Choice of time scale for analysis of recurrent events data.

Recurrent events refer to events that over time can occur several times for each individual. Full use of such data in a clinical trial requires a method that addresses the dependence between events. For modelling this dependence, there are two time scales to consider, namely time since start of the study (running time) or time since most recent event (gap time). In the multi-state setup, it is possible to estimate parameters also in the case, where the hazard model allows for an effect of both time scales, making this an extremely flexible approach. However, for summarizing the effect of a treatment in a transparent and informative way, the choice of time scale and model requires much more care. This paper discusses these choices both from a theoretical and practical point of view. This is supported by a simulation study showing that in a frailty model with assumptions covered by both time scales, the gap time approach may give misleading results. A literature dataset is used for illustrating the issues.

Lifespans of Twins: Does Zygosity Matter?

Studies with twins provide fundamental insights to lifespans of humans. We aim to clarify if monozygotic and dizygotic twin individuals differ in lifespan, that is, if zygosity matters. We investigate whether a possible difference in mortality after infancy between zygosities is stable in different age cohorts, and whether the difference remains when twins with unknown zygosity are taken into account. Further, we compare the distribution of long-livers, that is, the upper-tail of the lifespan distribution, between monozygotic and same-sex dizygotic twin individuals. The Danish Twin Registry provides a nationwide cohort of 109,303 twins born during 1870 to 1990 with valid vital status. Standard survival analysis is used to compare mortality in monozygotic and dizygotic twin individuals and twin individuals with unknown zygosity. The mortality of monozygotic and dizygotic twin individuals differs slightly after taking into consideration effects of birth- and age-cohorts, gender differences, and that twins are paired. However, no substantial nor systematic differences remain when taking twins with unknown zygosity into account. Further, the distribution of long-livers is very similar by zygosity, suggesting the same mortality process. The population-based and oldest twin cohort ever studied suggests that monozygotic and dizygotic twins have similar lifespans.

Influence of Disease Duration on Circulating Levels of miRNAs in Children and Adolescents with New Onset Type 1 Diabetes.

Circulating microRNAs (miRNAs) have been implicated in several pathologies including type 1 diabetes. In the present study, we aimed to identify circulating miRNAs affected by disease duration in children with recent onset type 1 diabetes. Forty children and adolescents from the Danish Remission Phase Cohort were followed with blood samples drawn at 1, 3, 6, 12, and 60 months after diagnosis. Pancreatic autoantibodies were measured at each visit. Cytokines were measured only the first year. miRNA expression profiling was performed by RT-qPCR. The effect of disease duration was analyzed by mixed models for repeated measurements adjusted for sex and age. Eight miRNAs (hsa-miR-10b-5p, hsa-miR-17-5p, hsa-miR-30e-5p, hsa-miR-93-5p, hsa-miR-99a-5p, hsa-miR-125b-5p, hsa-miR-423-3p, and hsa-miR-497-5p) were found to significantly change in expression (adjusted p-value < 0.05) with disease progression. Three pancreatic autoantibodies, ICA, IA-2A, and GAD65A, and four cytokines, IL-4, IL-10, IL-21, and IL-22, were associated with the miRNAs at different time points. Pathway analysis revealed associations with various immune-mediated signaling pathways. Eight miRNAs that were involved in immunological pathways changed expression levels during the first five years after diagnosis and were associated with variations in cytokine and pancreatic antibodies, suggesting a possible effect on the immunological processes in the early phase of the disease.

Is there a reasonable excuse for not providing post-operative analgesia when using animal models of peripheral neuropathic pain for research purposes?

