Glycation and lipid peroxidation in children and adolescents with type 1 diabetes mellitus with and without diabetic complications.

Glycation and oxidative stress lead to formation of compounds that have several biological effects and contribute to the development of the complications of diabetes mellitus. All steps of glycoxidation generate oxygen free radicals, some of them in common with lipid peroxidation pathways. Some oxidation or lipid peroxidation products may bind to proteins and amplify glycoxidation-generated lesions. The aim of this study was to measure glycation and lipid peroxidation parameters and examine the relationship between them in patients with type 1 diabetes mellitus (DM1) with (+DC) and without (-DC) diabetic complications. Fifty patients with DM1 aged from 7-19 years and with duration of DM1 (DD) at least 5 years were included. Twenty-four patients were -DC and 26 were +DC. Twelve healthy children formed a control group. There were significantly higher values of fructosamine (FAM), HbA(1c), serum advanced glycation endproducts (s-AGEs) and lipid peroxides (LPO) in the +DC group compared with -DC, and significantly higher values of HbA(1c), FAM and LPO in both diabetic groups than in controls. The s-AGEs level in the -DC group was similar to that in controls. In the total diabetic group, regardless of DC, there was a significant negative correlation between LPO and HDL-C (r = -0.379; p <0.05), and a positive correlation between LPO and triacylglycerol (TAG) (r = 0.852; p <<0.05), FAM (r = 0.414; p <0.05) and s-AGEs (r = 0.454; p <0.05). In the +DC group LPO correlated negatively with HDL-C (r = -0.392, p <0.05) and positively with TAG (r = 0.848; p <<0.05), FAM (r = 0.457; p = 0.02), and s-AGEs (r = 0.516, p = 0.02), whereas in the -DC group LPO correlated only with HDL-C (r = -0.441; p = 0.03) and TAG (r = 0.769; p <<0.05). We demonstrated a linkage between enhanced formation of AGEs and lipid peroxidation products and the presence of diabetic complications. Thus, the overproduction of glycation and lipid peroxidation products may take part in DC development as early as in childhood and adolescence.

HbA1c and serum levels of advanced glycation and oxidation protein products in poorly and well controlled children and adolescents with type 1 diabetes mellitus.

Diabetes mellitus is associated with hyperglycemia and with accelerated non-enzymatic glycation, increased oxidative stress and free radical production. The aim of the present study was to evaluate the levels of proteins glycation and oxidation parameters, compare them between poorly and well controlled children with type 1 diabetes mellitus, and determine the impact of glycemic control on these parameters. Blood and serum were obtained from 81 patients with type 1 diabetes mellitus (DM1) (20 patients had long-term good glycemic control [GGC], 61 patients had long-term poor glycemic control [PGC]). Thirty-one healthy children were used as controls. Fructosamine (FAM) was determined by a spectrophotometric method, HbA1c was measured by LPLC, serum advanced glycation end-products (s-AGEs) were determined fluorimetrically, and advanced oxidation protein products (AOPP) were measured spectrophotometrically. We observed significantly higher FAM, HbA1c, s-AGEs and AOPP levels in the patients with DM1 compared with controls, and significantly higher FAM, HbA1c and sAGEs levels in the PGC group compared with the GGC group. AOPP was higher in the PGC group than in the GGC group, but not significantly. In the PGC group we observed significant correlations between HbA1c and HDL-C (r = -0.306, p = 0.01), HbA1c and s-AGEs (r = 0.486, p < 0.001), and HbA1c and AOPP (r = 0.447, p < 0.01). s-AGEs significantly correlated with triacylglycerols (TAG) (r = 0.537, p < 0.001) and AOPP with HDL-C (r = -0.336, p < 0.05), TAG (r = 0.739, p < 0.001) and s-AGEs (r = 0.577, p < 0.001). In conclusion, our results showed both glycative and oxidative stress are increased in the PGC diabetic group compared with controls, they are linked with glycemic control, and probably contribute to the development of diabetic complications. We suggest that the measurement of not only HbA1c but also s-AGEs and AOPP may be useful to predict the risk of development of diabetic complications.

Identification of a novel beta-cell glucokinase (GCK) promoter mutation (-71G>C) that modulates GCK gene expression through loss of allele-specific Sp1 binding causing mild fasting hyperglycemia in humans.

OBJECTIVE: Inactivating mutations in glucokinase (GCK) cause mild fasting hyperglycemia. Identification of a GCK mutation has implications for treatment and prognosis; therefore, it is important to identify these individuals. A significant number of patients have a phenotype suggesting a defect in glucokinase but no abnormality of GCK. We hypothesized that the GCK beta-cell promoter region, which currently is not routinely screened, could contain pathogenic mutations; therefore, we sequenced this region in 60 such probands. RESEARCH DESIGN AND METHODS: The beta-cell GCK promoter was sequenced in patient DNA. The effect of the identified novel mutation on GCK promoter activity was assessed using a luciferase reporter gene expression system. Electrophoretic mobility shift assays (EMSAs) were used to determine the impact of the mutation on Sp1 binding. RESULTS: A novel -71G>C mutation was identified in a nonconserved region of the human promoter sequence in six apparently unrelated probands. Family testing established cosegregation with fasting hyperglycemia (> or = 5.5 mmol/l) in 39 affected individuals. Haplotype analysis in the U.K. family and four of the Slovakian families demonstrated that the mutation had arisen independently. The mutation maps to a potential transcriptional activator binding site for Sp1. Reporter assays demonstrated that the mutation reduces promoter activity by up to fourfold. EMSAs demonstrated a dramatic reduction in Sp1 binding to the promoter sequence corresponding to the mutant allele. CONCLUSIONS: A novel beta-cell GCK promoter mutation was identified that significantly reduces gene expression in vitro through loss of regulation by Sp1. To ensure correct diagnosis of potential GCK-MODY (maturity-onset diabetes of the young) cases, analysis of the beta-cell GCK promoter should be included.

CTLA-4 gene polymorphisms predispose to autoimmune endocrinopathies but not to celiac disease.

OBJECTIVES: The aim of the study was to determine the association of two CTLA-4 gene polymorphisms (CT60, +49 A/G) with Hashimoto thyroiditis (HT), type 1 diabetes mellitus (T1DM) and celiac disease (CD) as well as with the occurrence of multi-organ involvement by autoimmunity in children. METHODS: Genotyping was done by RFLP analysis in Slovak children with HT (n=63) and CD (n=120) and both Slovak and Slovene children with T1DM (n=320) and healthy controls (n=231). RESULTS: We found a significant association of the G allele of the CT60 polymorphism with HT (p<0,0005) in the Slovak population and T1DM in both Slovak (p<0.01) and Slovene populations (p<0.005). The G allele of the +49A/G polymorphism was significantly, though less strongly, associated with T1DM (p<0.05) and HT (p<0.05). Distribution of genotypes of CTLA-4 gene polymorphisms in CD patients did not differ significantly from controls. None of the polymorphisms was associated with multi-organ involvement by autoimmunity. CONCLUSION: The G allele of both examined CTLA-4 gene polymorphisms predisposes to HT and T1DM, but not to CD. No association with multi-organ involvement was found. The GG genotype of the CT60 polymorphism may identify CD patients at an increased risk for concomitant T1DM and HT. Further studies to assess the predictive value of CTLA-4 polymorphisms for the co-occurrence of HT and T1DM in CD patients are needed.