Poly(ADP-ribosylated) proteins in mononuclear cells from patients with type 2 diabetes identified by proteomic studies.

AIMS: In diabetes, hyperglycemia increases reactive oxygen species that induce DNA damage and poly(ADP-ribose)polymerase activation. The aim of this study is to characterize the proteomic profile and the role of poly(ADP-ribosylation) in patients with type 2 diabetes. METHODS: A proteomic platform based on 2DE and MALDI-ToF spectrometry was applied to peripheral blood mononuclear cells obtained from two different cohorts in which diabetic (n = 14) and normoglycemic patients (n = 11) were enrolled. RESULTS: Proteomic maps identified WD repeat protein, 78-kDa glucose-regulated protein precursor and myosin regulatory light chain 2, as unique proteins in diabetic patients; vimentin, elongation factor 2, annexin A1, glutathione S-transferase P, moesin and cofilin-1 as unique in the normoglycemic; and calreticulin, rho GDP-dissociation inhibitor 2, protein disulfide isomerase and tropomyosin alpha-4-chain as differentially expressed between the two cohorts. An enrichment in PARylation in diabetic patients was observed in particular, affecting GAPDH and α-Enolase leading to a decrease in their enzymatic activity. CONCLUSIONS: As the GAPDH and α-Enolase are involved in energy metabolism, protein synthesis and DNA repair, loss of their function or change in their activity can significantly contribute to the molecular mechanisms responsible for the development of type 2 diabetes. These data along with the proteomic profile associated with the disease may provide new insight into the pathophysiology of type 2 diabetes.

Could gestational diabetes mellitus be managed through dietary bioactive compounds? Current knowledge and future perspectives.

Gestational diabetes mellitus (GDM) is a serious problem growing worldwide that needs to be addressed with urgency in consideration of the resulting severe complications for both mother and fetus. Growing evidence indicates that a healthy diet rich in fruit, vegetables, nuts, extra-virgin olive oil and fish has beneficial effects in both the prevention and management of several human diseases and metabolic disorders. In this review, we discuss the latest data concerning the effects of dietary bioactive compounds such as polyphenols and PUFA on the molecular mechanisms regulating glucose homoeostasis. Several studies, mostly based on in vitro and animal models, indicate that dietary polyphenols, mainly flavonoids, positively modulate the insulin signalling pathway by attenuating hyperglycaemia and insulin resistance, reducing inflammatory adipokines, and modifying microRNA (miRNA) profiles. Very few data about the influence of dietary exposure on GDM outcomes are available, although this approach deserves careful consideration. Further investigation, which includes exploring the 'omics' world, is needed to better understand the complex interaction between dietary compounds and GDM.

Endothelial dysfunction markers as a therapeutic target for Sildenafil treatment and effects on metabolic control in type 2 diabetes.

OBJECTIVE: Endothelial dysfunction (ED) plays a role in diabetic cardiovascular complications. Hyperglycemia increases cytockines involved in vascular inflammation. Inhibition of phosphodiesterase type 5 (PDE5) exerts a relaxation on corpora cavernosa and has cardioprotective properties. The effect of chronic sildenafil treatment, on ED markers and metabolic parameters in a non-randomized study on men with type 2 diabetes (T2DM), was investigated. RESEARCH DESIGN AND METHODS: Twenty-eight T2DM patients (61.2 ± 7.8 years, hemoglobin A1c (HbA1c) 7.9 ± 1.3%, duration of diabetes 11.5 ± 7.8 years) were treated with sildenafil 100 mg/d for 3 months. Baseline and postprandial glycemia, insulin, HbA1c, HOMA index, lipids, glomerular filtration rate, homocysteine were assessed at each visit. P-selectin (CD62P), CD14/42b, CD14/41, ICAM (CD54), PECAM (CD31) and CD11b/CD18, were evaluated, after monocyte isolation with flow-cytometry, before and after treatment. RESULTS: After 3 months, sildenafil decreased P-selectin (p < 0.05), post-prandial glycemia (p < 0.01), HbA1c (p < 0.01), low-density lipoprotein cholesterol (p < 0.01) and increased high-density lipoprotein (p < 0.05). CONCLUSIONS: PDE5 inhibition, in T2DM patients, reduces the endothelial function marker P-selectin and exerts a beneficial effect on glycometabolic control.