INTRODUCTION: The induction of neuropathic pain-like behaviors in rodents often requires surgical intervention. This engages acute nociceptive signaling events that contribute to pain and stress post-operatively that from a welfare perspective demands peri-operative analgesic treatment. However, a large number of researchers avoid providing such care based largely on anecdotal opinions that it might interfere with model pathophysiology in the longer term. OBJECTIVES: To investigate effects of various peri-operative analgesic regimens encapsulating different mechanisms and duration of action, on the development of post-operative stress/welfare and pain-like behaviors in the Spared Nerve Injury (SNI)-model of neuropathic pain. METHODS: Starting on the day of surgery, male Sprague-Dawley rats were administered either vehicle (s.c.), carprofen (5.0mg/kg, s.c.), buprenorphine (0.1mg/kg s.c. or 1.0mg/kg p.o. in Nutella®), lidocaine/bupivacaine mixture (local irrigation) or a combination of all analgesics, with coverage from a single administration, and up to 72 hours. Post-operative stress and recovery were assessed using welfare parameters, bodyweight, food-consumption, and fecal corticosterone, and hindpaw mechanical allodynia was tested for assessing development of neuropathic pain for 28 days. RESULTS: None of the analgesic regimes compromised the development of mechanical allodynia. Unexpectedly, the combined treatment with 0.1mg/kg s.c. buprenorphine and carprofen for 72 hours and local irrigation with lidocaine/bupivacaine, caused severe adverse effects with peritonitis. This was not observed when the combination included a lower dose of buprenorphine (0.05mg/kg, s.c.), or when buprenorphine was administered alone (0.1mg/kg s.c. or 1.0mg/kg p.o.) for 72 hours. An elevated rate of wound dehiscence was observed especially in the combined treatment groups, underlining the need for balanced analgesia. Repeated buprenorphine injections had positive effects on body weight the first day after surgery, but depressive effects on food intake and body weight later during the first week. CONCLUSION: Post-operative analgesia does not appear to affect established neuropathic hypersensitivity outcome in the SNI model.

Circulating microRNA levels predict residual beta cell function and glycaemic control in children with type 1 diabetes mellitus.

AIMS/HYPOTHESIS: We aimed to identify circulating microRNA (miRNA) that predicts clinical progression in a cohort of 123 children with new-onset type 1 diabetes mellitus. METHODS: Plasma samples were prospectively obtained at 1, 3, 6, 12 and 60 months after diagnosis from a subset of 40 children from the Danish Remission Phase Cohort, and profiled for miRNAs. At the same time points, meal-stimulated C-peptide and HbA1c levels were measured and insulin-dose adjusted HbA1c (IDAA1c) calculated. miRNAs that at 3 months after diagnosis predicted residual beta cell function and glycaemic control in this subgroup were further validated in the remaining cohort (n = 83). Statistical analysis of miRNA prediction for disease progression was performed by multiple linear regression analysis adjusted for age and sex. RESULTS: In the discovery analysis, six miRNAs (hsa-miR-24-3p, hsa-miR-146a-5p, hsa-miR-194-5p, hsa-miR-197-3p, hsa-miR-301a-3p and hsa-miR-375) at 3 months correlated with residual beta cell function 6-12 months after diagnosis. Stimulated C-peptide at 12 months was predicted by hsa-miR-197-3p at 3 months (p = 0.034). A doubling of this miRNA level corresponded to a sixfold higher stimulated C-peptide level. In addition, a doubling of hsa-miR-24-3p and hsa-miR-146a-5p levels at 3 months corresponded to a 4.2% (p < 0.014) and 3.5% (p < 0.022) lower IDAA1c value at 12 months. Analysis of the remaining cohort confirmed the initial finding for hsa-miR-197-3p (p = 0.018). The target genes for the six miRNAs revealed significant enrichment for pathways related to gonadotropin-releasing hormone receptor and angiogenesis pathways. CONCLUSIONS/INTERPRETATION: The miRNA hsa-miR-197-3p at 3 months was the strongest predictor of residual beta cell function 1 year after diagnosis in children with type 1 diabetes mellitus.

Partial remission definition: validation based on the insulin dose-adjusted HbA1c (IDAA1C) in 129 Danish children with new-onset type 1 diabetes.

