Glibenclamide Mimics Metabolic Effects of Metformin in H9c2 Cells.

BACKGROUND: Sulfonylureas, such as glibenclamide, are antidiabetic drugs that stimulate beta-cell insulin secretion by binding to the sulfonylureas receptors (SURs) of adenosine triphosphate-sensitive potassium channels (KATP). Glibenclamide may be also cardiotoxic, this effect being ascribed to interference with the protective function of cardiac KATP channels for which glibenclamide has high affinity. Prompted by recent evidence that glibenclamide impairs energy metabolism of renal cells, we investigated whether this drug also affects the metabolism of cardiac cells. METHODS: The cardiomyoblast cell line H9c2 was treated for 24 h with glibenclamide or metformin, a known inhibitor of the mitochondrial respiratory chain. Cell viability was evaluated by sulforodhamine B assay. ATP and AMP were measured according to the enzyme coupling method and oxygen consumption by using an amperometric electrode, while Fo-F1 ATP synthase activity assay was evaluated by chemiluminescent method. Protein expression was measured by western blot. RESULTS: Glibenclamide deregulated energy balance of H9c2 cardiomyoblasts in a way similar to that of metformin. It inhibited mitochondrial complexes I, II and III with ensuing impairment of oxygen consumption and ATP synthase activity, ATP depletion and increased AMPK phosphorylation. Furthermore, glibenclamide disrupted mitochondrial subcellular organization. The perturbation of mitochondrial energy balance was associated with enhanced anaerobic glycolysis, with increased activity of phosphofructo kinase, pyruvate kinase and lactic dehydrogenase. Interestingly, some additive effects of glibenclamide and metformin were observed. CONCLUSIONS: Glibenclamide deeply alters cell metabolism in cardiac cells by impairing mitochondrial organization and function. This may further explain the risk of cardiovascular events associated with the use of this drug, alone or in combination with metformin.

Caveolin-1 is essential for metformin inhibitory effect on IGF1 action in non-small-cell lung cancer cells.

Metformin causes an AMP/ATP ratio increase and AMP-activated protein kinase (AMPK) activation. Since caveolin-1 (Cav-1) plays a role in AMPK activation and energy balance, we investigated whether Cav-1 could participate in metformin's inhibitory effect on IGF1 signaling. The effect of metformin was studied in two non-small-cell lung cancer (NSCLC) cell lines, Calu-1 and Calu-6, expressing higher and lower amounts of Cav-1, respectively. In Calu-1, but not in Calu-6 cells, metformin reduced phosphorylation of type 1 insulin-like growth factor receptor (IGF-IR) substrates Akt and Forkhead transcription factor 3a (FOXO3a), inhibited IGF1-dependent FOXO3a nuclear exit, and decreased IGF1-dependent cell proliferation. Here, we show that sensitivity of NSCLC cells to metformin was dependent on Cav-1 expression and that metformin required Cav-1 to induce AMPK phosphorylation and AMP/ATP ratio increase. Cav-1 silencing in Calu-1 and overexpression in Calu-6 reduced and improved, respectively, the inhibitory effect of metformin on IGF1-dependent Akt phosphorylation. Prolonged metformin treatment in Calu-6 cells induced a dose-dependent expression increase of Cav-1 and OCT1, a metformin transporter. Cav-1 and OCT1 expression was associated with the antiproliferative effect of metformin in Calu-6 cells (IC(50)=18 mM). In summary, these data suggest that Cav-1 is required for metformin action in NSCLC cells.

The plant hormone abscisic acid increases in human plasma after hyperglycemia and stimulates glucose consumption by adipocytes and myoblasts.