Short-term effects of glucagon-like peptide 1 (GLP-1) receptor agonists on fat distribution in patients with type 2 diabetes mellitus: an ultrasonography study.

AIMS: Glucagon-like peptide 1 receptor agonists (GLP-1 RA) induce weight loss and reduction in adipose tissue, but the effects of GLP-1 RA on the distribution of fat deposits have been poorly investigated. METHODS: In 25 patients with type 2 diabetes (16 females and 9 males, mean age 63.5 ± 8.8 years), treated with GLP-1 RA (exenatide, n. 12; liraglutide, n.13), both before and 3 months after starting treatment, an abdominal ultrasonographic scan, with Doppler of renal arteries, and echocardiography were performed. Subcutaneous fat width (peri-umbilical and sub-xiphoid), deep fat deposits (pre-aortic, peri-renal, and epicardial), and renal resistive index (RI) were evaluated. RESULTS: GLP-1 RA induced highly significant (p < 0.001) decrease in BMI and in fat thickness at all the assessed sites, without differences between exenatide and liraglutide treatment. A slight decrease in RI (p = 0.055) was also found. The percent changes of fat thickness was different between sites (p < 0.025), and the changes in subcutaneous deposits showed no significant correlation (p = 0.064) with those of deep fat deposits. CONCLUSIONS: A short course of treatment with GLP-1 RA, besides weight loss, induces a redistribution of adipose tissue deposits, possibly contributing to a better cardiovascular risk profile in patients with type 2 diabetes mellitus.

Lack of the QTc physiologic decrease during cardiac stress test in patients with type 2 diabetes treated with secretagogues.

Patients with type 2 diabetes are at increased susceptibility to a prolonged QT interval. Furthermore, insulin secretagogues, drugs used to treat diabetes, may prolong QT interval and provoke arrhythmias. We evaluated whether secretagogues can affect QTc interval during cardiac stress test in 20 patients with type 2 diabetes treated with secretagogues. ECG stress test was performed in all patients. QTc interval was calculated both before cardiac stress test (BCST) and at acme of cardiac stress test (ACST). Diabetic patients treated with secretagogues showed longer QTc-ACST values than those treated with metformin only. QTc-ACST values resulted shorter than QTc-BCST values in control group. Diabetic patients treated with secretagogues showed QTc-ACST values significantly longer than QTc-BCST values. In our study, diabetic patients treated with secretagogues did not show the QTc physiologic decrease that is a protective against arrhythmias. These results suggest to evaluate, in these patients, QT length, even during routine cardiac stress test.

Chronic Inhibition of cGMP phosphodiesterase 5A improves diabetic cardiomyopathy: a randomized, controlled clinical trial using magnetic resonance imaging with myocardial tagging.