OBJECTIVE: To validate the partial remission (PR) definition based on insulin dose-adjusted HbA1c (IDAA1c). SUBJECTS AND METHODS: The IDAA1c was developed using data in 251 children from the European Hvidoere cohort. For validation, 129 children from a Danish cohort were followed from the onset of type 1 diabetes (T1D). Receiver operating characteristic curve (ROC) analysis was used to evaluate the predictive value of IDAA1c and age on partial C-peptide remission (stimulated C-peptide, SCP > 300 pmol/L). RESULTS: PR (IDAA1c ≤ 9) in the Danish and Hvidoere cohorts occurred in 62 vs. 61% (3 months, p = 0.80), 47 vs. 44% (6 months, p = 0.57), 26 vs. 32% (9 months, p = 0.32) and 19 vs. 18% (12 months, p = 0.69). The effect of age on SCP was significantly higher in the Danish cohort compared with the Hvidoere cohort (p < 0.0001), likely due to higher attained Boost SCP, so the sensitivity and specificity of those in PR by IDAA1c ≤ 9, SCP > 300 pmol/L was 0.85 and 0.62 at 6 months and 0.62 vs. 0.38 at 12 months, respectively. IDAA1c with age significantly improved the ROC analyses and the AUC reached 0.89 ± 0.04 (age) vs. 0.94 ± 0.02 (age + IDAA1c) at 6 months (p < 0.0004) and 0.76 ± 0.04 (age) vs. 0.90 ± 0.03 (age + IDAA1c) at 12 months (p < 0.0001). CONCLUSIONS: The diagnostic and prognostic power of the IDAA1c measure is kept but due to the higher Boost stimulation in the Danish cohort, the specificity of the formula is lower with the chosen limits for SCP (300 pmol/L) and IDAA1c ≤9, respectively.

Effect of insulin analogues on risk of severe hypoglycaemia in patients with type 1 diabetes prone to recurrent severe hypoglycaemia (HypoAna trial): a prospective, randomised, open-label, blinded-endpoint crossover trial.

BACKGROUND: Insulin analogues have been developed to reduce the risk of hypoglycaemia in patients with diabetes who require insulin-based treatment, but their effect on this endpoint in patients with type 1 diabetes complicated by recurrent severe hypoglycaemia is unknown. We compared the occurrence of severe hypoglycaemic episodes in such patients during treatment with insulin analogues or human insulin. METHODS: In this investigator-initiated, prospective, randomised, open-label, blinded-endpoint crossover trial at seven medical centres in Denmark, we recruited patients (aged ≥18 years) with type 1 diabetes (diagnosed for >5 years) who had reported two or more episodes of severe hypoglycaemia in the preceding year. Patients were randomly assigned (1:1) using computer-generated site-specific randomisation lists in blocks of four to treatment with basal-bolus therapy with either analogue insulin (detemir and aspart) or human insulin (human neutral protamine Hagedorn and human regular) in a balanced crossover design. A 1-year plus 1-year treatment period was specified, consisting of two 3-month run-in periods, each followed by a 9-month maintenance period. The primary endpoint was the number of validated episodes of severe hypoglycaemia (defined by need for treatment assistance from others) reported during the maintenance periods, analysed by intention to treat. The study is registered with ClinicalTrials.gov, number NCT00346996. FINDINGS: Between May 9, 2007, and Oct 30, 2009, 159 patients were randomly assigned. 18 patients discontinued during the first run-in period, leaving 141 patients in the intention-to-treat population. 136 severe hypoglycaemic episodes were reported during treatment with human insulin and 105 episodes were reported during treatment with insulin analogues, resulting in an absolute rate reduction of 0.51 episodes (95% CI 0.19-0.84) per patient-year with insulin analogues. This result corresponds to a relative rate reduction of 29% (95% CI 11-48; p=0.010). INTERPRETATION: Treatment with insulin detemir and aspart in patients with type 1 diabetes and recurrent severe hypoglycaemia resulted in a clinically significant reduced rate of severe hypoglycaemia compared with human insulin. Patients with the greatest chance of benefitting from improved insulin therapy should be offered treatment with insulin analogues and be included in future trials of new insulins. FUNDING: Novo Nordisk A/S.