The plant hormone abscisic acid (ABA) is released from glucose-challenged human pancreatic ß cells and stimulates insulin secretion. We investigated whether plasma ABA increased during oral and intravenous glucose tolerance tests (OGTTs and IVGTTs) in healthy human subjects. In all subjects undergoing OGTTs (n=8), plasma ABA increased over basal values (in a range from 2- to 9-fold). A positive correlation was found between the ABA area under the curve (AUC) and the glucose AUC. In 4 out of 6 IVGTTs, little or no increase of ABA levels was observed. In the remaining subjects, the ABA increase was similar to that recorded during OGTTs. GLP-1 stimulated ABA release from an insulinoma cell line and from human islets, by ∼10- and 2-fold in low and high glucose, respectively. Human adipose tissue also released ABA in response to high glucose. Nanomolar ABA stimulated glucose uptake, similarly to insulin, in rat L6 myoblasts and in murine 3T3-L1 cells differentiated to adipocytes, by increasing GLUT-4 translocation to the plasma membrane. Demonstration that a glucose load in humans is followed by a physiological rise of plasma ABA, which can enhance glucose uptake by adipose tissues and muscle cells, identifies ABA as a new mammalian hormone involved in glucose metabolism.

Diabetes Resolution at 10 Years After Biliopancreatic Diversion in Overweight and Class 1 Obese Patients with Type 2 Diabetes.

BACKGROUND: Long-term anti-diabetic effects of BPD in overweight or class 1 obese T2DM patients were investigated reporting the results at 10 years after BPD performed in severely non-obese T2DM patients. MATERIAL AND METHODS: Thirty T2DM patients with BMI lower than 35 kg/m2 were investigated at 1, 5, and 10 years after BPD, and the results are compared with those of 30 T2DM patients followed for 10 years on pharmacological and/or behavioral conventional therapy. RESULTS: Mean levels of fasting blood glucose (FBG) and serum glycated hemoglobin (HbA1C) showed a marked reduction 1 year after BPD, values remaining slightly above the diabetic range throughout the entire follow-up. T2DM remission was observed in about 50% of the cases at 5 and 10 years after the operation. In 16 patients (53%), severe BPD-related complications developed, in ten cases requiring a surgical revision of the operation. In the BPD group, one patient died for malignant lymphoma and two patients after surgical revision. Within the control group, during the 10-year follow-up, no changes in the diabetic status were observed, being the FBG and HbA1C mean values higher than those recorded in the BPD patients at any follow-up time. All T2DM subjects of the control group were alive at the end of the 10-year follow-up. CONCLUSION: Despite satisfactory long-term metabolic outcomes, these data indicate that BPD should be used with caution as a metabolic procedure in the treatment of T2DM in overweight or class 1obese patients.

Effects of biliopanceratic diversion on type 2 diabetes in patients with BMI 25 to 35.

OBJECTIVE: Biliopancreatic diversion (BPD) resolves type 2 diabetes in near totality of morbidly obeses [BMI (body mass index) ≥35 kg/m]. However, studies of BPD effect in BMI range 25.0 to 34.9 kg/m, including about 90% of diabetic patients, are lacking. MATERIALS AND METHODS: If BPD effects are independent of weight changes, they should be maintained in patients who, being mildly obese or overweight, will lose little or no weight after operation. Thirty type 2 diabetic patients with BMI 25 to 34.9 were submitted to BPD and monitored 12 months. Thirty-eight diabetic patients selected from a large database, kept 1 year on medical therapy, served as controls. RESULTS: Nineteen male and 11 female. Mean age 56.4 ± 7.4 years, weight 84.8 ± 11.1 kg, BMI 30.6 ± 2.9 kg/m, waist circumference 104 ± 9.4 cm, diabetes duration 11.2 ± 6.9 years, HbA1c 9.3±1.5. Twelve patients on insulin. Fifteen (2 F) with BMI < 30 (mean: 28.1). No mortality or major adverse events occurred. BMI progressively decreased, stabilizing around 25 since the fourth month, without excessive weight loss. One year after BPD, mean HbA1c was 6.3%±0.8, with 25 patients (83%) controlled (HbA1c≤7%) on free diet, without antidiabetics, and the remaining improved. Acute insulin response to intravenous glucose had increased from 1.2 ± 2.9 to 4.2 ± 4.4 µIU/mL. Diabetes resolution correlated positively with BMI. HbA1c decreased at 1 year in the control group, along with an overall increased amount of antidiabetic therapy. CONCLUSIONS: BPD improves or resolves diabetes in BMI 25 to 35 without causing excessive weight loss, its action being on insulin sensitivity and beta-cell function. The strikingly different response between morbidly obese and low BMI patients might depend on different beta-cell defect. ClinicalTrials.gov Identifier: NCT00996294.

miR-126 Mimic Counteracts the Increased Secretion of VEGF-A Induced by High Glucose in ARPE-19 Cells.

