Alessandra Gallo

Rosiglitazone Reduces Glucose-Induced Oxidative Stress Mediated by NAD(P)H Oxidase via AMPK-Dependent Mechanism

Objective—Hyperglycemia is the main determinant of long-term diabetic complications, mainly through induction of oxidative stress. NAD(P)H oxidase is a major source of glucose-induced oxidative stress. In this study, we tested the hypothesis that rosiglitazone (RSG) is able to quench oxidative stress initiated by high glucose through prevention of NAD(P)H oxidase activation. Methods and Results—Intracellular ROS were measured using the fluoroprobe TEMPO-9-AC in HUVECs exposed to control (5 mmol/L) and moderately high (10 mmol/L) glucose concentrations. NAD(P)H oxidase and AMPK activities were determined by Western blot. We found that 10 mmol/L glucose increased significantly ROS production in comparison with 5 mmol/L glucose, and that this effect was completely abolished by RSG. Interestingly, inhibition of AMPK, but not PPAR&ggr;, prevented this effect of RSG. AMPK phosphorylation by RSG was necessary for its ability to hamper NAD(P)H oxidase activation, which was indispensable for glucose-induced oxidative stress. Downstream of AMPK activation, RSG exerts antioxidative effects by inhibiting PKC. Conclusions—This study demonstrates that RSG activates AMPK which, in turn, prevents hyperactivity of NAD(P)H oxidase induced by high glucose, possibly through PKC inhibition. Therefore, RSG protects endothelial cells against glucose-induced oxidative stress with an AMPK-dependent and a PPAR&ggr;-independent mechanism.

Correlation Between Baseline Characteristics and Clinical Outcomes in a Large Population of Diabetes Patients Treated with Liraglutide in a Real-World Setting in Italy

PURPOSE Treatment with liraglutide in randomized controlled trials is associated with significant reductions in glycated hemoglobin (HbA1c) and weight loss in type 2 diabetes patients. The aim of this retrospective observational study was to investigate correlations of glycemic control and weight outcomes with baseline characteristics of patients starting liraglutide in outpatient clinics in Italy. METHODS Type 2 diabetes patients were followed from baseline to 4, 8, and 12 months. Changes in glycemic parameters, weight, blood pressure, and lipids were assessed. Subanalyses were performed according to baseline characteristics. Multivariate linear and logistic regressions were used to assess correlations between glycemic efficacy, weight reduction, and liraglutide discontinuation after 12 months and baseline characteristics. FINDINGS Four hundred and eighty-one patients were included. Mean (SD) age at baseline was 57.3 (9.2) years, diabetes duration was 9.5 (6.8) years, weight was 106.7 (20.8) kg, body mass index (BMI; calculated as kg/m(2)) was 37.1 (6.6), HbA1c was 8.7% (1.3%), fasting plasma glucose was 168.5 (45.3) mg/dL; 38.2% were treated previously with insulin and 52.2% were treated with metformin alone. After 12 months, mean (SD) changes were HbA1c -1.2% (1.4%), fasting plasma glucose -28.3 (41.1) mg/dL, weight -3.5 (5.8) kg, BMI -1.3 (2.1), waist circumference -2.6 (6.7) cm (all, P < 0.001). Drop in weight and HbA1c did not differ between baseline BMI classes ≤30 or >30. Weight loss was unchanged among diabetes duration quartiles, and HbA1c reduction was significantly greater in patients with ≤4 years of diabetes duration (P = 0.01). Non-insulin-treated patients reached HbA1c ≤7% significantly more often than treated patients (44.2% vs 21.2%; odds ratio = 2.94; P < 0.001) and had significantly greater weight loss (-4.5 [8.2] kg vs -2.6 [5.4] kg; P = 0.03). Patients on metformin reached HbA1c target more frequently than others (43.1% vs 29.7%; odds ratio = 1.80; 95% CI, 1.05-3.07). Significant positive determinants for HbA1c reduction after 12 months were baseline HbA1c, age, and prior metformin monotherapy, and weight loss at 12 months was positively correlated with baseline weight, and negatively correlated with prior insulin treatment. Overall, 5.0% of patients interrupted liraglutide before the 12th month due to lack of glycemic control; they were less frequently treated with metformin only before liraglutide (29.2% vs 50.2%; P = 0.04). IMPLICATIONS Treatment with liraglutide in a real-world setting is associated with low therapy failure, good glycemic response, weight loss, and improvement in systolic blood pressure and lipid profile. The HbA1c drop did not differ among baseline BMI classes, indicating that efficacy is maintained in patients with lower BMI. The probability of reaching HbA1c ≤7% was significantly higher in patients previously treated with metformin alone and without any previous insulin. This could reinforce the hypothesis that better results with liraglutide could be achieved in patients after early metformin failure.

