Francesca Urbano

miR-296-3p, miR-298-5p and their downstream networks are causally involved in the higher resistance of mammalian pancreatic α cells to cytokine-induced apoptosis as compared to β cells

BackgroundThe molecular bases of mammalian pancreatic α cells higher resistance than β to proinflammatory cytokines are very poorly defined. MicroRNAs are master regulators of cell networks, but only scanty data are available on their transcriptome in these cells and its alterations in diabetes mellitus.ResultsThrough high-throughput real-time PCR, we analyzed the steady state microRNA transcriptome of murine pancreatic α (αTC1-6) and β (βTC1) cells: their comparison demonstrated significant differences. We also characterized the alterations of αTC1-6 cells microRNA transcriptome after treatment with proinflammatory cytokines. We focused our study on two microRNAs, miR-296-3p and miR-298-5p, which were: (1) specifically expressed at steady state in αTC1-6, but not in βTC1 or INS-1 cells; (2) significantly downregulated in αTC1-6 cells after treatment with cytokines in comparison to untreated controls. These microRNAs share more targets than expected by chance and were co-expressed in αTC1-6 during a 6–48 h time course treatment with cytokines. The genes encoding them are physically clustered in the murine and human genome. By exploiting specific microRNA mimics, we demonstrated that experimental upregulation of miR-296-3p and miR-298-5p raised the propensity to apoptosis of transfected and cytokine-treated αTC1-6 cells with respect to αTC1-6 cells, treated with cytokines after transfection with scramble molecules. Both microRNAs control the expression of IGF1Rβ, its downstream targets phospho-IRS-1 and phospho-ERK, and TNFα. Our computational analysis suggests that MAFB (a transcription factor exclusively expressed in pancreatic α cells within adult rodent islets of Langerhans) controls the expression of miR-296-3p and miR-298-5p.ConclusionsAltogether, high-throughput microRNA profiling, functional analysis with synthetic mimics and molecular characterization of modulated pathways strongly suggest that specific downregulation of miR-296-3p and miR-298-5p, coupled to upregulation of their targets as IGF1Rβ and TNFα, is a major determinant of mammalian pancreatic α cells resistance to apoptosis induction by cytokines.

Cardiovascular Risk Profile in Subjects With Prediabetes and New-Onset Type 2 Diabetes Identified by HbA 1c According to American Diabetes Association Criteria

OBJECTIVE We investigated the cardiovascular risk profile in subjects with prediabetes and new-onset type 2 diabetes identified by glycated hemoglobin A1c (HbA1c) according to the new American Diabetes Association criteria. RESEARCH DESIGN AND METHODS Arterial stiffness, intima-media thickness (IMT), soluble receptor for advanced glycation end products (sRAGEs), and oral glucose tolerance test (OGTT) were evaluated in 274 subjects without a previous history of diabetes. The subjects were stratified into three groups according to the HbA1c levels. RESULTS The subjects with prediabetes (n = 117, HbA1c 5.7–6.4% [39–46 mmol/mol]) showed a higher augmentation (Aug), augmentation index (AugI), and IMT compared with those with lower HbA1c; however, these values were similar to those of subjects with HbA1c >6.5% (48 mmol/mol). When we further analyzed the subjects with prediabetes but included only subjects with normal glucose tolerance (NT) in the analysis, AugI and IMT still remained significantly higher than their levels in control subjects with HbA1c <5.7% (39 mmol/mol). After multiple regression analyses including several cardiovascular risk factors, only HbA1c, age, and sRAGE were significantly correlated with the IMT, whereas age and 1-h postload glucose were the major determinants of AugI. CONCLUSIONS Our data show that subjects with prediabetes according to HbA1c, but with both NT according to the OGTT and normal fasting glycemia, have an altered IMT and AugI. These data suggest that a simple, reproducible, and less expensive marker such as HbA1c may be better able to identify prediabetic subjects at high cardiovascular risk compared with fasting glycemia or OGTT alone.

Beta and alpha cell function in metabolically healthy but obese subjects: Relationship with entero-insular axis†

Obesity is widely acknowledged as a critical risk factor for metabolic complications. Among obese subjects, there is a phenotype of metabolically healthy but obese (MHO) individuals that shows a favorable cardiometabolic risk profile. We aimed to evaluate the potential mechanisms underlying the metabolic profile of this subset, including alpha and beta cell function and entero‐insular axis.