Disease progression among 446 children with newly diagnosed type 1 diabetes located in Scandinavia, Europe, and North America during the last 27 yr.

OBJECTIVE: To clarify whether the rate of decline in stimulated C-peptide (SCP) from 2 to 15 months after diagnosis has changed over an interval of 27 yr. RESEARCH DESIGN AND METHODS: The rate of decline in SCP levels at 1, 2, 3, 6, 9, 12, and 15 months after diagnosis was compared in four paediatric cohorts from Scandinavian and European countries including 446 children with new onset type 1 diabetes (T1D, 1982-2004). Findings were evaluated against 78 children (2004-2009) from the TrialNet studies. RESULTS: The mean rate of decline [%/month (±SEM)] in SCP for a 10-yr-old child was 7.7%/month (±1.5) in the 1982-1985 Cohort, 6.3%/month (±1.7) in the 1995-1998 Cohort, 7.8%/month (±0.7) in the 1999-2000 Cohort, and 10.7%/month (±0.9) in the latest 2004-2005 Cohort (p = 0.05). Including the TrialNet Cohort with a rate of decline in SCP of 10.0%/month (±0.9) the differences between the cohorts are still significant (p = 0.039). The rate of decline in SCP was negatively associated with age (p < 0.0001), insulin antibodies (IA) (p = 0.003), and glutamic acid decarboxylase-65 (GAD65A) (p = 0.03) initially with no statistically significant effect of body mass index (BMI) Z-score at 3 months. Also, at 3 months the time around partial remission, the effect of age on SCP was significantly greater in children ≤5 yr compared with older children (p ≤ 0.0001). CONCLUSIONS: During the past 27 yr, initial C-peptide as well as the rate of C-peptide decline seem to have increased. The rate of decline was affected significantly by age, GAD65A, and IA, but not BMI Z-score or initial C-peptide.

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 [corrected]. 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.

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

OBJECTIVE: Proinsulin is a marker of beta-cell distress and dysfunction in type 2 diabetes and transplanted islets. Proinsulin levels are elevated in patients newly diagnosed with type 1 diabetes. Our aim was to assess the relationship between proinsulin, insulin dose-adjusted haemoglobin A1c (IDAA1C), glucagon-like peptide-1 (GLP-1), glucagon, and remission status the first year after diagnosis of type 1 diabetes. METHODS: Juvenile patients (n = 275) were followed 1, 6, and 12 months after diagnosis. At each visit, partial remission was defined as IDAA1C ≤ 9%. The patients had a liquid meal test at the 1-, 6-, and 12-month visits, which included measurement of C-peptide, proinsulin, GLP-1, glucagon, and insulin antibodies (IA). RESULTS: Patients in remission at 6 and 12 months had significantly higher levels of proinsulin compared to non-remitting patients (p < 0.0001, p = 0.0002). An inverse association between proinsulin and IDAA1C was found at 1 and 6 months (p = 0.0008, p = 0.0022). Proinsulin was positively associated with C-peptide (p < 0.0001) and IA (p = 0.0024, p = 0.0068, p < 0.0001) at 1, 6, and 12 months. Glucagon (p < 0.0001 and p < 0.02) as well as GLP-1 (p = 0.0001 and p = 0.002) were significantly lower in remitters than in non-remitters at 6 and 12 months. Proinsulin associated positively with GLP-1 at 1 month (p = 0.004) and negatively at 6 (p = 0.002) and 12 months (p = 0.0002). CONCLUSIONS: In type 1 diabetes, patients in partial remission have higher levels of proinsulin together with lower levels of GLP-1 and glucagon compared to patients not in remission. In new onset type 1 diabetes proinsulin level may be a sign of better residual beta-cell function.

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.

BACKGROUND: The 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. METHODS: The 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. RESULTS: A 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). CONCLUSIONS: The T allele of the C1858T variant is positively associated with proinsulin levels during the first 12 months in newly diagnosed type 1 diabetes children.