Vascular endothelial growth factor-A (VEGF-A) has a pathologic role in microvascular diabetic complication, such as diabetic retinopathy (DR). miR-126 plays an important role in vascular development and angiogenesis by regulating the expression of VEGF-A. Since levels of miR-126 have been found downregulated in diabetes, this study is aimed at investigating whether hyperglycemia affects expression of miR-126 in a retinal pigment epithelium cell line. ARPE-19 cells were transfected with miR-126 inhibitor or with miR-126 mimic and the respective scramble negative control. After 24 hours, medium was replaced and cells were cultured for 24 hours in normal (CTR) or diabetic condition (HG). Then, we analyzed mRNA levels of miR-126, VEGF-A, PI3KR2, and SPRED1. We also evaluated protein amount of HIF-1α, PI3KR2, and SPRED1 and VEGF-A secretion. The results showed that exposure of ARPE-19 cells to HG significantly decreased miR-126 levels; mRNA levels of VEGF-A and PI3KR2 were inversely correlated with those of miR-126. Overexpression of miR-126 under HG restored HIF-1α expression and VEGF-A secretion to the level of CTR cells. These results indicate that reduced levels of miR-126 may contribute to DR progression by increasing expression of VEGF-A in RPE cells. In addition, we provide evidence that upregulation of miR-126 in RPE cells counteracts the rise of VEGF-A secretion induced by hyperglycemia. In conclusion, our data support a role of miR-126 mimic-approach in counteracting proangiogenic effects of hyperglycemia.

Optimization of flow reserve measurement using SPECT technology to evaluate the determinants of coronary microvascular dysfunction in diabetes.

PURPOSE: The aim of this study was to validate a new method to measure regional myocardial perfusion reserve (MPR) with technetium-labelled tracers in patients with type 2 diabetes mellitus (DM2). METHODS: A total of 40 consecutive DM2 patients without history of coronary artery disease (CAD) and 7 control subjects were recruited. Dipyridamole myocardial blood flow index (MBF) was assessed by measuring first transit counts in the pulmonary artery and myocardial count rate from gated SPECT images using (99m)Tc-labelled tracers. The corresponding MBF index was estimated 2 h later according to the same procedure. Regional myocardial perfusion reserve (MPR) was defined as the ratio between dipyridamole and baseline MBF using a 17-segment left ventricular (LV) model. Coronary flow reserve (CFR) was estimated by transthoracic contrast echo Doppler monitoring of flow velocity in the left anterior descending coronary artery (LAD) during the same session. RESULTS: Estimated MPR was higher in control subjects than in patients (3.36 +/- 0.66 vs 1.91 +/- 0.61, respectively, p < 0.01). In patients, LAD CFR and LAD MPR were 2.01 +/- 0.78 vs 1.93 +/- 0.63, respectively (p = ns). The agreement between the two techniques was documented by their close correlation (r = 0.92, p < 0.001) and confirmed by the Bland-Altman analysis. Reversible perfusion defects occurred in 13 patients (32%) who showed similar MPR values as the remaining 27 (2.10 +/- 0.71 vs 1.83 +/- 0.71, respectively, p = ns). Finally, MPR was closely correlated with age (r = -0.50, p < 0.01) and time elapsed from the diagnosis of DM2 (r = -0.51, p < 0.01). CONCLUSION: LV regional MPR can be accurately estimated with the broadly available single photon technology. Application of this method to DM2 patients documents the presence of a microvascular dysfunction homogeneously distributed throughout the LV walls and most frequently not associated with reversible perfusion defects.

Antiapolipoprotein A-1 Autoantibody Positivity Is Associated with Threatened Abortion.