Chronic Exposure to GLP-1 Increases GLP-1 Synthesis and Release in a Pancreatic Alpha Cell Line (α-TC1): Evidence of a Direct Effect of GLP-1 on Pancreatic Alpha Cells

Aims/Hypothesis Incretin therapies, which are used to treat diabetic patients, cause a chronic supra-physiological increase in GLP-1 circulating levels. It is still unclear how the resulting high hormone concentrations may affect pancreatic alpha cells. The present study was designed to investigate the effects of chronic exposure to high GLP-1 levels on a cultured pancreatic alpha cell line. Methods α-TC1-6 cell line was cultured in the presence or absence of GLP-1 (100 nmol/l) for up to 72 h. In our model GLP-1 receptor (GLP-1R) was measured. After the cells were exposed to GLP-1 the levels of glucagon secretion were measured. Because GLP-1 acts on intracellular cAMP production, the function of GLP-1R was studied. We also investigated the effects of chronic GLP-1 exposure on the cAMP/MAPK pathway, Pax6 levels, the expression of prohormone convertases (PCs), glucagon gene (Gcg) and protein expression, glucagon and GLP-1 production. Results In our model, we were able to detect GLP-1R. After GLP-1 exposure we found a reduction in glucagon secretion. During further investigation of the function of GLP-1R, we found an activation of the cAMP/MAPK/Pax6 pathway and an increase of Gcg gene and protein expression. Furthermore we observed a significant increase in PC1/3 protein expression, GLP-1 intracellular content and GLP-1 secretion. Conclusions/Interpretation Our data indicate that the chronic exposure of pancreatic alpha cells to GLP-1 increases the ability of these cells to produce and release GLP-1. This phenomenon occurs through the stimulation of the transcription factor Pax6 and the increased expression of the protein convertase PC1/3.

Separate impact of metabolic syndrome and altered glucose tolerance on early markers of vascular injuries

OBJECTIVE We investigated the separate impact of metabolic syndrome (MS) and altered glucose tolerance on early markers of vascular injuries. METHODS Intima-media thickness (IMT) and pulse wave analysis (PWA), were evaluated in 132 overweight or obese subjects, with (MS(+)) or without (MS(-)) MS; subjects were further classified as normotolerant (NT) or with altered glucose tolerance (AGT) according to a 2 h oral glucose tolerance test (OGTT). RESULTS In MS(+) patients, IMT was higher than in the MS(-) group, and PWA revealed higher augmentation pressure (Aug, the contribution that wave reflection makes to systolic arterial pressure) and lower subendocardial viability ratio (SEVR, an estimate of myocardial perfusion). When analyzed according to glucose tolerance, IMT was higher in MS(+)NT subjects and AGT patients with and without MS, vs. MS(-)NT subjects. Logistic regression modeling showed that both AGT and MS were independently associated with increased IMT. However, only MS remained associated with IMT after adjustment for age. SEVR was reduced only in MS(+) patients, independently of glucose tolerance. In both groups, Aug and AugI were higher in the AGT group, but the correlation with 2 h-plasma glucose disappeared when corrected for age. CONCLUSION Both MS and AGT altered IMT, but the effect of AGT disappears when age is added to the multiple regression model. In contrast, arterial stiffness was affected differently in the two categories: in subjects with MS, the subendocardial viability ratio (an estimate of myocardial perfusion) was impaired, while in subjects with AGT, both Aug and AugI were increased. These data suggest that applying the definition of MS might help to better characterize cardiovascular risk in subjects with altered glucose tolerance or obesity.

Low Endogenous Secretory Receptor for Advanced Glycation End-Products Levels Are Associated With Inflammation and Carotid Atherosclerosis in Prediabetes.

