Diabetes and Cardiovascular Disease (D&CVD) EASD Study Group Annual Meeting; Milan, Italy, 22nd – 24th June 2017, Milan, Italy

Comparative proteomic analysis identifies novel peptides in the cellular model of glucose variability: a preliminary study in HUVECs

L. La Sala1, D. Capitanio2, S. Genovese1, C. Gelfi2, A. Ceriello1,3
1) IRCCS MultiMedica, Lab Diabetes and CVD, Milan, Italy
2) University of Milan, Milan, Italy
3) IDIBAPS, Barcelona, Spain

Background and aims: Experimental findings and clinical observations suggest that glucose variability (GV) is considered an independent risk factor for cardiovascular complication of diabetes. Although it has been suggested that delay, or prevention, of complications is closely related to a glycaemic control, the identifications of proteins responsible for the failure in glucoregulation are still not fully elucidated. In this work, we sought to study the changes of protein signature in in vitro model of GV, employing proteomic approach.

Materials and methods: 2-DE coupled with matrix assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF/TOF-MS) was used as proteomic technique in order to investigate differentially expressed proteins in a cellular model of GV. HUVECs cultured in i) NG, normal glucose (5 mmol/l), ii) OG, oscillating glucose (5 – 25 mmol/l), and iii) HG, high glucose (25 mmol/l) for 3 weeks were used.

Results: 292 differentially expressed protein spots were detected by 2D-DIGE, and 89 spots were analyzed in MALDI-TOF/TOF-MS. Our proteome data reflect a large portion of proteins, which have not been reported before for our model. By comparing proteomic results with respect to normal glucose, 39 protein isoforms showed the same upregulation in OG and HG, whereas 46 proteins were downregulated in both conditions. Only 3 proteins showed differential expression between OG and HG. Specifically, the upregulation was found in OG, concomitantly with the downregulation in HG. The founded proteins were categorized into groups based on their function and the most of them belonged to energy and metabolism pathway, cell structure, inflammation, redox and defense categories. Differential expression of key proteins was also crosschecked at transcriptional level by using qRT-PCR.

Conclusions: The comparative proteome analysis conducted here revealed the involvement of novel key proteins in our model of GV, and further investigation will be required to establish mechanistic biochemistry related to this cellular phenotype, that could assist in the discovery of new strategies for innovative therapies.


Hyperglycaemia-induced miR-21 is linked to ROS generation: a comparison between in vitro and ex vivo studies

L. La Sala, E. Sangalli, S. Mrakic-Sposta, A. Uccellatore, C. Specchia, G. Spinetti, S. Lupini, P. de Candia, S. Genovese, A. Ceriello

Oxidative stress is the main pathogenic cause of diabetes. MicroRNAs (miRs), small non-coding RNA of 22 nucleotides, are emerged as epigenetic molecules able to regulate gene expression, detectable in tissues, cells and in biological fluids suggesting their diagnostic potential. MiR-21 is one of the miRNAs that contribute to the development of cardiovascular diseases such as cardiac hypertrophy and cardiovascular remodelling. Recently, miR-21 has been related with diabetes but its function is poorly elucidated. Since reactive oxygen species (ROS) generation has been suggested to be involved in the appearance of diabetic complications, we explored a possible relationship between miR-21 and oxidative stress.

Here, the effects of different models of hyperglycaemia in cellular model, constant high glucose (resembling diabetes) and glucose variability (resembling impaired glucose tolerance, IGT) were tested on oxidative stress generation and miR-21 levels in human endothelial cells. We also sought to monitor and compare the plasma levels of miR-21 and 4-hydroxynonenal (4-HNE) as a marker of oxidative stress among a cohort of subjects with IGT and newly diagnosed, drug naïve T2DM.