BACKGROUND: cGMP phosphodiesterase type 5 protein is upregulated in myocardial hypertrophy. However, it has never been ascertained whether phosphodiesterase type 5 inhibition exerts an antiremodeling effect in nonischemic heart disease in humans. We explored the cardioreparative properties of a selective phosphodiesterase type 5 inhibitor, sildenafil, in a model of diabetic cardiomyopathy. METHODS AND RESULTS: Fifty-nine diabetic men (60.3 ± 7.4 years) with cardiac magnetic resonance imaging consistent with nonischemic, nonfailing diabetic cardiomyopathy (reduced circumferential strain [σ], -12.6 ± 3.1; increased left ventricular [LV] torsion [θ], 18.4 ± 4.6°; and increased ratio of LV mass to volume, 2.1 ± 0.5 g/mL) were randomized to receive sildenafil or placebo (100 mg/d). At baseline, the metabolic indices were correlated with torsion, strain, N-terminal pro-B-type natriuretic peptide, vascular endothelial growth factor, monocyte chemotactic protein-1, and blood pressure. After 3 months, sildenafil produced a significant improvement compared with placebo in LV torsion (Δθ: sildenafil, -3.89 ± 3.11° versus placebo, 2.13 ± 2.35°; P<0.001) and strain (Δσ: sildenafil, -3.30 ± 1.86 versus placebo, 1.22 ± 1.84; P<0.001). Sildenafil-induced improvement of LV contraction was accompanied by consistent changes in chamber geometry and performance, with a 6.5 ± 11 improvement in mass-to-volume ratio over placebo (P=0.021). Monocyte chemotactic protein-1 and transforming growth factor-ß were the only markers affected by active treatment (Δmonocyte chemotactic protein-1: -75.30 ± 159.28 pg/mL, P=0.032; Δtransforming growth factor-ß: 5.26 ± 9.67 ng/mL, P=0.009). No changes were found in endothelial function, afterload, or metabolism. CONCLUSIONS: The early features of diabetic cardiomyopathy are LV concentric hypertrophy associated with altered myocardial contraction dynamics. Chronic phosphodiesterase type 5 inhibition, at this stage, has an antiremodeling effect, resulting in improved cardiac kinetics and circulating markers. This effect is independent of any other vasodilatory or endothelial effects and is apparently exerted through a direct intramyocardial action.

Transaminase levels in the upper normal range are associated with oral hypoglycemic drug therapy failure in patients with type 2 diabetes.

Incident diabetes and the worsening of diabetes have recently been linked to hepatic steatosis. Aim of our study was to determine whether oral hypoglycemic agent failure is associated with higher transaminase levels (valid measure of liver steatosis). We selected 200 patients, attenders (3 consecutive annual evaluations) in our clinic, with type 2 diabetes among which 100 with oral hypoglycemic agents failure and 100 who were still responsive to oral therapy. Failure to therapy was defined as glycated hemoglobin >7.5% despite maximal-dose oral therapy. We analyzed patient histories and laboratory data. Compared with oral-therapy-responsive patients, those with failure had a significantly higher level mostly of alanine aminotransferase at the time of therapy failure and 2 years before. They were more likely to have had symptoms of hyperglycemia at the time of diabetes diagnosis. Regression analysis indicated that each 5-unit increase in transaminase levels independently increased the risk for oral hypoglycemic agents failure by 1.70. Higher liver transaminase levels, especially in patients who had symptomatic hyperglycemia at diabetes diagnosis, associate with oral hypoglycemic agent failure. The possible pathogenetic link between transaminase and declining islet function might consist of insulin resistance and increased circulating fatty acid levels, in turn causing liver steatosis and beta-cell dysfunction.

Atorvastatin downregulates monocyte CD36 expression, nuclear NFkappaB and TNFalpha levels in type 2 diabetes.

AIM: Type 2 diabetes increases the risk for cardiovascular disease, and 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors (statins) reduce cardiovascular events in these patients. The benefits of statin therapy cannot be explained only by the lipid-lowering effect. The aim of this study was to test the effect of atorvastatin therapy on CD36 scavenger receptor expression, nuclear factor-kappaB (NFkappaB) levels and markers of inflammation (C-reactive protein, CRP, Tumor Necrosis Factor-alpha, TNF-alpha) in circulating monocytes from diabetic patients. METHODS: Twenty-two type 2 diabetic patients were treated for 8 weeks with atorvastatin (20 mg/day). At baseline and after treatment a blood sample was collected for measurement of glucose, lipid profile (total cholesterol, HDL, LDL cholesterol, triglycerides), glycated hemoglobin (HbA1c), CRP and for isolation of monocytes. RESULTS: Atorvastatin decreased total (p<0.0001) and LDL (p<0.01), and incresased HDL choles-terol (p<0.02). CD36 surface protein expression (anti-CD36 fluorescein isothiocyanate-FITC) was reduced in circulating monocytes after atorvastatin therapy (p<0.02) while immunoblot analysis showed reduced nuclear and increased cytoplasm NFkappaB levels (p<0.05). Finally, TNFalpha production in lipopolysaccharide-activated monocytes from patients treated with atorvastatin was reduced (p<0.05). CONCLUSION: These results suggest that atorvastatin therapy, beside lowering serum cholesterol levels, could exert anti-atherogenic and anti-inflammatory effects in type 2 diabetic patients.