BACKGROUND: Autoantibodies against apolipoprotein A-1 (anti-ApoA-1 IgG) were demonstrated to be associated with cardiovascular outcomes in several inflammatory diseases. As balanced inflammation is critical for uncomplicated pregnancy, we aimed to investigate the prevalence of anti-ApoA-1 IgG and anti-c-terminal ApoA-1 autoantibodies (Ac-terAA1 IgG) in a cohort of pregnant women and their potential relationship with threatened abortion (TA). METHODS: Between 2012 and 2014, 371 consecutive outpatient pregnant women were included in this study and followed until delivery. Anti-ApoA-1 and anti-Ac-terAA1 IgG were measured by ELISA technique on serum samples collected between the 24th and 26th week of pregnancy. Associations with TA were tested using linear regression analysis and C-statistics. RESULTS: Median age was 34 with a prevalence of the Caucasian ethnicity (90.5%). TA occurred in 10 women (2.7%). C-statistics indicated that anti-ApoA-1 and anti-Ac-terAA1 IgG levels upon study inclusion were predictive of TA (0.73, 95% confidence interval [CI] 0.69-0.78, p < 0.001 and 0.76, 95% CI 0.71-0.80, p < 0.001 and 0.76, 95% CI 0.71-0.80, p < 0.001 and 0.76, 95% CI 0.71-0.80, p < 0.001 and 0.76, 95% CI 0.71-0.80. CONCLUSION: Anti-ApoA-1 and anti-Ac-terAA1 IgG are independently associated with TA during pregnancy with an appealing NPV. The causal biological mechanisms underlying this association as well as the possible clinical relevance of these findings require further investigations.

Insulin analogues: fears, facts and fantasies.

IGF-I and insulin, acting through both IGF-I and insulin receptors, have been studied widely to evaluate their oncogenic and teratogenic properties. These two properties need to be studied for each new insulin analogue, in addition to measurements of their metabolic and pharmacodynamic features. This editorial critiques a study in this issue of the journal of several insulin analogues in their action in vitro on several cancer-related cell lines. The conclusions and limitations of these studies are highlighted, especially as they influence guidelines for using these analogues patients.

Glycosylated haemoglobin (A1c) best values for type 2 diabetes in the battlefield much ado about nothing? (apparently).

Despite intensive research, therapy of diabetes mellitus type 2 (T2DM) is far from be effective. The most important unresolved issue is to establish a safe glycosylated hemoglobin C (A1c) value well balanced between benefit and side effects. As a result different guidelines suggest different A1c targets generating confusion for patients and clinicians. Here we report two observations which might support a relaxed A1c as suggested by American college of physician (ACP).

Adipose Tissue Composition in Obesity and After Bariatric Surgery.

The adipose tissue is a complex organ that regulates food intake and energy expenditure as well as induces low-grade inflammation. This review deals with changes in the composition and activity of the adipose organ after bariatric surgery, focusing on epicardial and ectopic fat and on relationships between white and brown adipose tissues. Postoperative improvements of ectopic fat and epicardial fat size and composition account for the metabolic recovery and the decreased cardiovascular risk. Following Roux-en-Y gastric bypass or biliopancreatic diversion, a proportional increase in the size and activity of the metabolically active brown adipose tissue was observed, most likely related to the postoperative rearrangement of the entero-hormonal pattern with an increase of GLP-1 production: this aspect would promote the postoperative weight loss and maintenance of post-surgery benefits.

Advanced Glycation End-Products and Hyperglycemia Increase Angiopoietin-2 Production by Impairing Angiopoietin-1-Tie-2 System.

The angiopoietin-Tie-2 system plays a crucial role in the maintenance of endothelial integrity. Hyperglycemia and advanced glycation end-products (AGEs) are involved in endothelial cell dysfunction responsible of the pathogenesis of microvascular complications of diabetes. Here, we investigated whether glycated serum (GS) or hyperglycemia (HG) affect the angiopoietin-Tie-2 system in the microvascular endothelial cells HMEC-1. We found that culture for 5 days in the presence of AGEs and HG (alone or in combination) decreased cell proliferation, increased reactive oxygen species (ROS) production, and reduced ratio between the oxidized and the reduced form of glutathione. Since angiopoietin-1 (Ang-1) signaling regulates angiopoietin-2 (Ang-2) expression through inactivation of the forkhead transcription factor FoxO1, we investigated intracellular signaling of Ang-1 and expression of Ang-2. HG and AGEs reduced phosphorylation of Akt and abrogated phosphorylation of FoxO1 induced by Ang-1 without affecting neither Tie-2 expression nor its activation. Furthermore, AGEs and/or HG induced nuclear translocation of FoxO1 and increased Ang-2 production. In conclusion, we demonstrated that both hyperglycemia and AGEs affect the angiopoietin-Tie-2 system by impairing Ang-1/Tie-2 signaling and by increasing Ang-2 expression. These results suggest that therapeutic strategies useful in preventing or delaying the onset of diabetic vascular complications should be aimed to preserve Ang-1 signaling.