We found in HUVECs a pronounced oxidative stress, evaluated by electronic paramagnetic resonance (EPR), concomitant with significant miR-21 elevation, and the involvement of the mitochondria redox imbalance. All these effects high-glucose-mediated, were reverted with anti-miR-21 inhibitor, suggesting the contribution of miR-21 in mitochondrial redox imbalance of diabetes. We also tested plasma of miR-21 (c-miR-21) and of 4-hydroxynonenal (4-HNE), as oxidative stress marker, in a selected cohort (n = 81) of subjects enrolled in the DIAPASON (Diabetes Prediction And Screening Observational study) screened on ADA criteria for 2hPG and grouped in IGT and newly diagnosed, drug naïve T2DMs (T2D). C-miR-21 expressions were significantly elevated in IGT and T2D subjects, were positively correlated with glycaemic parameter, 2hPG, and the diagnostic accuracy of c-miR-21 was high and accurate. Also significant accumulation of 4-HNE was found in IGT and T2D subjects. These data suggest that miR-21 elevated in circulation may represent the degree of damage, ROS-induced. Therapies and behavioural changing capacities to reduce the impact of oxidative stress may be beneficial to deplete diabetes-associated interventions.


High level of circulating microRNA-15a is associated with impaired glucose tolerance: data from the DIAPASON study

G. Spinetti1, A. Uccellatore1, C. Specchia1,2, E. Sangalli1, S. Gasparino1, S. Lupini1, P. de Candia1, P. Madeddu3, A. Ceriello1, S. Genovese1
1) IRCCS MultiMedica, Milan, Italy
2) University of Brescia, Brescia, Italy
3) University of Bristol, Bristol, UK

Background: The asymptomatic period before the onset of type 2 diabetes mellitus (T2DM) presents opportunities for prevention of the disease and its vascular complications. MicroRNAs (miRs), small non-coding RNAs that regulate gene expression, are easily detectable in body fluids and may represent valuable disease biomarkers. High levels of circulating miR-15a are associated with T2DM micro- and macrovascular complications including retinopathy and limb ischaemia.

Aim: Analyze whether quantification of plasmatic miR-15a is able to provide with useful information about the early phases of T2DM.

Methods: Subjects at high risk for diabetes were selected among those enrolled in a clinical study for the prediction and early diagnosis of diabetes mellitus (DIAPASON: DIAbetes Prediction And Screening Observational study). Three groups were identified according to diagnostic procedures: 1) newly diagnosed, drug naïve (T2DM, n = 25), 2) patients with impaired glucose tolerance (IGT, n = 25), and 3) subjects with normal glucose tolerance (NGT, n = 26). MiR-15a relative quantity to Cel-miR-39 has been measured in plasma by RTqPCR. MiR levels have been compared among groups and the correlation with clinical parameters associated with T2DM was assessed.

Results: Abundance of miR-15a significantly increased in plasma of patients with newly diagnosed T2DM and with IGT compared to NGT subjects also after adjustment for gender and age. Significant positive correlation was observed between miR-15a quantity and fasting glucose levels, HbA1c percentage, insulinaemia, HOMA index, and body mass index.

Conclusions: Prevention of the complications is the primary objective of treating people with T2DM. Our data demonstrate the association of elevated blood circulating miR-15a with early phases of T2DM. Future studies to understand the role of miR-15a in delivering a pathological signal predisposing to diabetic vasculopathy are ongoing.


Altered heart function in high sucrose-fed overweight rats: in vivo and in vitro investigations

E. Arioglu-Inan1, A. Durak2, Y. Olgar2, E. Tuncay2, G. Kayki-Mutlu1, I. Karaomerlioglu1, V. M. Altan3, B. Turan2
1) Department of Pharmacology, Faculty of Pharmacy, Ankara University, Ankara, Turkey
2) Department of Biophysics, Faculty of Medicine, Ankara University, Ankara, Turkey
3) Department of Pharmacology, Faculty of Pharmacy, Bezmi Alem Vakif University, Istanbul, Turkey

Aims: Mechanical activity of the heart is adversely affected by metabolic syndrome (MetS), which is characterized by increased body mass and insulin resistance. Thus, we aimed to investigate the effects of high carbohydrate intake on cardiac functional abnormalities through evaluating in situ heart work, heart rate and electrocardiograms (ECG) in rats.