Effect of insulin on airway responsiveness in patients with type 2 diabetes mellitus: a cohort study.

BACKGROUND: The correlation between low insulin levels and a decreased sensitivity of the muscarinic receptor has been shown on induced-diabetes animal models. We designed a cohort study with the aim of evaluating the effects of insulin therapy on airway responsiveness (AR) in human patients with type 2 diabetes mellitus. METHODS: We enrolled 92 patients with type 2 diabetes who had switched from oral anti-diabetic therapy to treatment by insulin subcutaneous injection. Patients were administered the methacholine challenge test (MCT) at time 0 (pre-insulin therapy) and at intervals of 15, 30, 90, 180, and 360 days after insulin treatment. The decline of forced expiratory volume in 1 second (FEV(1))% from baseline (Delta FEV(1)) in response to inhaled methacholine (MCH) was determined to assess airway hyper-responsiveness (AHR). RESULTS: A total of 81 patients (18 women and 63 men) completed the study. Their mean age was 58 +/- 7 years and the mean duration of disease was 13.5 +/- 7.7 years. The mean decrease of FEV(1) at pre-insulin assessment was 2.96 +/- 2.6%. Compared with the pre-insulin value, a significant increase of Delta FEV(1) was observed at 15, 30, and 90 days after treatment (6.25%, CI 95% 5.4 to 7.2, p = 0.0005; 7.64%, CI 95% 6.6 to 8.1, p < 0.001; 6.45%, CI 95% 5.5 to 7.3, p = 0.0004, respectively), while after 180 and 360 days AR was similar to pre-insulin values (Delta FEV(1), 3.62%, CI 95% 2.7 to 3.5 and 3.11%, CI 95% 7.9 to 9.3, respectively). CONCLUSIONS: The finding of an increased AR in patients with type 2 diabetes during the first 3 months of insulin therapy may underline the importance of monitoring pulmonary function and respiratory symptoms in patients switching from oral anti-diabetic drugs to insulin therapy, especially in the subset of individuals with respiratory disorders.

Poor glycemic control is an independent risk factor for low HDL cholesterol in patients with type 2 diabetes.

OBJECTIVE To determine whether the association observed between poor glycemic control and low HDL cholesterol in type 2 diabetes is dependent on obesity and/or hypertriglyceridemia. RESEARCH DESIGN AND METHODS We performed a cross-sectional study of 1,819 patients with type 2 diabetes and triglycerides <400 mg/dl enrolled at three diabetes centers in Italy. The risk for low HDL cholesterol was analyzed as a function of A1C levels. Odds ratios (ORs) were calculated after adjustment for confounding factors. RESULTS A 1% increase in A1C significantly increased the risk for low HDL cholesterol (OR 1.17 [95% CI 1.1-1.2], P = 0.00072); no changes were observed when age, sex, smoking, and lipid-lowering therapy were included in the model (1.17 [1.1-1.2], P = 0.00044). The association remained strong after adjustments for obesity and hypertriglyceridemia in multivariate analysis (1.12 [1.05-1.18], P = 0.00017). CONCLUSIONS Poor glycemic control appears to be an independent risk factor for low HDL cholesterol in type 2 diabetes.

Metabolic syndrome is not a risk factor for kidney dysfunction in obese non-diabetic subjects.

