Estudio de los mecanismos moleculares de estrés oxidativo, disfunción mitocondrial, estrés de retículo endoplasmático y autofagia en leucocitos de pacientes con diabetes tipo 2

Introducción: La diabetes mellitus tipo 2 (DM2) se presenta como un cuadro clínico que incluye diversas alteraciones en el metabolismo, incluyendo hiperglicemia, resistencia a insulina, dislipidemia e inflamación crónica de bajo grado. Supone un gran problema a nivel mundial dada su creciente incidencia y las complicaciones deletéreas que conlleva, principalmente en el sistema cardiovascular. El principal factor determinante de DM2 es la obesidad. A medida que aumenta el índice de masa corporal se desarrolla resistencia a la insulina lo que finalmente originará una pérdida del control homeostático de la glucosa. Entre las principales complicaciones vasculares de la diabetes están por una parte la enfermedad cerebrovascular, la enfermedad arterial periférica y la cardiopatía isquémica, que representan el grupo de complicaciones macrovasculares y suman el 52% de las muertes en pacientes diabéticos. Por otra parte, la nefropatía, la retinopatía y la neuropatía diabética constituyen el conjunto de complicaciones microvasculares, dentro de las cuales la nefropatía diabética es la más común afectando aproximadamente al 40% de los pacientes. El control glicémico es clave para prevenir la aparición de estas complicaciones en los pacientes con DM2, ya que el principal desencadenante de daño vascular es la hiperglicemia. La inflamación sistémica es una de las características más representativa de la DM2, caracterizada por unos elevados niveles sistémicos de citoquinas proinflamatorias y una activación crónica del sistema inmune. A nivel local en las paredes vasculares, el proceso inflamatorio contribuye al inicio de la aterosclerosis debido a que convergen la disfunción endotelial, la activación de los leucocitos y el aumento de señales atrayentes (citoquinas y moléculas de adhesión) que favorecen la migración de los leucocitos hacia la pared endotelial. En este contexto es interesante el estudio de la fisiopatología de la DM2 en leucocitos de sangre periférica, ya que estas células participan tanto en la defensa inmune como en el proceso de aterosclerosis e influyen en la aparición de problemas cardiovasculares. De entre los mecanismos subyacentes a la DM2 que contribuyen al desarrollo de complicaciones vasculares el estrés oxidativo juega un rol principal, así como la disfunción mitocondrial. De hecho, se ha especulado mucho acerca de las rutas de señalización que conectan la situación de hiperglicemia con el incremento de especies reactivas de oxígeno (ERO) y su papel en la inducción de enfermedad cardiovascular. Otros procesos a parte del estrés oxidativo y la disfunción mitocondrial han sido relacionados con la DM2 en los últimos años, como el estrés de retículo endoplasmático (RE) y la autofagia. Sin embargo, el grado de conocimiento acerca del papel que juegan éstos en la DM2 es bastante menor. Además se precisan más estudios acerca de la relación de estos procesos con el proceso inflamatorio y de adhesión de los leucocitos a las paredes vasculares, estableciendo así su posible influencia en la aparición de complicaciones vasculares. Objetivos: En la presente Tesis Doctoral nos planteamos evaluar el papel de la disfunción mitocondrial en la activación de los leucocitos en la DM2, así como su relación con el incremento de interacciones leucocito-endotelio y la correlación de estos factores con el desarrollo de cardiopatía isquémica silente (CIS). Por otra parte, quisimos estudiar el papel de la mieloperoxidasa (MPO) en la generación de ERO en la DM2 y evaluar la posible asociación de la MPO con la función endotelial y el desarrollo de nefropatía diabética. También nos propusimos evaluar la influencia del control glicémico de la DM2 sobre la inducción de estrés oxidativo y estrés de RE y el papel que juega el estrés de RE en la inducción de interacciones leucocito-endotelio. Por último nos planteamos determinar si el proceso de autofagia está alterado en leucocitos de pacientes con DM2, así como su posible relación con el estrés oxidativo y el estrés de RE. Metodología: Se reclutaron pacientes con DM2 y sujetos control y se determinaron parámetros antropométricos (peso, talla, IMC, índice cintura/cadera) y bioquímicos (en muestras sanguíneas). Se realizó el diagnóstico de la CIS mediante electrocardiograma y estudio coronariográfico seguido en los pacientes con CIS por SPECT (Single Photon Emission Computed Tomography) con 123-I-MIBG (Metayodobencilguanidina) mediante gammacámara. La nefropatía diabética se confirmó en los pacientes que presentaban un ratio albúmina:creatinina en orina mayor a 30mg/g. Para la determinación de MPO, citoquinas y moléculas de adhesión se conservaron alícuotas de suero a -80ºC. La concentración de MPO se determinó mediante un inmunoensayo tipo ELISA, mientras que las citoquinas y las moléculas de adhesión se determinaron mediante la tecnología X-MAP de LUMINEX. La evaluación del estrés oxidativo, la disfunción mitocondrial, el estrés de RE y la autofagia se hizo a partir de leucocitos aislados de sangre periférica mediante gradiente de Ficoll. Los leucocitos se incubaron con los siguientes fluorocromos para la valoración del estado redox: DCFH-DA, indicativo de la producción total de ERO; Mitosox Red, indicativo de la producción superóxido mitocondrial; TMRM, indicativo del potencial de membrana