Dario Iafusco

Zonulin Upregulation Is Associated With Increased Gut Permeability in Subjects With Type 1 Diabetes and Their Relatives

Zonulin, a protein that modulates intestinal permeability, is upregulated in several autoimmune diseases and is involved in the pathogenesis of autoimmune diabetes in the BB/Wor animal model of the disease. To verify the association between serum zonulin levels and in vivo intestinal permeability in patients with type 1 diabetes, both parameters were investigated in different stages of the autoimmune process. Forty-two percent (141 of 339) of the patients had abnormal serum zonulin levels, as compared with age-matched control subjects. The increased zonulin levels correlated with increased intestinal permeability in vivo and changes in claudin-1, claudin-2, and myosin IXB genes expression, while no changes were detected in ZO1 and occludin genes expression. When tested in serum samples collected during the pre–type 1 diabetes phase, elevated serum zonulin was detected in 70% of subjects and preceded by 3.5 ± 0.9 years the onset of the disease in those patients who went on to develop type 1 diabetes. Combined, these results suggest that zonulin upregulation is associated with increased intestinal permeability in a subgroup of type 1 diabetic patients. Zonulin upregulation seems to precede the onset of the disease, providing a possible link between increased intestinal permeability, environmental exposure to non–self antigens, and the development of autoimmunity in genetically susceptible individuals.

Seven mutations in the human insulin gene linked to permanent neonatal/infancy-onset diabetes mellitus

Permanent neonatal diabetes mellitus (PNDM) is a rare disorder usually presenting within 6 months of birth. Although several genes have been linked to this disorder, in almost half the cases documented in Italy, the genetic cause remains unknown. Because the Akita mouse bearing a mutation in the Ins2 gene exhibits PNDM associated with pancreatic beta cell apoptosis, we sequenced the human insulin gene in PNDM subjects with unidentified mutations. We discovered 7 heterozygous mutations in 10 unrelated probands. In 8 of these patients, insulin secretion was detectable at diabetes onset, but rapidly declined over time. When these mutant proinsulins were expressed in HEK293 cells, we observed defects in insulin protein folding and secretion. In these experiments, expression of the mutant proinsulins was also associated with increased Grp78 protein expression and XBP1 mRNA splicing, 2 markers of endoplasmic reticulum stress, and with increased apoptosis. Similarly transfected INS-1E insulinoma cells had diminished viability compared with those expressing WT proinsulin. In conclusion, we find that mutations in the insulin gene that promote proinsulin misfolding may cause PNDM.

High prevalence of glucokinase mutations in Italian children with MODY. Influence on glucose tolerance, first-phase insulin response, insulin sensitivity and BMI

Aims/hypothesis. The aim of this study was to assess the prevalence of glucokinase gene mutations in Italian children with MODY and to investigate genotype/phenotype correlations of the mutants. Methods. Screening for sequence variants in the glucokinase gene was performed by denaturing gradient gel electrophoresis and direct sequencing in 132 children with maturity onset diabetes of the young (MODY) and in 9 children with chronic fasting hyperglycaemia but without laboratory evidence for Type I (insulin-dependent) diabetes mellitus and with normoglycaemic parents (“non-classical” MODY). Results. Altogether 54 mutations were identified in the MODY group (54/132 or 41 %) and 3 among the “non-classical” MODY individuals (3/9 or 33 %). Paternity testing indicated that the latter mutations have arisen de novo. Mean fasting plasma glucose concentrations of the children with the mutant glucokinase was in the expected impaired fasting glucose range. In contrast, results of the oral glucose tolerance test showed a wide range from normal glucose tolerance (Group 1: 2-h OGTT = 6.7 ± 1.1 mmol/l; 11 patients) to diabetes (Group 2: 2-h OGTT = 11.5 ± 0.5 mmol/l; 9 patients), with the remaining in the impaired glucose tolerance range. Disruptive mutations (i. e. nonsense, frameshifts, splice-site) were equally represented in Groups 1 and 2 and were not clearly associated with an impaired first-phase insulin response. Surprisingly, 5 out of 11 children (or 45 %) in Group 1 were found to be overweight but no children in Group 2 were overweight. Sensitivity index (SI), calculated by a recently described method, was found to be significantly lower in Group 2 than in Group 1 (SI Group 2 = 0.0013 ± 0.0009 ml Kg–1 min–1/μU/ml; SI Group 1 = 0.0068 ± 0.0048, p < 0.0035). Conclusion/interpretation. Mutations in glucokinase are the first cause of MODY among Italian children selected through a low threshold limit of fasting plasma glucose (i. e. > 5.5 mmol). The lack of correlation between the molecular severity of glucokinase mutations, insulin secretion at intravenous glucose tolerance test and differences in glucose tolerance suggests that factors outside the beta cell are also involved in determining post-load glucose concentrations in these subjects. Our results seem to indicate that the differences observed in the 2-h responses at the OGTT among children with MODY 2 could be related to individual differences in insulin sensitivity. [Diabetologia (2001) 44: 898–905]

Celiac disease in children and adolescents with type I diabetes: importance of hypoglycemia.