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

BACKGROUND: To 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 METHODS: In 261 newly diagnosed children with type 1 diabetes, we measured residual ß-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. RESULTS: Twenty-four patients (9.1%) tested AAB negative after one month. Patients, who were AAB-negative throughout the 12-month period, had higher residual ß-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. CONCLUSION: GAD, 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.

New definition for the partial remission period in children and adolescents with type 1 diabetes.

OBJECTIVE To find a simple definition of partial remission in type 1 diabetes that reflects both residual beta-cell function and efficacy of insulin treatment. RESEARCH DESIGN AND METHODS A total of 275 patients aged <16 years were followed from onset of type 1 diabetes. After 1, 6, and 12 months, stimulated C-peptide during a challenge was used as a measure of residual beta-cell function. RESULTS By multiple regression analysis, a negative association between stimulated C-peptide and A1C (regression coefficient -0.21, P < 0.001) and insulin dose (-0.94, P < 0.001) was shown. These results suggested the definition of an insulin dose-adjusted A1C (IDAA1C) as A1C (percent) + [4 x insulin dose (units per kilogram per 24 h)]. A calculated IDAA1C < or =9 corresponding to a predicted stimulated C-peptide >300 pmol/l was used to define partial remission. The IDAA1C < or =9 had a significantly higher agreement (P < 0.001) with residual beta-cell function than use of a definition of A1C < or =7.5%. Between 6 and 12 months after diagnosis, for IDAA1C < or =9 only 1 patient entered partial remission and 61 patients ended partial remission, for A1C < or =7.5% 15 patients entered partial remission and 53 ended, for a definition of insulin dose < or =0.5 units . kg(-1) . 24 h(-1) 5 patients entered partial remission and 66 ended, and for stimulated C-peptide (>300 pmol/l) 9 patients entered partial remission and 49 ended. IDAA1C at 6 months has good predictive power for stimulated C-peptide concentrations after both 6 and 12 months. CONCLUSIONS A new definition of partial remission is proposed, including both glycemic control and insulin dose. It reflects residual beta-cell function and has better stability compared with the conventional definitions.

Variation within the PPARG gene is associated with residual beta-cell function and glycemic control in children and adolescents during the first year of clinical type 1 diabetes.

CONTEXT: Conflicting evidence exists as to whether the Pro12Ala single nucleotide polymorphism of the type 2 diabetes susceptibility gene peroxisome proliferator-activated receptor gamma (PPARG) also confers risk for type 1 diabetes (T1D). OBJECTIVE: The objective of this study was to investigate the PPARG gene in relation to residual beta-cell function and glycemic control in newly diagnosed T1D. DESIGN: Prospective, non-interventional, 12-month follow-up study, conducted in 18 centers in 15 countries. PATIENTS: Two hundred and fifty-seven children and adolescents (aged <16 yr) with newly diagnosed T1D. MAIN OUTCOME MEASURES: Beta-cell function was determined as 90-min meal-stimulated C-peptide (Boost test) 1, 6, and 12 months after diagnosis. Hemoglobin A1c (HbA1c) and daily insulin dose (IU/kg/d) were recorded at 1, 3, 6, 9, and 12 months after diagnosis. Haplotypes within PPARG were estimated by SNPHap program. Statistical analyses were performed in a repeated measurements model. RESULTS: Five haplotypes within PPARG were generated (h1, 68.4%; h2, 16.3%; h3, 8.3%; h4, 3.5%; and hx, 3.5%). Compared with the most frequent h1 haplotype, the haplotypes h3 and h4 of the PPARG associated with residual beta-cell function during the first year of clinical disease: h3 with a 27% lower C-peptide (p = 0.02) and h4 with a 39% lower C-peptide (p = 0.01). Haplotype h4 also associated with a 0.86% (absolute) higher HbA1c, after adjustment for the insulin dose (p = 0.02). CONCLUSION: Variation in the PPARG locus may influence disease progression during the first year after the presentation of T1D.