mitocondrial; NAO, indicativo de la masa mitocondrial y Hoescht, indicativo de la morfología nuclear. La fluorescencia emitida por los leucocitos se cuantificó bajo un microscopio de fluorescencia (IX81; Olympus) acoplado al software de citometría estática “ScanR”. La respiración mitocondrial se evaluó mediante un electrodo de O2 tipo Clark. Las interacciones leucocito-endotelio se evaluaron mediante una cámara de flujo paralelo. En este sistema se perfundió una suspensión de leucocitos sobre células endoteliales humanas cultivadas en monocapa (aisladas a partir de cordón umbilical mediante un tratamiento con colagenasa) en condiciones que simulan las del flujo sanguíneo. La interacción de los leucocitos con la monocapa endotelial se observó a través de un microscopio invertido de contraste de fases (Nikon Eclipse TE2000-S) y las imágenes se grabaron con una cámara acoplada (Sony Exware HAD). Se estimaron los procesos de rodamiento y adhesión firme leucocitaria. Se extrajo RNA y proteínas totales de los leucocitos para evaluar la expresión de los distintos marcadores de estrés de RE y de autofagia mediante Real Time PCR y Western Blotting. El análisis estadístico se realizó mediante los programas Graphpad Prism y SPSS. Resultados y conclusiones: Comparamos pacientes con DM2 de al menos 2 años de duración con sujetos control ajustados por edad y sexo. Los pacientes con DM2 presentaron un aumento en el IMC, la circunferencia de cintura y la presión arterial. Además mostraron mayores niveles de glucosa y de insulina en ayunas y por tanto un incremento en el índice HOMA-IR, así como un aumento de la HbA1c. Se observó a su vez un aumento de triglicéridos y unos niveles de colesterol-HDL reducidos en los pacientes diabéticos. Los marcadores de inflamación PCR, IL6 y TNFα mostraron niveles incrementados en el suero de los pacientes con DM2 además de un aumento significativo de las moléculas E-selectina y VCAM-1. Estas observaciones sugieren que existe un desequilibrio metabólico en los pacientes con DM2, que junto con la obesidad abdominal que presentan favorece la aparición de una inflamación sistémica de bajo grado y de origen metabólico. En cuanto a los leucocitos, como consecuencia de la situación de inflamación crónica en los pacientes con DM2, manifestaron una menor velocidad de rodamiento sobre el endotelio, un mayor flujo de rodamiento y más adhesión. Cuando estas interacciones están incrementadas se favorece la disfunción endotelial y el daño vascular que precede a múltiples enfermedades cardiovasculares. Uno de los mecanismos que podría estar favoreciendo este fenotipo protrombótico en los leucocitos es el estrés oxidativo y la disfunción mitocondrial, y de hecho lo pudimos evidenciar a partir de nuestras observaciones. En los pacientes diabéticos, los leucocitos presentaron una excesiva producción de radicales libres junto con una disminución de las defensas antioxidantes, una menor masa mitocondrial, una reducción en el potencial de membrana y una menor tasa de respiración mitocondrial. Estas características se manifestaron mucho más aumentadas en los pacientes que además presentaban CIS, y a su vez la producción de ERO mitocondriales mostró una fuerte correlación con el aumento en las interacciones leucocito-endotelio pudiendo por tanto indicar que la disfunción mitocondrial sería un factor determinante en la inducción de complicaciones vasculares como la CIS. Por otra parte observamos unos niveles aumentados de MPO en suero de pacientes con DM2, que fueron mayores en los pacientes que sufrían nefropatía, habiendo además una correlación positiva entre la concentración de MPO y el ratio albúmina/creatinina. Los pacientes con nefropatía mostraron una mayor frecuencia de contacto entre leucocitos y endotelio, además de un mayor incremento en los niveles de TNFα y E-selectina. Estos resultados plantean que la MPO participaría en la generación excesiva de ERO en leucocitos de pacientes con DM2 y podría tener un papel importante en el desarrollo de nefropatía mediado por la disfunción endotelial. En cuanto a la inducción de estrés de RE en los pacientes con DM2, vimos que los niveles de GRP78 se aumentaron en los leucocitos de pacientes con DM2 de manera general. Sin embargo, el control glicémico de los pacientes parecía estar influenciando el patrón de expresión de los marcadores de estrés de RE, siendo adaptativo en los pacientes con un buen control de los niveles de glucosa, y proapoptótico en los pacientes mal controlados. También en este grupo de pacientes con mal control de la glicemia se mostraron mayores niveles de PCR, TNFα y E-selectina, así como un mayor flujo de rodamiento sobre el endotelio. La relación entre el estrés de RE y el proceso de interacción de los leucocitos con el endotelio se pudo intuir al observar que los niveles de GRP78 y sXBP1 se correlacionaban negativavente con la velocidad de rodamiento y los de CHOP exhibían una correlación positiva con el grado de adhesión leucocitaria, sugiriendo que bajo un buen control de la glicemia durante la DM2 los leucocitos tienden a frenar sobre las paredes endoteliales sin adherirse, en cambio cuando no hay un buen control de los niveles de glucosa la adhesión leucocitaria se acentúa. Por último, evaluamos el papel de la autofagia en la DM2, determinando los niveles de LC3-II y Beclina 1 en leucocitos. Observamos que en los pacientes con DM2 estos marcadores de autofagia estaban aumentados junto con un patrón de condensación