Methods: MetS was induced in Wistar male rats by adding 32 % sucrose to their drinking water for 22 – 24 weeks. Heart work was evaluated through PV loop analysis. Beta adrenergic responsiveness was measured by Langendorff heart preperation. ECGs were recorded in situ through the animals’ paws using two home-made electrodes. Histological examination was made with Masson’s Trichrome staining. Total oxidant/antioxidant status and cAMP levels were measured with proper kits.

Results: MetS is confirmed with a pattern characterized by increased body weight, blood glucose and insulin, insulin resistance, elevated systolic and diastolic blood pressures, and furthermore, significant left ventricular integrity loss and increased connective tissue around myofibrils. Analysis of in situ ECG recordings showed markedly shortened QT interval and depressed QRP with increased heart rate together with markedly high beta adrenergic responsiveness. We also observed augmented oxidative stress and decreased antioxidant defense characterized by decreases in serum total thiol level and attenuated paraoxonase and arylesterase activities.

Conclusion: Increased heart rate, shortened QT interval, increased left ventricular developed pressure mediated by beta adrenergic receptor stimulation and decreased cAMP levels together indicate a compensative mechanism in MetS rats. MetS is known to result further in obesity and insulin resistance, thus, alterations in heart function in MetS should be interpreted carefully as it could lead to cardiac dysfunction gradually.

Grant support: This study is supported by TUBITAK (SBAG-214S254).


The effects of sitagliptin treatment on beta 3 adrenergic receptor mediated relaxation responses in streptozotocine-induced diabetic rat aorta

A. E. Muderrisoglu, B. R. Erdogan, Z. E. Yesilyurt, C. Uyar-Boztas, I. Karaomerlioglu, V. M. Altan, E. Arioglu-Inan

Aims: Diabetes mellitus is a chronic metabolic disease which increases the cardiovascular risk. It is well known that diabetes leads to endothelial dysfunction as a result of decreased endothelium mediated relaxation reponses. DPP4 inhibitors, being used for diabetes treatment, have been recently shown to have vasoprotective effects as they improve vascular relaxation responses. It has been shown that beta 3 adrenergic receptors are located on the vascular endothelium and they contribute to nitric oxide (NO) mediated vasodilatation. However, it has not been studied whether DPP4 inhibitors affect beta 3 adrenergic receptor mediated vasorelaxation. Thus, we aimed to investigate the possible effects of sitagliptin, a DPP4 inhibitor, on beta 3 adrenoceptor mediated relaxation responses in streptozotocine-induced diabetic rat aorta.

Methods: 8-week old male Sprague-Dawley rats were divided into 3 groups; control (C), diabetic (D), sitagliptin treated diabetic group (S). Diabetes was induced by single dose streptozotocine injection (35 mg/kg, i. p.). At the end of 10-week-diabetes period, some of the diabetic rats were treated with sitagliptin (10 mg/kg/day, orally) for 28 days. At the end of the treatment, thoracic aorta was dissected under ether anesthesia, connective and fat tissue were removed. Then 5 mm aorta rings were attached into the organ bath containing Krebs solution. The temperature was set at 37 °C and the bath was continously oxygenated with 95 % O2 and 5 % CO2. After 1-hour incubation period, dose response curves of phenylephrine, isoprenaline and CL 316,243 were obtained. Then protein expression of beta 3 adrenergic receptors, eNOS and phosphorylated eNOS (peNOS) was evaluated with western blot analysis.

