Estudio de los mecanismos de estrés oxidativo, activación del inflamasoma NLRP3 y las interacciones leucocito-endotelio en la Diabetes: Efectos del papel de la Empagliflozina

La diabetes mellitus (DM) presenta un problema de salud pública a nivel mundial. La DM es un grupo de enfermedades metabólicas heterogéneas asociadas a alteraciones en el metabolismo glucídico, lipídico y proteico y inflamación sistémica crónica de bajo grado. Además, está caracterizada por un estado de hiperglucemia secundaria a un defecto absoluto o relativo en la secreción de insulina por parte de las células-β del páncreas. Tanto en la DM tipo 1 como en la DM de tipo 2 (DM1 y DM2) la hiperglucemia, la inflamación sistémica y el estrés oxidativo, son unos de los mayores factores predisponentes a la disfunción endotelial y a las enfermedades cardiovasculares. En los últimos años se han desarrollado como tratamiento farmacológico de la DM otro grupo de fármacos entre los que se incluyen los inhibidores del cotransportador SGLT-2 (iSGLT2). Gracias a su mecanismo de acción, los i-SGLT2 favorecen un mejor control glucémico modulando la disfunción mitocondrial, la cual es asociada a la activación del inflamasoma NLRP3 en la DM2. A tal fin, en la presente tesis doctoral nos planteamos investigar sobre diversos aspectos donde existen vacíos en el conocimiento. Por lo tanto, hemos establecido los siguientes objetivos específicos: 1. Evaluar las interacciones leucocitos-endotelio y explorar tal relación y su correlación con la producción ROS y la disfunción mitocondrial en leucocitos de pacientes con DM1 y sus respectivos controles. 2. Evaluar el papel del control glicémico en la activación del inflamasoma NLRP3 en pacientes con DM2. 3. Evaluar el efecto del iSGLT2 empagliflozina sobre el perfil inflamatorio y la respuesta antioxidante en leucocitos procedentes de pacientes con DM2 y sus respectivos controles. En la presente tesis doctoral, hemos observado que la disfunción mitocondrial, el estrés oxidativo y el control glucémico pueden modular las dinámicas y la función de los leucocitos en los pacientes con DM1 y DM2, favoreciendo las interacciones entre los leucocitos y las células endoteliales vasculares provocando junto a la inflamación de bajo grado danos vasculares. Además, los resultados apoyan la hipótesis que los pacientes DM2 tratados con Empagliflozina mejoran el perfil inflamatorio. En conclusión, los datos obtenidos sugieren que un buen manejo del control glucémico y de la inflamación junto al utilizo de los iSGLT2 podría prevenir también otras comorbilidades asociadas a la DM2.Diabetes mellitus (DM) has been recognized as one of the four major non-communicable diseases widespread worldwide. Its multifactorial aetiology leads to impaired carbohydrate and fat metabolism, prolonged and inappropriate hyperglycaemia, chronic low-grade inflammation, as well as defects in insulin action and/or secretion. Accumulating evidence suggests that hyperglycaemia, systemic inflammation and oxidative stress are some of the main common features of both type 1 and 2 diabetes (T1D and T2D), thus acting as key factors in the development of DM associated macro- and microvascular complications. Over the last few years, inhibitors of sodium-glucose co-transporter type 2 (iSGLT2) have received growing attention as new oral antidiabetic drugs. Their unique mechanism of action results in improved glucose control by modulating mitochondrial dysfunction, which is associated with NLRP3 inflammosome activation in T2D. With this aim in mind, we established the following objectives: 1. To assess the relationship between the inflammatory/adhesive process in the vascular wall and intracellular mechanisms in T1D patients, and explore its relevance in ROS production and mitochondrial impairment. 2. To explore the role played by glycaemic control in NLRP3 inflammasome activation in T2D patients. 3. To evaluate beneficial effects of iSGLT2 Empagliflozin treatment on glycaemic state, systemic inflammation and antioxidant properties in leukocytes in the peripheral blood (PMNs) in T2D patients. The findings of the current doctoral thesis reveal that mitochondrial dysfunction, oxidative stress and glycaemic control affect leukocyte dynamics and function in both T1D and T2D patients by promoting the interaction between these cells and the vascular endothelium and therefore vessels damage due to low grade inflammation. Moreover, our data support the hypothesis that treatment of T2D patients with Empagliflozin ameliorates the disease’s inflammatory profile. Together, these findings suggest that adequate management of the glycaemic state and inflammation and the use of iSGLT2 can prevent the onset of the atherosclerotic process and, consequently, delay the development of DM-associated vascular complications