Type 2 Diabetes Remission and Control in Overweight and in Mildly Obese Diabetic Patients at Long-Term Follow-Up After Biliopancreatic Diversion.

BACKGROUND: In severely obese patients with type 2 diabetes (T2DM), the metabolic benefits after biliopancreatic diversion (BPD) are due to mechanisms independent of weight loss. Therefore, the anti-diabetic effect of BPD in overweight or mildly obese T2DM patients was investigated. METHODS: Ninety T2DM patients with BMI 25-35 underwent BPD and were evaluated 1 and 5 years after the operation (follow-up rate 100 and 83%, respectively). RESULTS: T2DM control (Hb1Ac < 7%) and remission (Hb1Ac < 6 without antidiabetics) was observed in 86.6 and 65% of cases at 1 year and 64.0% and 26.5% at 5 years, respectively. The long-term T2DM remission was predicted by baseline BMI value. Both before BPD and throughout the follow-up period, HOMA values were similar in the metabolically successful and unsuccessful subjects, while C-peptide normalized for FBG value as a marker of beta cell mass and insulin secretion increased progressively only in the former from 1.06 ± 0.64 to 1.44 ± 1.08 mcg/l ml/dl-1 * 100 (p < 0.002). CONCLUSIONS: In T2DM patients with BMI of 25-35, a positive metabolic outcome is less frequent than in their counterparts with morbid obesity. In T2DM overweight patients, in spite of a short-term normalization of FBG and HbA1c levels and a well-sustained increase of insulin sensitivity, a long-term T2DM relapse occurs in the majority of the cases. While the surgically obtained decrease in insulin resistance leads to T2DM control in half of the patients, the increase in insulin secretion is mandatory for T2DM stable remission.

Baseline neutrophil-to-lymphocyte ratio is associated with long-term T2D remission after metabolic surgery.

AIMS: Metabolic surgery is considered as a therapeutic option for obese patients with type 2 diabetes (T2D). In order to identify novel laboratory variables that could improve the selection of patients who might greatly benefit from a surgical approach, we focused on the neutrophil-to-lymphocyte ratio (NLR) as a predictor of long-term T2D remission following metabolic surgery. METHODS: Thirty-one obese patients with T2D included in this pilot study underwent Roux-en-Y gastric bypass or biliopancreatic diversion (BPD) at the Surgical Department of Genoa University, IRCCS Ospedale Policlinico San Martino in Genoa (Italy). Before surgery, serum samples were collected to evaluate blood count, glycemic profile, and circulating neutrophil degranulation products. RESULTS: The median age was 56 years, median body mass index (BMI) was 32.37 kg/m2, and median glycated hemoglobin was 8.4%. White blood cell count was in a range of normality, with a median NLR of 1.97. By a receiver operating characteristic curve analysis, NLR has been found to be significantly associated with T2D remission at 1, 3, and 5 years and the best cutoff of ≤ 1.97 has been identified by Youden index. When comparing study groups according to NLR cutoff, those with NLR ≤ 1.97 were older and underwent more often BPD. By a logistic regression analysis, NLR ≤ 1.97 has been found to predict T2D remission across 5 years, irrespective of baseline BMI. CONCLUSIONS: A baseline low NLR is associated with long-term T2D remission in obese patients undergoing metabolic surgery, suggesting that circulating inflammatory cells (i.e., neutrophils) might negatively impact on T2D remission.