CONTEXT Prediabetes is associated with atherosclerotic vascular damage. OBJECTIVE We investigated the correlation of endogenous secretory receptor for advanced glycation end-products (esRAGE), total soluble RAGE (sRAGE) and markers of inflammation, with early cardiovascular disease in subjects with prediabetes. We particularly focused on individuals with prediabetes identified only by glycated hemoglobin A1c (HbA1c) (5.7–6.4%) who had normal fasting glucose and were normotolerant after oral glucose tolerance test. DESIGN This was a cross-sectional study. SETTING The study was conducted in the Department of Clinical and Molecular Medicine, University of Catania, Italy. MAIN OUTCOME MEASURE sRAGE, esRAGE, carboxymethyl-lysine, S100A12, HbA1c, fasting glycemia, oral glucose tolerance test, pulse wave velocity, and intima-media thickness were evaluated in subjects with prediabetes. PATIENTS Three hundred eighty subjects without previous history of diabetes were stratified into three groups: controls (n = 99), prediabetes (n = 220), and new-onset type 2 diabetes (n = 61). RESULTS Subjects with prediabetes exhibited the following: lower esRAGE (0.29 ± 0.18 vs 0.45 ± 0.26 ng/mL; P < .05) and higher S100A12 levels than controls. RT-PCR analysis in mononuclear cells revealed that the mRNA expression level of the esRAGE splice variant progressively decreased in patients with prediabetes and type 2 diabetes with respect to controls. No difference was observed in sRAGE and carboxymethyl-lysine plasma levels between the groups. After multiple regression analyses, only age, HbA1c, and hs-CRP were independently associated with esRAGE levels. Age, HbA1c, and esRAGE were the major determinants of intima-media thickness, whereas S100A12 and systolic blood pressure were the major determinants of pulse wave velocity. When we analyzed the subjects with HbA1c prediabetes (normal fasting glucose/normotolerant and HbA1c 5.7–6.4%), esRAGE and inflammatory markers plasma levels still remained significantly different in respect to controls. CONCLUSIONS Subjects with HbA1c prediabetes exhibited significantly reduced esRAGE levels and increased levels of markers of inflammation. These alterations are associated with early markers of cardiovascular disease.

Altered expression of uncoupling protein 2 in GLP-1-producing cells after chronic high glucose exposure: implications for the pathogenesis of diabetes mellitus

Glucagon-like peptide-1 (GLP-1) is a gut L-cell hormone that enhances glucose-stimulated insulin secretion. Several approaches that prevent GLP-1 degradation or activate the GLP-1 receptor are being used to treat type 2 diabetes mellitus (T2DM) patients. In T2DM, GLP-1 secretion has been suggested to be impaired, and this defect appears to be a consequence rather than a cause of impaired glucose homeostasis. However, although defective GLP-1 secretion has been correlated with insulin resistance, little is known about the direct effects of chronic high glucose concentrations, which are typical in diabetes patients, on GLP-1-secreting cell function. In the present study, we demonstrate that glucotoxicity directly affects GLP-1 secretion in GLUTag cells chronically exposed to high glucose. Our results indicate that this abnormality is associated with a decrease in ATP production due to the elevated expression of mitochondrial uncoupling protein 2 (UCP2). Furthermore, UCP2 inhibition using small interfering RNA (siRNA) and the application of glibenclamide, an ATP-sensitive potassium (KATP(+)) channel blocker, reverse the GLP-1 secretion defect induced by chronic high-glucose treatment. These results show that glucotoxicity diminishes the secretory responsiveness of GLP-1-secreting cells to acute glucose stimulation. We conclude that the loss of the incretin effect, as observed in T2DM patients, could at least partially depend on hyperglycemia, which is typical in diabetes patients. Such an understanding may not only provide new insight into diabetes complications but also ultimately contribute to the identification of novel molecular targets within intestinal L-cells for controlling and improving endogenous GLP-1 secretion.