Voters\u2019 preferences and electoral systems: the EuroVotePlus experiment in Italy

Motivated by the need to understand voting behaviour under different electoral rules, Laslier et al. (Eur Union Polit, 16(4):601\u2013615, 2015) have conducted an online experiment, the EuroVotePlus experiment, focusing on the effects of the different rules adopted to elect members of the European parliament on voters\u2019 behaviour. The experiment took place in several European countries in the 3 weeks before the 2014 elections for the European Parliament. This paper focuses on the Italian data. Firstly, we show that the behaviour of Italian respondents is consistent with the empirical findings at the European level. Then, we exploit the change from open list to closed list elections implemented in Italy in 1993 to investigate whether and how preferences over institutions are affected by experience. We find that respondents who voted using the open list system in Italy are more likely to prefer closed list systems, and that the effect is stronger the higher the number of open list elections the respondents have faced

The Mitochondria-Targeted Antioxidant MitoQ Modulates Mitochondrial Function and Endoplasmic Reticulum Stress in Pancreatic β Cells Exposed to Hyperglycaemia.

BACKGROUND/AIMS: Mitochondria-targeted antioxidants such as mitoquinone (MitoQ) have demonstrated protective effects against oxidative damage in several diseases. The increase in reactive oxygen species (ROS) production during glucose metabolism in β cells can be exacerbated under hyperglycaemic conditions such as type 2 diabetes (T2D), thus contributing to β cell function impairment. In the present work, we aimed to evaluate the effect of MitoQ on insulin secretion, oxidative stress, endoplasmic reticulum (ER) stress and nuclear factor kappa B (NFκB) signalling in a pancreatic β cell line under normoglycaemic (NG, 11.1 mM glucose), hyperglycaemic (HG, 25 mM glucose) and lipidic (palmitic acid (PA), 0.5mM) conditions. METHODS: We incubated the pancreatic β cell line INS-1E with or without MitoQ (0.5µM) under NG, HG and PA conditions. We then assessed the following parameters: glucose-induced insulin secretion, O₂ consumption (with a Clark-type electrode); mitochondrial function, oxidative stress parameters and calcium levels (by fluorescence microscopy); ER stress markers and NFκB-p65 protein levels (by western blotting). RESULTS: MitoQ increased insulin secretion and prevented the enhancement of ROS production and O₂ consumption and decrease in GSH levels that are characteristic under HG conditions. MitoQ also reduced protein levels of ER stress markers (GRP78 and P-eIF2α) and the proinflammatory nuclear transcription factor NFκB-p65, both of which increased under HG. MitoQ did not significantly alter ER stress markers under lipidic conditions. CONCLUSION: Our findings suggest that treatment with MitoQ modulates mitochondrial function, which in turn ameliorates endoplasmic reticulum stress and NFκB activation, thereby representing potential benefits for pancreatic β cell function

Downregulation of miR-31 in Diabetic Nephropathy and its Relationship with Inflammation