Genetic variation and activity of the renin-angiotensin system and severe hypoglycemia in type 1 diabetes.

BACKGROUND: The deletion-allele of the angiotensin-converting enzyme (ACE) gene and elevated ACE activity are associated with increased risk of severe hypoglycemia in type 1 diabetes. We explored whether genetic and phenotypic variations in other components of the renin-angiotensin system are similarly associated. METHODS: Episodes of severe hypoglycemia were recorded in 171 consecutive type 1 diabetic outpatients during a 1-year follow-up. Participants were characterized at baseline by gene polymorphisms in angiotensinogen, ACE, angiotensin-II receptor types 1 (AT1R) and 2 (AT2R), and by plasma angiotensinogen concentration and serum ACE activity. RESULTS: Three risk factors for severe hypoglycemia were identified: plasma angiotensinogen concentration in the upper quartile (relative rate [RR] vs. lower quartile 3.1, 95% confidence interval [CI,] 1.4-6.8), serum ACE activity in the upper quartile (RR vs. lower quartile 2.9, 95% CI, 1.3-6.2), and homo- or hemizygosity for the A-allele of the X chromosome-located AT2R 1675G/A polymorphism (RR vs. noncarriers 2.5, 95% CI, 1.4-5.0). The three risk factors contributed independently to prediction of severe hypoglycemia. A backward multiple regression analysis identified a high number of renin-angiotensin system-related risk factors and reduced ability to perceive hypoglycemic warning symptoms (impaired hypoglycemia awareness) as predictors of severe hypoglycemia. CONCLUSIONS: High renin-angiotensin system activity and the A-allele of the AT2R 1675G/A polymorphism associate with high risk of severe hypoglycemia in type 1 diabetes. A potential preventive effect of renin-angiotensin system blocking drugs in patients with recurrent severe hypoglycemia merits further investigation.

Meal-stimulated glucagon release is associated with postprandial blood glucose level and does not interfere with glycemic control in children and adolescents with new-onset type 1 diabetes.

CONTEXT: The role of glucagon in hyperglycemia in type 1 diabetes is unresolved, and in vitro studies suggest that increasing blood glucose might stimulate glucagon secretion. OBJECTIVE: Our objective was to investigate the relationship between postprandial glucose and glucagon level during the first 12 months after diagnosis of childhood type 1 diabetes. DESIGN: We conducted a prospective, noninterventional, 12-month follow-up study conducted in 22 centers in 18 countries. PATIENTS: Patients included 257 children and adolescents less than 16 yr old with newly diagnosed type 1 diabetes; 204 completed the 12-month follow-up. SETTING: The study was conducted at pediatric outpatient clinics. MAIN OUTCOME MEASURES: We assessed residual beta-cell function (C-peptide), glycosylated hemoglobin (HbA(1c)), blood glucose, glucagon, and glucagon-like peptide-1 (GLP-1) release in response to a 90-min meal stimulation (Boost) at 1, 6, and 12 months after diagnosis. RESULTS: Compound symmetric repeated-measurements models including all three visits showed that postprandial glucagon increased by 17% during follow-up (P = 0.001). Glucagon levels were highly associated with postprandial blood glucose levels because a 10 mmol/liter increase in blood glucose corresponded to a 20% increase in glucagon release (P = 0.0003). Glucagon levels were also associated with GLP-1 release because a 10% increase in GLP-1 corresponded to a 2% increase in glucagon release (P = 0.0003). Glucagon levels were not associated (coefficient -0.21, P = 0.07) with HbA(1c), adjusted for insulin dose. Immunohistochemical staining confirmed the presence of Kir6.2/SUR1 in human alpha-cells. CONCLUSION: Our study supports the recent in vitro data showing a stimulation of glucagon secretion by high glucose levels. Postprandial glucagon levels were not associated with HbA(1c), adjusted for insulin dose, during the first year after onset of childhood type 1 diabetes.

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.