de la cromatina acentuado indicativo de muerte celular. Estos mismos leucocitos mostraban signos de disfunción mitocondrial, con una menor masa mitocondrial y una mayor producción de ERO, así como signos de estrés de RE, con un aumento en los marcadores GRP78, p-eIF2α y ATF6. Además, los niveles de Beclina 1 se correlacionan positivamente con la producción de ERO y también Beclina 1 y LC3-II se correlacionan positivamente con los distintos marcadores de estrés de RE estudiados. Estas observaciones sugieren que la DM2 altera la función autofágica en los leucocitos, y que esta función está regulada de igual forma que el estrés oxidativo y el estrés de RE durante la DM2. En conjunto, estas evidencias ponen de manifiesto que los mecanismos moleculares de disfunción mitocondrial, estrés oxidativo, estrés de RE y autofagia ocurren en leucocitos de pacientes con DM2 y potencian la interacción de estas células con el endotelio, pudiendo ser determinantes en la inducción de complicaciones en el sistema cardiovascular.Introduction: Type 2 diabetes (T2D) is a metabolic disorder characterized by hyperglycemia, insulin resistance, dyslipidemia and chronic low-grade inflammation. T2D is becoming a major public health issue due to its prevalence, which is increasing worldwide, and deleterious associated complications in the cardiovascular system. Obesity is the main determinant in the development of T2D. As body mass index (BMI) increases, insulin resistance develops and leads to an imbalance in glucose homeostasis. Among the main vascular complications of T2D, cerebrovascular disease, peripheral artery disease and myocardial ischemia are macrovascular complications and represent 52% of deaths in T2D population. On the other hand, nephropathy, retinopathy and diabetic neuropathy are microvascular complications of the disease, with nephropathy being the most common, as it develops in approximately 40% of diabetic patients. Adequate glycemic control is the key to preventing these complications, as hyperglycemia is the main inducer of vascular damage. Systemic inflammation is one of the main features of T2D and is characterized by enhanced circulatory levels of proinflammatory cytokines and chronic activation of the immune system. In the vascular wall, this inflammatory process contributes to the onset of atherosclerosis, as endothelial dysfunction, leukocyte activation and an increase in cytokines and adhesion molecules converge and promote leukocyte migration to the endothelial wall. In this context, it is interesting to study the physiopathology of T2D in peripheral blood cells such as leukocytes, since they participate in immunity and in the development of atherosclerosis, which can promote cardiovascular disease. Oxidative stress and mitochondrial dysfunction play a main role in the development of vascular complications in T2D. In fact, a growing number of studies have described different signalling pathways that connect the hyperglycaemic situation with the induction of cardiovascular diseases, in which oxidative stress is the major contributor. Other mechanisms besides oxidative stress and mitochondrial dysfunction have been related with T2D in recent years, such as endoplasmic reticulum (ER) stress and autophagy. However, less is known about how these processes participate in the physiopathology of T2D. Moreover, further research is needed to assess the relationship between the inflammatory/adhesive process in the vascular walls and intracellular mechanisms, such as oxidative and ER stress or autophagy in leukocytes, that can eventually lead to vascular complications. Objectives: In the current Doctoral Thesis we have aimed to evaluate the role of mitochondrial dysfunction in the activation of leukocytes in T2D, as well as its relationship with the interactions between leukocytes and endothelial cells, and the potential correlation among these factors and the onset of silent myocardial ischemia (SMI). On the other hand, our goal was to assess the potential participation of myeloperoxidase (MPO) in reactive oxygen species (ROS) generation in patients with T2D, and to evaluate is potential relationship with endothelial function and the development of diabetic nephropathy. We also decided to assess in T2D patients with different extent of glycemic control, the induction of oxidative stress and ER stress and the role of the latter in the promotion of leukocyte-endothelial interactions. Finally, we aimed to determine whether the process of autophagy is altered in the leukocytes of T2D patients, as well as exploring its connection with oxidative and ER stress. Methods: T2D patients and healthy subjects were recruited and anthropometric parameters (weight, height, BMI, waist and hip circumferences, blood pressure) and biochemical assessments (from blood samples) were determined. SMI diagnosis was confirmed by electrocardiogram and a coronary angiography test and SPECT (Single Photon Emission Computed Tomography). Diabetic nephropathy was confirmed when a urinary albumin excretion over 30 mg/g creatinine was detected in T2D patients. To assess the concentration of MPO, cytokines and adhesion molecules, serum aliquots were stored at -80ºC after collection. MPO concentration was determined by an ELISA immunoassay, whereas cytokines and adhesion molecules were studied by X-MAP technology in LUMINEX equipment. Oxidative stress, mitochondrial dysfunction, ER stress and autophagy were evaluated following isolation of