Background Symptomatic hypoglycemia is an unavoidable problem in the treatment of type I diabetes. Celiac disease is associated with malabsorption and may therefore represent an important risk factor. Methods The frequency of symptomatic hypoglycemia in patients with type I diabetes and celiac disease (cases) was compared with those of patients who had diabetes without celiac disease (controls). For this purpose, each case was matched for age, sex, and duration of disease with one to two control patients. Indices of metabolic control (hemoglobin [Hb]A1c, frequency of hypoglycemia, and total insulin requirement) were retrieved for the 18 months before and after diagnosis of celiac disease. Results Eighteen patients (6 males and 12 females) had diagnosed celiac disease and were matched with 26 control patients (10 males and 16 females). There was no difference in age (11.0 years; range, 1.8–21.9 vs. 13.1 years; range, 2.3–22;P = 0.3) and duration of disease (8.4 years; range, 1.2–19.3 vs. 8.3 years; range, 1.1–18.7;P = 0.3) between the two groups. During the 6 months before and after diagnosis of celiac disease the cases had significantly more hypoglycemic episodes than the controls (means ± SD; 4.5 ± 4 vs. 2.0 ± 2.2 episodes/months, P = 0.01). This was reflected by a progressive reduction in insulin requirement over the 12 months before diagnosis reaching a nadir at time 0 (0.6 ± 0.2 vs. 0.9 ± 0.3, P = 0.05). Conclusion These data suggest that underlying celiac disease is associated with an increased risk of symptomatic hypoglycemia and that the introduction of a gluten-free diet with normalization of the intestinal mucosa may reduce its frequency.

Maturity-onset diabetes of the young in children with incidental hyperglycemia: a multicenter Italian study of 172 families

OBJECTIVE To investigate the prevalence of maturity-onset diabetes of the young (MODY) in Italian children with incidental hyperglycemia. RESEARCH DESIGN AND METHODS Among 748 subjects age 1–18 years with incidental hyperglycemia, minimal diagnostic criteria for MODY were met by 172 families. Mutational analyses of the glucokinase (GCK) and hepatocyte nuclear factor 1α (HNF1Α) genes were performed. RESULTS We identified 85 GCK gene mutations in 109 probands and 10 HNF1Α mutations in 12 probands. In GCK patients, the median neonatal weight and age at the first evaluation were lower than those found in patients with HNF1A mutations. Median fasting plasma glucose and impaired fasting glucose/impaired glucose tolerance frequency after oral glucose tolerance testing were higher in GCK patients, who also showed a lower frequency of diabetes than HNF1A patients. CONCLUSIONS GCK mutations are the prevailing cause of MODY (63.4%) when the index case is recruited in Italian children with incidental hyperglycemia.

Insulin gene mutations as cause of diabetes in children negative for five type 1 diabetes autoantibodies

OBJECTIVE—Heterozygous, gain-of-function mutations of the insulin gene can cause permanent diabetes with onset ranging from the neonatal period through adulthood. The aim of our study was to screen for the insulin gene in patients who had been clinically classified as type 1 diabetic but who tested negative for type 1 diabetes autoantibodies. RESEARCH DESIGN AND METHODS—We reviewed the clinical records of 326 patients with the diagnosis of type 1 diabetes and identified seven probands who had diabetes in isolation and were negative for five type 1 diabetes autoantibodies. We sequenced the INS gene in these seven patients. RESULTS—In two patients whose diabetes onset had been at 2 years 10 months of age and at 6 years 8 months of age, respectively, we identified the mutation GB8S and a novel mutation in the preproinsulin signal peptide (ASignal23S). CONCLUSIONS—Insulin gene mutations are rare in absolute terms in patients classified as type 1 diabetic (0.6%) but can be identified after a thorough screening of type 1 diabetes autoantibodies.