Heme oxygenase-1 is an important modulator in limiting glucose-induced apoptosis in human umbilical vein endothelial cells.

Hyperglycaemia is associated with oxidative stress. The inducible isoform of heme oxygenase (HO-1) is an effective system to counteract oxidative stress, yet it is unclear how hyperglycaemia affects HO-1. In this study, we explored: 1) the HO-1 protein content and HO activity in human umbilical vein endothelial cells (HUVECs) exposed to different glucose concentrations, and 2) the mechanisms which account for the high glucose-induced effects on HO-1. We evaluated HO-1 protein expression, HO activity, apoptosis and reactive oxygen species (ROS) in HUVECs treated for 48 h with 5.5, 10 and 20 mM glucose. A dose-dependent production of reactive oxygen species was observed. At 10 mM glucose, an increase of HO-1 protein expression and HO activity was observed, whereas at 20 mM, there was no change in protein content and activity relative to at 5.5 mM glucose. HO-1 protein expression in HUVECs exposed to 20 mM of glucose was increased in the presence of 20 U/ml superoxide dismutase (SOD). HO-1 gene silencing augments ROS production both at 5.5 and 10 mM glucose, leading to an increased apoptosis. We conclude that, in endothelial cells, the regulation of HO-1 by glucose is dependent upon levels of glucose itself. Lack of homeostatic HO-1 upregulation fails to protect from oxidative damage and results in a higher rate of apoptotic cell death.

Antibodies to post-translationally modified mitochondrial peptide PDC-E2(167–184) in type 1 diabetes

BACKGROUND Mitochondria play a role in type 1 diabetes (T1D) particularly in the treatment and prevention of disorder consequences. Due to their demonstrated role in diabetes pathology, mitochondrial proteins can be an interesting starting point to study candidate antigens in T1D. We investigated the role of relevant post-translational modifications (PTM) on a synthetic mitochondrial peptide as putative antigen. METHODS The antibody response in T1D was evaluated by solid phase-ELISA using a collection of synthetic peptides bearing different PTMs. We investigated the role of lipoylation, phosphorylation, and glycosylation. The PTMs were introduced at position 173 of the mitochondrial pyruvate dehydrogenase E2 complex peptide PDC-E2(167-184) and at position 7 of a structure-based designed β-turn peptide as an irrelevant sequence to investigate the role of the specific PDC-E2 peptide sequence. RESULTS IgM titres in 31 T1D patients were higher than IgGs to all the synthetic PTM peptides. Results demonstrated the crucial role of lysine lipoamide, serine O-phosphorylation, and O-glycosylation into the PDC-E2(167-184) peptide sequence for IgM antibody recognition. CONCLUSIONS Results highlight the importance of immune dysregulation in T1D, furthermore, if confirmed in a large number of patients, they will contribute to add novel diagnostic markers for the understanding the physiopathology of the disease.

Adult-Onset Type 1 Diabetes and Pregnancy: Three Case Reports

From 5% to 10% of diabetic patients have type 1 diabetes. Here we describe three cases of adult-onset type 1 diabetes in pregnancy treated at our clinic between 2009 and 2012. Two patients came for specialist examination during pregnancy, the third after pregnancy. These women had no prior overt diabetes and shared certain characteristics, that is, no family diabetes history, age over 35, normal prepregnancy BMI, need for insulin therapy as of the early weeks of pregnancy, and high-titer anti-GAD antibody positivity. The patients had persistent diabetes after delivery, suggesting that they developed adult-onset type 1 diabetes during pregnancy. About 10% of GDM patients become pancreatic autoantibody positive and the risk of developing overt diabetes is higher when two or more autoantibodies are present (particularly GAD and ICA). GAD-Ab shows the highest sensitivity for type 1 diabetes prediction. We need to bear in mind that older patients might conceivably develop an adult-onset type 1 diabetes during or after pregnancy. So we suggest that women with GDM showing the described clinical features shall be preferably tested for autoimmunity. Pregnant patients at risk of type 1 diabetes should be identified to avoid the maternal and fetal complications and the acute onset of diabetes afterwards.