peripheral blood leukocytes by Ficoll gradient. Leukocytes were incubated with the following fluorescent probes to evaluate redox balance: DCFH-DA, a marker of total ROS production; Mitosox Red, an indicator of mitochondrial ROS production; TMRM, a marker of mitochondrial membrane potential; NAO, an indicator of mitochondrial mass; and Hoescht, a nuclear morphology probe. Fluorescence was quantified using a fluorescence microscope (IX81; Olympus) coupled to the static cytometry software “ScanR”. The mitochondrial respiration rate was assessed by a Clark-type oxygen electrode. Leukocyte-endothelial interactions were estimated using a parallel flow chamber in which leukocytes were drawn across an endothelial cell monolayer (isolated from human umbilical cords) at a controlled flow rate similar to the in vivo vascular system. Recordings of this process were obtained using a video camera (Sony Exware HAD) coupled to a phase contrast microscope (Nikon Eclipse TE2000-S) and finally, the processes of leukocyte rolling and adhesion were estimated. To assess the levels of ER stress and autophagy markers, the RNA and proteins from leukocytes were isolated and Real-Time PCR and Western Blotting were performed, respectively. Statistical analyses were performed by means of Graphpad Prism and SPSS software. Results and conclusions: We compared T2D patients (with at least 2 years of disease duration) with age and sex matched control subjects. T2D patients showed increases in BMI, waist circumference and blood pressure. Furthermore, they displayed increased fasting glucose and insulin levels and, therefore, an enhanced HOMA-IR index, as well as elevated HbA1c levels. A rise in triglycerides and reduced HDL content was also observed in T2D patients. The inflammatory parameters PCR, IL6 and TNFα were increased in the serum of T2D patients, as were the adhesion molecules E-selectin and VCAM-1. These results suggest there is an imbalanced metabolism in T2D patients, which, together with abdominal obesity, promotes a low-grade inflammatory state of metabolic origin. As a consequence of the chronic inflammatory condition in T2D, leukocytes display diminished rolling velocity and enhanced rolling flux and adhesion over the endothelium. When these properties are increased, endothelial dysfunction is promoted and leads to the vascular damage that precedes cardiovascular diseases. One of the mechanisms that promotes this prothrombotic state in leukocytes may be oxidative stress and mitochondrial dysfunction, as our results confirm. Leukocytes from T2D patients showed enhanced ROS production, impaired antioxidant response, less mitochondrial mass, reduced membrane potential and a reduction in the oxygen consumption rate when compared to those from the control group. These features were even more pronounced in patients with SMI. At the same time, mitochondrial ROS production was strongly correlated with the increase in leukocyte-endothelial interactions, suggesting that mitochondrial dysfunction is a determinant factor in the induction of SMI. On the other hand, we observed a higher MPO concentration in serum from T2D patients, which was even higher in those with diabetic nephropathy. Furthermore, MPO levels correlated positively with the albumin/creatinine ratio. The interaction between leukocytes and the endothelium was more frequent in diabetic patients with nephropathy, and was accompanied by a rise in TNFα and E-selectin levels. These findings indicate that MPO contributes to an enhanced ROS generation in the leukocytes of T2D patients, which could have an important role in the development of diabetic nephropathy mediated by endothelial dysfunction. Regarding the potential activation of ER stress in T2D, we observed a rise in GRP78 levels in general. However, glycemic control seemed to influence the pattern of expression of several ER stress markers, and appeared to follow an adaptive pattern in well-controlled patients and a proapoptotic pattern in poorly controlled T2D patients. Moreover, the group of poorly controlled patients displayed enhanced levels of inflammatory markers and increased rolling flux. A relationship between ER stress activation and leukocyte-endothelial interactions was apparent, since the adaptive markers GRP78 and sXBP1 were negatively correlated with rolling velocity and the proapoptotic marker CHOP was positively correlated with leukocyte adhesion. These findings indicate that, when T2D is well controlled, the velocity of leukocytes along the endothelium slows down and, when there is poor control of the disease, leukocyte adhesion is exacerbated. Finally, we assessed the role of autophagy in T2D by determining LC3-II and Beclin1 levels in leukocytes. We detected enhanced levels of these autophagic markers together with a chromatin condensation increase that indicated ongoing cell death. The same leukocytes displayed signs of mitochondrial dysfunction, including reduced mitochondrial mass, enhanced ROS production, and increased ER stress with a rise in GRP78, p-eIF2α and ATF6 levels. Furthermore, Beclin1 levels were positively associated with ROS production, while Beclin1 and LC3-II were associated with markers of ER stress. These results suggest that T2D alters autophagic function in leukocytes, and that this function is regulated in parallel with oxidative and ER stresses during T2D. Overall, our findings reveal that mitochondrial dysfunction, oxidative stress, ER stress and autophagy oc

