = 0.03) for the T allele carriers the first year after onset. We also found a significant positive association between proinsulin and IA levels (est.: 1.12, p = 0.002), which did not influence the association between PTPN22 and proinsulin (est.: 1.28, p = 0.03).ConclusionsThe T allele of the C1858T variant is positively associated with proinsulin levels during the first 12 months in newly diagnosed type 1 diabetes children.

Partial Remission Definition: Validation based on the insulin dose-adjusted HbA1c (IDAA1C) in 129 Danish Children with New-Onset Type 1 Diabetes †

To validate the partial remission (PR) definition based on insulin dose‐adjusted HbA1c (IDAA1c).

Complex multi-block analysis identifies new immunologic and genetic disease progression patterns associated with the residual β-cell function 1 year after diagnosis of type 1 diabetes

The purpose of the present study is to explore the progression of type 1 diabetes (T1D) in Danish children 12 months after diagnosis using Latent Factor Modelling. We include three data blocks of dynamic paraclinical biomarkers, baseline clinical characteristics and genetic profiles of diabetes related SNPs in the analyses. This method identified a model explaining 21.6% of the total variation in the data set. The model consists of two components: (1) A pattern of declining residual β-cell function positively associated with young age, presence of diabetic ketoacidosis and long duration of disease symptoms (P = 0.0004), and with risk alleles of WFS1, CDKN2A/2B and RNLS (P = 0.006). (2) A second pattern of high ZnT8 autoantibody levels and low postprandial glucagon levels associated with risk alleles of IFIH1, TCF2, TAF5L, IL2RA and PTPN2 and protective alleles of ERBB3 gene (P = 0.0005). These results demonstrate that Latent Factor Modelling can identify associating patterns in clinical prospective data – future functional studies will be needed to clarify the relevance of these patterns.

Association between autoantibodies to the Arginine variant of the Zinc transporter 8 (ZnT8) and stimulated C-peptide levels in Danish children and adolescents with newly diagnosed type 1 diabetes

The zinc transporter 8 (ZnT8) was recently identified as a common autoantigen in type 1 diabetes (T1D) and inclusion of ZnT8 autoantibodies (ZnT8Ab) was found to increase the diagnostic specificity of T1D.

Proinsulin, GLP-1, and glucagon are associated with partial remission in children and adolescents with newly diagnosed type 1 diabetes

Kaas A, Max Andersen ML, Fredheim S, Hougaard P, Buschard K, Petersen JS, de Beaufort C, Robertson KJ, Hansen L, Mortensen HB, Nielsen LB, On behalf of The Hvidoere Study Group on childhood diabetes. Proinsulin, GLP‐1, and glucagon are associated with partial remission in children and adolescents with newly diagnosed type 1 diabetes.

Disease progression and search for monogenic diabetes among children with new onset type 1 diabetes negative for ICA, GAD- and IA-2 Antibodies.

BackgroundTo investigate disease progression the first 12 months after diagnosis in children with type 1 diabetes negative (AAB negative) for pancreatic autoantibodies [islet cell autoantibodies(ICA), glutamic acid decarboxylase antibodies (GADA) and insulinoma-associated antigen-2 antibodies (IA-2A)]. Furthermore the study aimed at determining whether mutations in KCNJ11, ABCC8, HNF1A, HNF4A or INS are common in AAB negative diabetes.Materials and methodsIn 261 newly diagnosed children with type 1 diabetes, we measured residual β-cell function, ICA, GADA, and IA-2A at 1, 6 and 12 months after diagnosis. The genes KCNJ11, ABCC8, HNF1A, HNF4A and INS were sequenced in subjects AAB negative at diagnosis. We expressed recombinant K-ATP channels in Xenopus oocytes to analyse the functional effects of an ABCC8 mutation.ResultsTwenty-four patients (9.1%) tested AAB negative after one month. Patients, who were AAB-negative throughout the 12-month period, had higher residual β-cell function (P = 0.002), lower blood glucose (P = 0.004), received less insulin (P = 0.05) and had lower HbA1c (P = 0.02) 12 months after diagnosis. One patient had a heterozygous mutation leading to the substitution of arginine at residue 1530 of SUR1 (ABCC8) by cysteine. Functional analyses of recombinant K-ATP channels showed that R1530C markedly reduced the sensitivity of the K-ATP channel to inhibition by MgATP. Morover, the channel was highly sensitive to sulphonylureas. However, there was no effect of sulfonylurea treatment after four weeks on 1.0-1.2 mg/kg/24 h glibenclamide.ConclusionGAD, IA-2A, and ICA negative children with new onset type 1 diabetes have slower disease progression as assessed by residual beta-cell function and improved glycemic control 12 months after diagnosis. One out of 24 had a mutation in ABCC8, suggesting that screening of ABCC8 should be considered in patients with AAB negative type 1 diabetes.