Background/Aims: There is a lack of reliable biological markers for the early diagnosis of diabetic nephropathy (DN) during type 2 diabetes. In this pilot study we aim to assess whether miR-31 levels are modulated by the presence of DN and whether the expression of this miRNA is related to leukocyte-endothelial interactions and inflammation. Methods: Thirty-one T2D patients were enrolled in this pilot study; 18 with no diabetic complications and 13 with diabetic nephropathy. 24 non-diabetic subjects and 13 T2D patients with retinopathy (absent of other complications) were included to test the specificity of miR-31. Following anthropometric and biochemical evaluation, serum miR-31 levels were assessed by Real Time-PCR. Leukocyte-endothelial interactions were evaluated by a parallel flow chamber in vitro model. Serum TNFα, IL-6 and ICAM-1 levels were determined by XMAP-technology in a flow cytometry-based Luminex 200 instrument. Results: Serum miR-31 levels were similar between control and T2D subjects. However, T2D patients with DN displayed reduced levels of miR-31 with respect to patients without complications. This decrease in miR-31 was more pronounced in patients with macroalbuminuria than in those with microalbuminuria and was specific for DN, since patients with retinopathy displayed unaltered miR-31 levels. The presence of DN involved a lower leukocyte rolling velocity and an increased rolling flux and adhesion. miR-31 levels were positively correlated with leukocyte rolling velocity and negatively associated to leukocyte adhesion, TNFα, IL-6 and ICAM-1 levels. Conclusion: Serum miR-31 may be a biomarker for DN in T2D patients. The regulation of this miRNA seems to be related to the recruitment of leukocytes to vascular walls induced by pro-inflammatory and adhesion molecules

Mechanisms of action of metformin in type 2 diabetes: Effects on mitochondria and leukocyte-endothelium interactions

Type 2 diabetes (T2D) is a very prevalent, multisystemic, chronic metabolic disorder closely related to atherosclerosis and cardiovascular diseases. It is characterised by mitochondrial dysfunction and the presence of oxidative stress. Metformin is one of the safest and most effective anti-hyperglycaemic agents currently employed as first-line oral therapy for T2D. It has demonstrated additional beneficial effects, unrelated to its hypoglycaemic action, on weight loss and several diseases, such as cancer, cardiovascular disorders and metabolic diseases, including thyroid diseases. Despite the vast clinical experience gained over several decades of use, the mechanism of action of metformin is still not fully understood. This review provides an overview of the existing literature concerning the beneficial mitochondrial and vascular effects of metformin, which it exerts by diminishing oxidative stress and reducing leukocyte-endothelium interactions. Specifically, we describe the molecular mechanisms involved in metformin's effect on gluconeogenesis, its capacity to interfere with major metabolic pathways (AMPK and mTORC1), its action on mitochondria and its antioxidant effects. We also discuss potential targets for therapeutic intervention based on these molecular actions.This work was funded by grants PI16/00090, PI19/00838, PI19/0437,PI19/01266 and CIBERehd CB06/04/0071 by Carlos III Health Institute and by the European Regional Development Fund (ERDF ‘‘A way to build Europe’‘); by PROMETEO/2019/027 by Ministry of Education of the Valencian Regional Government; by RTI2018-096748-B-100 (Spanish Ministry of Science, Innovation and Universities), by Andalusian Ministry of Economy, Innovation, Science and Employment (CTS-6264), Andalusian Ministry of Equality, Health and Social Policies (PI-0198-2016) and by an unrestricted grant from Menarini S.A. M.R and VM.V are recipients of contracts from the Ministry of Health of the Valencian Regional Government and Carlos III Health Institute (CPII16/00037 and CES10/030, respectively) while AG is supported by the Foundation “Juan Esplugues”

Association between Proinflammatory Markers, Leukocyte–Endothelium Interactions, and Carotid Intima–Media Thickness in Type 2 Diabetes: Role of Glycemic Control