Metformin and its redox-related mechanisms of action in type 2 diabetes

Type 2 diabetes (T2D) is a long-term metabolic disease characterized by progressive β-cell functional decline and insulin resistance, which increases the risk of cardiovascular complications as well as associated-morbidity and mortality. Evidence suggests a strong relationship between hyperglycaemia, oxidative stress and the development and progression of T2D. Indeed, a hyperglycaemic state can reduce the activity of antioxidant enzymes and increase lipid peroxidation and protein oxidation products, as well as DNA damage. At present, metformin is the recommended first-line glucose-lowering agent in patients with T2D. Despite the vast clinical experience gained over several decades of use, several mechanisms of action of metformin have yet to be fully elucidated. This review provides an overview of the existing literature concerning the complicated interplay between oxidative stress and T2D and the molecular mechanisms underlying the redox-related mechanisms of action of metformin, which include (but are not limited to) interaction with AMP-activated protein kinase (AMPK)-dependent and AMPK-independent mechanisms, inhibition of gluconeogenesis and action on leukocyte–endothelium interactions

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

MicroRNAs in diabetes and its vascular complications

Diabetes is a complex, metabolic disorder that is characterised by chronic hyperglycaemia and is widely recognised as a major health threat worldwide. The major causes of morbidity and mortality in diabetes result from vascular complications. These can be both microvascular, resulting in retinopathy, nephropathy and neuropathy, and macrovascular, affecting the heart and peripheral vessels. In this chapter, the roles of microRNAs in the development of diabetes are described. In addition, we show how microRNA-mediated mechanisms in diabetes result in an impaired vascular reparative potential, acting via the bone marrow and both stem and progenitor cells. Finally, we discuss the roles of microRNAs in the development of specific microvascular and macrovascular complications in the context of diabetes, and describe the potential of microRNAs as circulating biomarkers of diabetic cardiovascular disease

Mitochondria, the NLRP3 Inflammasome, and Sirtuins in Type 2 Diabetes: New Therapeutic Targets.