Results: Contractile response and Emax value at 10 μM in the presence of phenylephrine, an alpha 1 adrenergic receptor agonist, were increased significantly in D group compared to C group, however, no marked difference was observed in S group. Isoprenaline, a nonselective beta adrenergic receptor agonist, mediated relaxation response and E max value at 10 μM were decreased significantly in D and S group compared to C. There was no difference between S and D group. CL 316,243, beta 3 AR agonist, mediated relaxation response and Emax value at 1 μM did not differ between the groups. Beta 3 adrenergic receptor protein expression was not changed in C, D and S groups. The protein expression of eNOS was increased in D and S groups, however it did not reach the statistical difference. The protein expression of peNOS was decreased in D group and increased in S group. The ratio of peNOS/eNOS was decreased in D group compared to C group. This ratio was increased significantly in S group compared to D group.

Conclusion: Sitagliptin did not improve attenuated beta 3 adrenergic receptor mediated relaxation response in diabetic aorta. Beta 3 adrenergic receptor mediated relaxation response was not different in diabetic aorta, and furthermore, sitagliptin had no significant effect on this reponse. These results are supported with the finding that beta 3 adrenergic receptor protein expression was not changed in any group. However, peNOS protein expression and peNOS/eNOS ratio was increased markedly in sitagliptin treated group which points the effect of the drug on eNOS pathway. Further studies are needed to explain its effect on this pathway.

Grant support: This study was supported by Ankara University BAP (17L0237002) and TUBITAK (115 S 564).


Dysregulation of plasma microRNome as read-out of metabolic imbalance in pre-diabetes

P. de Candia1, G. Spinetti1, C. Specchia1,2, E. Sangalli1, L. La Sala1, A. Uccellatore1, S. Lupini1, G. Matarese3,4, S. Genovese1, A. Ceriello1,5,6
1) Department of Cardiovascular and Metabolic Diseases, IRCCS MultiMedica, Milan, Italy
2) University of Brescia, Italy
3) Laboratorio di Immunologia, Istituto di Endocrinologia e Oncologia Sperimentale, Consiglio Nazionale delle Ricerche (IEOS-CNR), Naples, Italy
4) Dipartimento di Medicina Molecolare e Biotecnologie Mediche, Università di Napoli "Federico II", Naples, Italy
5) Insititut d‘Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS), Barcelona, Spain
6) Centro de Investigación Biomédica en Red de Diabetes y Enfermedades Metabólicas Asociadas (CIBERDEM), Barcelona, Spain

Objective: Type 2 diabetes (T2D) is a complex metabolic disorder. A major unmet medical need is an accurate prediction of the disease in individuals who already show glucose tolerance impairment (IGT), but are not yet diagnosed as diabetic. We here propose the possibility to predict/monitor the progression of IGT subjects to T2D though the quantification of blood circulating miRNAs, a highly suitable source of disease biomarkers that may be used to assess cell activity at distance.

Methods: Circulating miRNAs were retrospectively quantified by RT qPCR in plasma of subjects with i) normal glucose tolerance (NGT, n = 9), ii) impaired glucose tolerance (IGT, n = 9), divided into non progressors (NP, n = 5) and progressors (P, n = 4) based on subsequent diabetes outbreak, and iii) type 2 diabetes (T2D, n = 9). We performed miRNA differential expression between groups and miRNA quantity correlation with relevant clinical parameters.

Results: We found that i) the global miRNA quantity progressively increased in IGT and T2D subjects compared to NGT, and correlated with glucose level; ii) NP IGT group showed the majority of differentially expressed miRNAs and clusterized by itself, leading to the identification of a "non progressor miRNA signature"; iii) a subset of differentially expressed miRNAs was also correlated with clinical parameters of glucose homeostasis and anthropometric measurements of obesity.

Conclusions: Several miRNAs found differentially expressed in IGT were previously described as playing a role in glucose metabolism, suggesting that, beside being a readout of metabolic imbalance, their dysregulation may also have an impact on glucose homeostasis.



Erschienen in: Diabetes, Stoffwechsel und Herz, 2017; 26 (4) Seite 233-235