Sulfonylurea treatment outweighs insulin therapy in short-term metabolic control of patients with permanent neonatal diabetes mellitus due to activating mutations of the KCNJ11 (KIR6.2) gene

To the Editor, Activating missense mutations in the gene encoding potassium inwardly rectifying channel, subfamily J, member 11 (KCNJ11) represent the most common cause (40 to 64%, depending on populations) of permanent neonatal diabetes mellitus in patients diagnosed in the first 6 months of life [1, 2]. In addition, KCNJ11 activating mutations can lead to transient/relapsing neonatal diabetes [3, 4]. The KCNJ11 gene encodes the pore-forming subunit (also known as KIR6.2) of the pancreatic beta cell ATP-sensitive potassium channel (KATP), which exerts a pivotal role in glucose-regulated insulin release. In the beta cell, KIR6.2 forms a hetero-octameric complex (4:4) with the sulfonylurea receptor subtype 1 (SUR1); binding to SUR1 by sulfonylureas determines channel closure and insulin secretion [2]. In previously published cases, seven patients have been reported to respond well to the transfer from insulin to oral hypoglycaemic agents [4–8]. Here we report on the replacement of insulin with sulfonylureas in ten Italian children who have mutations in KCNJ11 (R50P, V59M [x4], K170R, R201C and R201H [x3]) and were followed in nine Diabetologia (2006) 49:2210–2213 DOI 10.1007/s00125-006-0329-x

Large incidence variation of Type I diabetes in central-southern italy 1990-1995 : lower risk in rural areas

Aims/hypothesis. To evaluate the relation between the incidence of childhood Type I (insulin-dependent) diabetes mellitus and the degree of urbanization in the central-southern part of Italy. Methods. The incidence was determined in two areas: area A encompasses 3 regions of central-eastern Italy (Marche, Abruzzo, Umbria), whereas area B encompasses one southern region (Campania). During 1990–1995, 706 children aged 14 or under with insulin-dependent diabetes mellitus of recent onset were registered. The completeness of the case ascertainment in the registries analysed separately for each region was high, ranging from 96.3 % to 99 %. Results. The age-standardized incidence was higher in area A (9.6 per 100 000 person per year; 95 % confidence interval: 8.5–10.8) than in area B (5.4 per 100 000 person per year; 95 % confidence interval: 4.9–6.0). In both areas the standardized incidence ratios increased with the degree of urbanization (chi-squared for trend: area A = 140, p < 0.0001; area B = 79, p < 0.0001). The highest standardized incidence ratios were in the most urban communities. Conclusion/interpretation. This study showed a statistically significant difference in incidence of childhood insulin-dependent diabetes mellitus among different areas of the continental peninsula of Italy. People living in the rural communities appear to have a lower risk. [Diabetologia (1999) 42: 789–792]

Use of integrated real-time continuous glucose monitoring/insulin pump system in children and adolescents with type 1 diabetes: A 3-year follow-up study

BACKGROUND Insulin pumps and real-time continuous glucose monitoring devices have recently been combined into the sensor-augmented pump (SAP) system. The objective of this study was the evaluation of the clinical use of SAP in a large series of children with type 1 diabetes using insulin pump therapy. METHODS A questionnaire was administered in all pediatric diabetologic centers in Italy; data were analyzed only regarding patients 18 years old or younger and using SAP for 6 months or more. RESULTS Among all patients using an insulin pump, 129 (13.5 ± 3.8 years old, with a disease duration of 6.3 ± 3.4 years) have been using SAP for 1.4 ± 0.7 years. Four hundred ninety-three patients (12.9 ± 3.4 years old, with a disease duration of 6.2 ± 3.3 years) using conventional insulin pump therapy for 1.7 ± 0.5 years have been evaluated as the control group. After 0.5-3 years of using SAP or conventional insulin pump therapy, glycosylated hemoglobin significantly improved (8.0 ± 1.5% vs. 7.4 ± 0.8% [P = 0.002] and 8.0 ± 1.6% vs. 7.7 ± 1.1% [P = 0.006], respectively); the improvement was higher with SAP (P = 0.005). Insulin requirement showed a significant decrease only in SAP patients (0.88 ± 0.25 vs. 0.7 ± 0.23 U/kg/day, P = 0.003). Body mass index did not change during the observation period. No diabetic ketoacidosis episodes were observed during the follow-up, and severe hypoglycemia significantly decreased in SAP patients (P = 0.04). CONCLUSIONS The increased availability of continuous glucose sensors is likely to have a significant impact on pediatric diabetes therapy and education in the near future. In daily settings, patients using SAP can achieve a better control than patients using conventional insulin pump.

Blood ketone bodies in patients with recent‐onset type 1 diabetes (a multicenter study)

Background:  Insulin deficiency with glucagon excess leads to the release of ketone bodies (KBs) by the liver and excretion in the urine. So far, only KB monitoring in urine has been used during assessment of children with diabetes. Currently used nitroprusside strips for urine KB detection react only with acetoacetate (AcAc) and not with the most prevalent KB moiety – 3β‐hydroxybutyrate (3HB) – that is in equilibrium with AcAc (up to 10:1 ratio).