OBJECTIVE: To investigate whether insulin resistance (IR) and the metabolic syndrome (MS) are associated with kidney dysfunction in obese non-diabetic (OND) subjects. METHODS AND PROCEDURES: Three-hundred and eighty (113M/267F; age = 41 +/- 14 years) OND subjects (BMI > or = 30 kg/m(2); range = 43 +/- 8 kg/m(2)) were studied. Anthropometric measures, blood pressure, fasting glucose, insulin, lipid profile, and serum creatinine were evaluated. Glomerular filtration rate (GFR) was estimated (e-GFR) with the Modification of Diet in Renal Disease equation. Chronic kidney disease (CKD) was defined as e-GFR <60 ml/min/1.73 m(2). RESULTS: e-GFR was associated with gender (being lower in women) (P = 0.001) and age (P < 0.0001). CKD was present in 32 subjects (8.4%), who were older (P < 0.0001) and more frequently affected by hypertension (P = 0.04) as compared to subjects without CKD. MS was present in 212 (55.8%) subjects. They were older (P< 0.001), had lower e-GFR (P = 0.02) and were more frequently affected by CKD (odds ratio (OR), 95% confidence interval (CI) = 2.3, 1.1-5.1) than those without MS. However, differences in e-GFR values and in the risk of CKD were no longer statistically significant after adjusting for age (P = 0.99 for e-GFR and OR, 95% CI = 1.2, 0.5-2.8 for the risk of CKD, respectively). Homeostasis model assessment of IR (HOMA(IR)) index was neither higher in subject with CKD (P = 0.1) nor inversely correlated with e-GFR (r = 0.1, P = 0.1). DISCUSSION: In OND individuals the risk of CKD is independent of the MS and related abnormalities. This suggests that these individuals are not susceptible to a further deleterious role on kidney function on the top of that played by obesity itself.

Endothelial dysfunction and oxidative stress in type 1 and type 2 diabetic patients without clinical macrovascular complications.

AIMS: To evaluate the relationship between oxidative stress and endothelial dysfunction (ED) in diabetic patients without clinical macrovascular complications. METHODS: In 27 type 1, 56 type 2 diabetic patients and 35 healthy controls the redox state (GSH, GSSG; enzymatic method), endothelin-1 (ET-1; ELISA) and von Willebrand factor (vWF; ELISA) plasma levels, urinary vascular endothelial growth factor (VEGF; ELISA) were measured. RESULTS: Decreased GSH levels (p<0.05, type 1 and type 2), GSH/GSSG ratio (p<0.05 type 1, p<0.001 type 2) and elevated vWF levels (p<0.001, type 1 and type 2) were observed in diabetic patients in comparison with controls. A negative correlation between GSH and vWF (p<0.02 and p<0.001, in type 1 and type 2, respectively) and GSH and BMI (p<0.02 in type 1 and type 2) was observed. ET-1 was positively correlated to age (p<0.05) and diabetes duration (p<0.03) in type 1, while vWF was correlated to systolic blood pressure (p<0.05) in type 2 diabetic patients. Urinary VEGF was higher in type 2 (p<0.05) in comparison with type 1 diabetic patients and was correlated to glycemia (p<0.05) and systolic blood pressure (p<0.05). CONCLUSIONS: These data might indicate that markers of oxidative stress and ED are altered in diabetic patients without clinical macrovascular complications.

Antioxidant treatment associated with sildenafil reduces monocyte activation and markers of endothelial damage in patients with diabetic erectile dysfunction: a double-blind, placebo-controlled study.

OBJECTIVE: To investigate the synergic effect of propionyl L-carnitine (PLC) plus sildenafil in reducing monocyte oxidative activity and endothelial dysfunction markers in diabetic patients with erectile dysfunction (ED). METHODS: Thirty-two type 2 diabetic patients with ED (according to the International Index of Erectile Function-5 [IIEF-5]) were randomized to receive PLC (2 g/d) alone (n=8) or combined with sildenafil (50 mg/d twice weekly) (n=8), sildenafil alone (50 mg/d twice weekly) (n=8), or placebo (n=8) in a double-blind, fixed-dose study. Monocyte oxidative activity (stimulation index [SI]), intercellular adhesion molecule-1 [ICAM-1], P-selectin, advanced glycation end product (AGE) levels, Doppler sonography (recording peak systolic velocity [PSV]; end diastolic velocity [EDV]; systolic wave time [SWT]; resistive index [RI]), and IIEF score were evaluated before and after 12 wk of treatment; IIEF-5 was evaluated again 4 wk posttreatment. RESULTS: SI was reduced by treatment with PLC alone or combined with sildenafil (p<0.05). In patients treated with PLC plus sildenafil, a decrease in ICAM-1, P-selectin, and EDV values was observed compared with patients treated with sildenafil alone (p<0.05, p<0.01, p<0.001, respectively). IIEF-5 improved in all patients treated with PLC plus sildenafil or sildenafil alone (p<0.03, p<0.05, respectively). Four weeks posttreatment, patients treated with PLC plus sildenafil maintained the improvement of the IIEF-5 compared with patients on sildenafil alone (p=0.05). In patients on PLC treatment (with or without sildenafil), SI was correlated with IIEF-5 (p<0.001), glycemia with STW (p<0.03), and AGEs with IIEF-5 (p<0.01). CONCLUSION: PLC plus sildenafil was more effective in reducing SI and endothelial dysfunction markers in patients with type 2 diabetes and ED.