Caveolin-1 down-regulation inhibits insulin-like growth factor-I receptor signal transduction in H9C2 rat cardiomyoblasts.

Caveolin (Cav)-1, the major caveolar protein, directly interacts with IGF-I receptor (IGF-IR) and its intracellular substrates. To determine the role of Cav-1 in IGF-IR signaling, we transfected H9C2 cells with small interfering RNA specific for Cav-1-siRNA. The selective down-regulation of Cav-1 (90%) was associated with a smaller reduction of Cav-2, whereas Cav-3 expression was unaffected. A significant reduction of IGF-IR tyrosine phosphorylation in Cav-1-siRNA H9C2 cells was found compared with H9C2 control cells (Ctr-siRNA). The reduced IGF-IR autophosphorylation resulted in a decrease of insulin receptor substrate-1, Shc, and Akt activation. In addition, in Cav-1-siRNA H9C2 cells, IGF-I did not prevent apoptosis, suggesting that Cav-1 is required to mediate the antiapoptotic effect of IGF-I in cardiomyoblasts. The down-regulation of Cav-1 decreased IGF-IR activation and affected the ability of IGF-I to prevent apoptosis after serum withdrawal also in human umbilical vein endothelial cells. These results demonstrate that: 1) Cav-1 down-regulation negatively affects IGF-IR tyrosine phosphorylation; 2) this effect causes a reduced activation of insulin receptor substrate-1, Shc, and Akt; and 3) Cav-1 is involved in IGF-IR antiapoptotic signaling after serum deprivation.

Levels of serum uric acid at admission for hypoglycaemia predict 1-year mortality.

AIMS: Hypoglycaemia represents a critical burden with clinical and social consequences in the management of diabetes. Serum uric acid (SUA) has been associated with cardiovascular diseases (CVD), but no conclusive findings are available nowadays in patients suffering from hypoglycaemia. We investigated whether SUA levels at the time of hypoglycaemia could predict all-cause mortality after 1-year follow-up. METHODS: In total, 219 patients admitted to the Emergency Department (ED) of Ospedale Policlinico S. Martino of Genoa (Italy) have been enrolled between January 2011 and December 2014. The primary endpoint of the study consisted in determining whether SUA levels at the time of ED admission could predict the occurrence of death after 1 year. RESULTS: The majority of patients were diabetic, especially type 2. CVD and chronic kidney disease were prevalent comorbidities. By a cut-off value obtained by the receiver operating characteristic curve analysis, a Kaplan-Meier analysis demonstrated that patients with SUA levels > 5.43 mg/dL were more prone to death after 1 year compared to those with lower SUA levels. The risk of death increased with high SUA levels both in the univariate and the multivariate models including estimated glomerular filtration rate, C-reactive protein, type of diabetes, and age-adjusted Charlson comorbidity index. CONCLUSIONS: SUA could be useful as a predictor of 1-year mortality in hypoglycaemic patients, irrespective of severe comorbidities notably increasing the risk of death in these frail patients.

Skin and diabetes: an experts' opinion from the Italian diabetologists and dermatologists of the DiaDex group.

The metabolic changes associated with diabetes mellitus (DM) affect a variety of organs and systems, including the skin. Skin lesions are frequently observed in patients with DM, resulting from a complex interaction among biochemical, vascular, immune, and metabolic changes. Cutaneous manifestations may develop at any time in the course of DM. They can be the first sign of the disease, possibly helping in diagnosis, or represent a marker of poor glycemic control. Given the high prevalence of cutaneous manifestations in DM, their possible role in favoring DM early diagnosis, and their relationship with the patient's metabolic control, a group of Italian dermatologists and diabetologists, the DiaDex expert group, jointly formulated a few basic statements aimed at favoring a stricter interdisciplinary cooperation in order to improve patients' management. Deeper knowledge of the skin lesions most commonly associated with DM, their early identification, and prompt reciprocal referral, when appropriate, are the pivotal points of these statements and should represent the pillars of such desired cooperation. The dermatologists and diabetologists of the DiaDex group believe that their different diagnostic and therapeutic skills put together may significantly benefit the many DM patients with cutaneous complications and hope that this paper may provide some guidance on how to achieve this goal.