Type 2 diabetes mellitus and hyperglycemia can lead to the development of comorbidities such as atherosclerosis and microvascular/macrovascular complications. Both type 2 diabetes and its complications are related to mitochondrial dysfunction and oxidative stress. Type 2 diabetes is also a chronic inflammatory condition that leads to inflammasome activation and the release of proinflammatory mediators, including interleukins (ILs) IL-1β and IL-18. Moreover, sirtuins are energetic sensors that respond to metabolic load, which highlights their relevance in metabolic diseases, such as type 2 diabetes. Recent Advances: Over the past decade, great progress has been made in clarifying the signaling events regulated by mitochondria, inflammasomes, and sirtuins. Nod-like receptor family pyrin domain containing 3 (NLRP3) is the best characterized inflammasome, and the generation of oxidant species seems to be critical for its activation. NLRP3 inflammasome activation and altered sirtuin levels have been observed in type 2 diabetes. Critical Issue: Despite increasing evidence of the relationship between the NLRP3 inflammasome, mitochondrial dysfunction, and oxidative stress and of their participation in type 2 diabetes physiopathology, therapeutic strategies to combat type 2 diabetes that target NLRP3 inflammasome and sirtuins are yet to be consolidated. In this review article, we attempt to provide an overview of the existing literature concerning the crosstalk between mitochondrial impairment and the inflammasome, with particular attention to cellular and mitochondrial redox metabolism and the potential role of the NLRP3 inflammasome and sirtuins in the pathogenesis of type 2 diabetes. In addition, we discuss potential targets for therapeutic intervention based on these molecular interactions. Antioxid. Redox Signal. 29, 749-791

Mitochondrial dynamics in type 2 diabetes: Pathophysiological implications

Mitochondria play a key role in maintaining cellular metabolic homeostasis. These organelles have a high plasticity and are involved in dynamic processes such as mitochondrial fusion and fission, mitophagy and mitochondrial biogenesis. Type 2 diabetes is characterised by mitochondrial dysfunction, high production of reactive oxygen species (ROS) and low levels of ATP. Mitochondrial fusion is modulated by different proteins, including mitofusin-1 (MFN1), mitofusin-2 (MFN2) and optic atrophy (OPA-1), while fission is controlled by mitochondrial fission 1 (FIS1), dynamin-related protein 1 (DRP1) and mitochondrial fission factor (MFF). PARKIN and (PTEN)-induced putative kinase 1 (PINK1) participate in the process of mitophagy, for which mitochondrial fission is necessary. In this review, we discuss the molecular pathways of mitochondrial dynamics, their impairment under type 2 diabetes, and pharmaceutical approaches for targeting mitochondrial dynamics, such as mitochondrial division inhibitor-1 (mdivi-1), dynasore, P110 and 15-oxospiramilactone. Furthermore, we discuss the pathophysiological implications of impaired mitochondrial dynamics, especially in type 2 diabetes. Keywords: Mitochondrial dynamics, Type 2 diabetes, Redox biology, Oxidative stres

Is Glycemic Control Modulating Endoplasmic Reticulum Stress in Leukocytes of Type 2 Diabetic Patients?

Oxidative and endoplasmic reticulum (ER) stress is related to type 2 diabetes (T2D), but the influence of glycemic control on these parameters and its relationship with leukocyte-endothelial interactions is not known. In our study population consisting of 164 diabetic patients, (102 with glycated hemoglobin [HbA1c] 7%) and 84 nondiabetic controls, we have verified a common anthropometric and metabolic pattern of T2D with dyslipidemia. Inflammatory parameters (high-sensitive C-reactive protein [hs-CRP] and tumor necrosis factor alpha [TNFα]) and E-selectin levels were enhanced in the HbA1c >7% group with regard to controls. O2 consumption and mitochondrial membrane potential were lower in diabetic patients than in controls. Reactive oxygen species (ROS) production was enhanced in diabetic patients than in controls and positively correlated with HbA1c levels. GRP78 levels were higher in both diabetic groups. However, HbA1c 7% patients exhibited preferentially enhanced levels of CHOP (CCAAT/enhancer binding protein [C/EBP] homologous protein) and activating transcription factor 6 (ATF6). Reduced leukocyte rolling velocity and increased rolling flux and adhesion were observed in diabetic patients. Our findings lead to the hypothesis of an association between poor glycemic control in T2D and increased leukocyte ROS production and chronic ER stress that could finally promote leukocyte-endothelial interactions, which, in turn, poses a risk of vascular complications for these patient