Glycated hemoglobin monitorization could be a tool for maintaining type 2 diabetes (T2D) under control and delaying the appearance of cardiovascular events. This cross-sectional study was designed to assess the role of glycemic control in modulating early-stage markers of cardiovascular complications. One hundred and eight healthy controls and 161 type 2 diabetic patients were recruited and distributed according to their glycemic control, setting the threshold at 6.5% (good control). Biochemical and anthropometrical parameters were registered during the initial visit, and peripheral blood was extracted to obtain polymorphonuclear cells and analyze inflammatory markers, adhesion molecules, leukocyte–endothelium interactions, and carotid intima–media thickness. Correlations between these parameters were explored. We found that inflammatory markers and adhesion molecules were augmented in type 2 diabetic subjects with poor glycemic control. Polymorphonuclear leukocytes interacted more with the endothelium in the diabetic population, and even more significantly in the poorly controlled subjects. In parallel, carotid intima–media thickness was also increased in the diabetic population, and the difference was greater among poorly controlled subjects. Finally, correlation measurement revealed that carotid intima–media thickness was related to glycemic control and lipid metabolism in diabetic patients. Our results suggest that glycemic control delays the onset of cardiovascular comorbidities in diabetic subjects

Inhibition of transcription by dactinomycin reveals a new characteristic of immunogenic cell stress

Abstract Chemotherapy still constitutes the standard of care for the treatment of most neoplastic diseases. Certain chemotherapeutics from the oncological armamentarium are able to trigger pre‐mortem stress signals that lead to immunogenic cell death (ICD), thus inducing an antitumor immune response and mediating long‐term tumor growth reduction. Here, we used an established model, built on artificial intelligence to identify, among a library of 50,000 compounds, anticancer agents that, based on their molecular descriptors, were predicted to induce ICD. This algorithm led us to the identification of dactinomycin (DACT, best known as actinomycin D), a highly potent cytotoxicant and ICD inducer that mediates immune‐dependent anticancer effects in vivo. Since DACT is commonly used as an inhibitor of DNA to RNA transcription, we investigated whether other experimentally established or algorithm‐selected, clinically employed ICD inducers would share this characteristic. As a common leitmotif, a panel of pharmacological ICD stimulators inhibited transcription and secondarily translation. These results establish the inhibition of RNA synthesis as an initial event for ICD induction

Does Empagliflozin Modulate Leukocyte–Endothelium Interactions, Oxidative Stress, and Inflammation in Type 2 Diabetes?

Sodium-glucose co-transporter 2 inhibitors (iSGLT2) have been linked to cardiovascular risk reduction in patients with type 2 diabetes (T2D). However, their underlying molecular mechanisms remain unclear. This study aimed to evaluate the effects of empagliflozin, a novel potent and selective iSGLT-2, on anthropometric and endocrine parameters, leukocyte–endothelium interactions, adhesion molecules, ROS production, and NFkB-p65 transcription factor expression. According to standard clinical protocols, sixteen T2D patients receiving 10 mg/day of empagliflozin were followed-up for 24 weeks. Anthropometric and analytical measurements were performed at baseline, 12 weeks, and 24 weeks. Interactions between polymorphonuclear leukocytes and human umbilical vein endothelial cells (HUVECs), serum levels of adhesion molecules (P-Selectin, VCAM-1 and ICAM-1) and pro-inflammatory cytokines (TNF-α, IL-1β and IL-6), mitochondrial ROS levels, antioxidant enzymes (SOD1 and GPX1), and NFkB-p65 were measured. We observed a decrease in body weight, BMI, and HbA1C levels from 12 weeks of treatment, which became more pronounced at 24 weeks and was accompanied by a significant reduction in waist circumference and glucose. Leukocyte–endothelium interactions were reduced due to an enhancement in the leukocyte rolling velocity from 12 weeks onwards, together with a significant decrease in leukocyte rolling flux and adhesion at 24 weeks. Accordingly, a significant decrease in ICAM-1 levels, mitochondrial ROS levels, and IL-6 and NFkB-p65 expression was observed, as well as an increase in SOD1. This pilot study provides evidence of the anti-inflammatory and antioxidant properties of empagliflozin treatment in humans, properties which may underlie its beneficial cardiovascular effects