Update on pre-diabetes: Focus on diagnostic criteria and cardiovascular risk

Pre-diabetes, which is typically defined as blood glucose concentrations higher than normal but lower than the diabetes threshold, is a high-risk state for diabetes and cardiovascular disease development. As such, it represents three groups of individuals: Those with impaired fasting glucose (IFG), those with impaired glucose tolerance (IGT) and those with a glycated haemoglobin (HbA1c) between 39-46 mmol/mol. Several clinical trials have shown the important role of IFG, IGT and HbA1c-pre-diabetes as predictive tools for the risk of developing type 2 diabetes. Moreover, with regard to cardiovascular disease, pre-diabetes is associated with more advanced vascular damage compared with normoglycaemia, independently of confounding factors. In view of these observations, diagnosis of pre-diabetes is mandatory to prevent or delay the development of the disease and its complications; however, a number of previous studies reported that the concordance between pre-diabetes diagnoses made by IFG, IGT or HbA1c is scarce and there are conflicting data as to which of these methods best predicts cardiovascular disease. This review highlights recent studies and current controversies in the field. In consideration of the expected increased use of HbA1c as a screening tool to identify individuals with alteration of glycaemic homeostasis, we focused on the evidence regarding the ability of HbA1c as a diagnostic tool for pre-diabetes and as a useful marker in identifying patients who have an increased risk for cardiovascular disease. Finally, we reviewed the current evidence regarding non-traditional glycaemic biomarkers and their use as alternatives to or additions to traditional ones.

Atorvastatin but Not Pravastatin Impairs Mitochondrial Function in Human Pancreatic Islets and Rat β-Cells. Direct Effect of Oxidative Stress

Statins are a class of drugs widely prescribed as frontline therapy for lowering plasma LDL-cholesterol in cardiovascular risk prevention. Several clinical reports have recently suggested an increased risk of type 2 diabetes associated with chronic use of these drugs. The pathophysiology of this effect remains to be fully elucidated but impaired β-cell function constitutes a potential mechanism. The aim of this study was to explore the effect of a chronic treatment with lipophilic and hydrophilic statins on β-cell function, using human pancreatic islets and rat insulin-secreting INS-1 cells; we particularly focused on the role of mitochondria and oxidative stress. The present study demonstrates, for the first time, that atorvastatin (lipophilic) but not pravastatin (hydrophilic) affected insulin release and mitochondrial metabolism due to the suppression of antioxidant defense system and induction of ROS production in pancreatic β-cell models. Mevalonate addition and treatment with a specific antioxidant (N-AcetylCysteine) effectively reversed the observed defects. These data demonstrate that mitochondrial oxidative stress is a key element in the pathogenesis of statin-related diabetes and may have clinical relevance to design strategies for prevention or reduction of statin induced β-cell dysfunction and diabetes in patients treated with lipophilic statins.

HbA1c Identifies Subjects With Prediabetes and Subclinical Left Ventricular Diastolic Dysfunction

Context Prediabetes is associated with subclinical cardiac changes associated with heart failure development. Objective We investigated diastolic function and its association with markers of glycation and inflammation related to cardiovascular disease in patients with prediabetes. We focused on individuals with prediabetes identified only by glycated hemoglobin A1c [HbA1c; 5.7% to 6.4% and normal fasting glucose (NFG) and normal glucose tolerance (NGT) after an oral glucose tolerance test (OGTT)]. Design Cross-sectional study. Setting Departments of Clinical and Experimental Medicine and Cardiology, University of Catania, Catania, Italy. Main Outcome Measures HbA1c, OGTT, Doppler echocardiography, soluble receptor for advanced glycation end products (sRAGEs), and endogenous secretory RAGE (esRAGE) were evaluated. Patients We recruited 167 subjects with NFG/NGT who were stratified according to HbA1c level: controls (HbA1c <5.7%) and HbA1c prediabetes (HbA1c 5.7% to 6.4%). Results Patients with HbA1c prediabetes (n = 106) showed a lower peak mitral inflow in early diastole (E wave) to late diastolic atrial filling velocity (A wave) ratio (E/A ratio) than controls (n = 61) (1.10 ± 0.24 vs 1.18 ± 0.23; P < 0.05). They showed a higher left atrium volume (LAV) (28.4 ± 5 vs 22.1 ± 3; P < 0.05) and sphericity index (SI) (0.6 ± 0.06 vs 0.5 ± 0.05; P < 0.05). After multiple regression analyses, HbA1c, sRAGE, and esRAGE were the major determinants of E/A ratio, LAV, and SI. Conclusions Subjects with HbA1c prediabetes exhibited subclinical cardiac alterations associated with sRAGE, esRAGE, and HbA1c. These subjects would not have been classified as having prediabetes on the basis of fasting glycemia or post-OGTT values.