Circulating monocyte oxidative activity is increased in patients with type 2 diabetes and erectile dysfunction.

PURPOSE: We investigated the relationship between oxidative stress and diabetic erectile dysfunction. MATERIALS AND METHODS: A total of 23 patients with a mean +/- SD age of 56.7 +/- 5.6 years, a history of type 2 diabetes for 10.0 +/- 8.3 years and erectile dysfunction, as tested by the International Index of Erectile Function questionnaire, but without vascular and neurological complications, and 15 age matched patients with diabetes without erectile dysfunction were recruited. Circulating monocyte oxidative activity by cytofluorometry, and endothelin-1, intercellular adhesion molecule-1, plasminogen activator inhibitor-1 by enzyme linked immunosorbent assay were evaluated in all patients in the study. RESULTS: Monocyte free radical production, and total and low density lipoprotein cholesterol were higher in patients with than in those without erectile dysfunction (p <0.03, <0.02 and <0.05, respectively). In all patients the International Index of Erectile Function score inversely correlated with low density lipoprotein (p <0.05), while in patients with erectile dysfunction it negatively correlated with age (p <0.03), body mass index (p <0.02), endothelin-1 (p <0.02) and intercellular adhesion molecule-1 (p <0.05). Endothelin-1, intercellular adhesion molecule-1 and plasminogen activator inhibitor-1 were not different in patients with diabetes with and without erectile dysfunction. CONCLUSIONS: In men with type 2 diabetes who have erectile dysfunction but are asymptomatic for cardiovascular disease oxidative activation of monocytes is increased and it is related to other risk factors of endothelial dysfunction.

Poly(ADP-ribose)polymerase activity is reduced in circulating mononuclear cells from type 2 diabetic patients.

Poly(ADP-ribose)polymerase (PARP-1), a nuclear enzyme activated by DNA strand breaks, is involved in DNA repair, aging, inflammation, and neoplastic transformation. In diabetes, reactive oxygen and nitrogen species occurring in response to hyperglycemia cause DNA damages and PARP-1 activation. Because circulating mononuclear cells (MNCs) are involved in inflammation mechanisms, these cells were chosen as the experimental model to evaluate PARP-1 levels and activity in patients with type 2 diabetes. MNCs were isolated from 25 diabetic patients (18 M, 7 F, age, 63.5 +/- 10.2 years, disease duration 17.7 +/- 8.2 years) and 11 age and sex matched healthy controls. PARP-1 expression and activity were analyzed by semi-quantitative PCR, Western and activity blot, and immunofluorescence microscopy. PARP-1-mRNA expression was increased in MNCs from all diabetic patients versus controls (P < 0.01), whereas PARP-1 content and activity were significantly lower in diabetic patients (P < 0.0001). To verify whether low PARP-1 levels and activity were due to a proteolytic effect of caspase-3 like, the latter activation was measured by a fluorimetric assay. Caspase-3 activity in MNCs was significantly higher in diabetic patients versus control subjects (P < 0.0001). The different PARP-1 behavior in MNCs from patients with type 2 diabetes could therefore be responsible for the abnormal inflammation and infection responses in diabetes.