Antioxidant and neuroprotective actions of IGF-II against glucocorticoid-induced toxicity in dopaminergic neurons.

The neurodegenerative Parkinson’s disease (PD) affects 1–3% of the population aged over 65. A wide range of pathways and mechanisms are involved in its pathogenesis, such as oxidative stress, mitochondrial dysfunction, inflammation and neuronal glucocorticoid-induced toxicity, which ultimately produce a progressive loss of nigral dopamine neurons. Insulin-like growth factor II (IGF-II) has shown antioxidant and neuroprotective effects in some neurodegenerative disorders. Therefore, our aim was to study IGF-II protective effects against oxidative damage on a cellular combined model of PD and mild to moderate stress, based on corticosterone (CORT), an endocrine response marker to stress, and the dopaminergic neurotoxin 1-methyl-4-phenylpyridinium (MPP+). The dopaminergic neuronal cell line SN4741 (RRID:CVCL_S466) derived from mouse substantia nigra were exposed to 200 μM MPP+, 0.5 μM CORT or both, with or without 25 ng/mL IGF-II, for 2.5 or 6 h. Cell viability, oxidative stress parameters, mitochondrial and dopamine markers and intracellular signaling pathways were evaluated. The administration of MPP+ or CORT individually led to cell damage compared to control situations, whereas the combination of both drugs produced very considerable toxic synergistic effect. IGF-II counteracts the mitochondrial-oxidative damage, protecting dopaminergic neurons from death and neurodegeneration. IGF-II maintained the tyrosine hydroxylase expression and promotes nuclear factor (erythroid-derived 2)-like 2 antioxidant response in a glucocorticoid receptor-dependent pathway, preventing oxidative cell damage and maintaining mitochondrial function. This work revealed the potential neuroprotective role of IGF-II to protect nigral dopamine neurons against mitochondrial-oxidative damage induced by CORT and MPP+ was demonstrated. Thus, IGF-II is a potential therapeutic tool for prevention and treatment of PD patients suffering mild to moderate emotional stress.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech

RPL13A and EEF1A1 Are Suitable Reference Genes for qPCR during Adipocyte Differentiation of Vascular Stromal Cells from Patients with Different BMI and HOMA-IR

Real-time or quantitative PCR (qPCR) is a useful technique that requires reliable reference genes for data normalization in gene expression analysis. Adipogenesis is among the biological processes suitable for this technique. The selection of adequate reference genes is essential for qPCR gene expression analysis of human Vascular Stromal Cells (hVSCs) during their differentiation into adipocytes. To the best of our knowledge, there are no studies validating reference genes for the analyses of visceral and subcutaneous adipose tissue hVSCs from subjects with different Body Mass Index (BMI) and Homeostatic Model Assessment of Insulin Resistance (HOMA-IR) index. The present study was undertaken to analyze this question. We first analyzed the stability of expression of five potential reference genes: CYC, GAPDH, RPL13A, EEF1A1, and 18S ribosomal RNA, during in vitro adipogenic differentiation, in samples from these types of patients. The expression of RPL13A and EEF1A1 was not affected by differentiation, thus being these genes the most stable candidates, while CYC, GAPDH, and 18S were not suitable for this sort of analysis. This work highlights that RPL13A and EEF1A1 are good candidates as reference genes for qPCR analysis of hVSCs differentiation into adipocytes from subjects with different BMI and HOMA-IR.Instituto de Salud Carlos III (PI10/01947, PI13/02628) with Fondos FEDER and the Consejería de Economía e Innovación, Ciencia y Empleo, Junta de Andalucía (CTS-7895) with Fondos FEDER. R. El Bekay is under a contract Miguel Servet type II (CPII13/00041) from the Instituto de Salud Carlos III. F-JB-S is a recipient of a "Miguel Servet II" research contract (CPII13/00042) and also belongs to the regional "Nicolás Monardes" research program of the Consejería de Salud (C-0070-2012; Junta de Andalucía, Spain). This work was supported by the FIS-Thematic Networks and Co-Operative Research Centres RIRAAF (RD07-0064). JM is under the Programa de Intensificación de la Actividad Investigadora del Sistema Nacional de Salud. AV-R is under a contract Proyectos de I+D+i para jóvenes investigadores from the Ministerio de Economía y Competitividad (SAF2014-60649-JIN). S-YR-Z is recipient of a post-doctoral contract from Consejería de Salud de la Junta de Andalucía (RH-0070-2013)

Neuronal Metabolism and Neuroprotection: Neuroprotective Effect of Fingolimod on Menadione-Induced Mitochondrial Damage

Imbalance in the oxidative status in neurons, along with mitochondrial damage, are common characteristics in some neurodegenerative diseases. The maintenance in energy production is crucial to face and recover from oxidative damage, and the preservation of different sources of energy production is essential to preserve neuronal function. Fingolimod phosphate is a drug with neuroprotective and antioxidant actions, used in the treatment of multiple sclerosis. This work was performed in a model of oxidative damage on neuronal cell cultures exposed to menadione in the presence or absence of fingolimod phosphate. We studied the mitochondrial function, antioxidant enzymes, protein nitrosylation, and several pathways related with glucose metabolism and glycolytic and pentose phosphate in neuronal cells cultures. Our results showed that menadione produces a decrease in mitochondrial function, an imbalance in antioxidant enzymes, and an increase in nitrosylated proteins with a decrease in glycolysis and glucose-6-phosphate dehydrogenase. All these effects were counteracted when fingolimod phosphate was present in the incubation media. These effects were mediated, at least in part, by the interaction of this drug with its specific S1P receptors. These actions would make this drug a potential tool in the treatment of neurodegenerative processes, either to slow progression or alleviate symptoms.This research was supported by the following projects: PS13/14: Study of the non immunological mechanisms of action of Gilenya (Fingolimod) as therapeutic tool in Multiple Sclerosis and/or other neurodegenerative diseases, Novartis Farmacéutica S.A.; CTS507 and CTS156 from Consejería de Economía Innovación Ciencia y Empresa, Junta de Andalucía. N. Valverde was supported by Proyectos I+D+I-Programa Operativo FEDER Andalucía 2014-2020 (UMA18-FEDERJA 004) Junta de Andalucía and Fondo Social Europeo (EU).Ye

IGF-II treatment prevents the oxidative damage derived by MPP+/MPTP administration in a cellular and animal model of Parkinson’s disease.

El factor de crecimiento de la insulina-II (IGF-II) ha mostrado efectos antioxidantes y neuroprotectores en algunos trastornos neurodegenerativos, como es la Enfermedad de Parkinson (EP). Analizamos el efecto de IGF-II y la implicación de la esfingosina kinasa (SPHK), en la citoarquitectura/función mitocondrial tras provocar daño oxidativo con la administración de la neurotoxina MPTP y su metabolito activo. La línea celular SN4741 se trató con MPP+ solo/en presencia de IGF-II (2 h). Los tratamientos fueron reemplazados por medio/IGF-II (2h), respectivamente. Se estudió: morfología (microscopía electrónica, EM), tasa de consumo de oxígeno (OCR) y muerte celular (LDH). MPTP/probenecid(p) por vía parenteral (35d) indujo un daño progresivo en la vía nigroestriatal dopaminérgica (DA-NSP) de los animales. En los días 36-44, se inyectó vehículo. El grupo control recibió vehículo siguiendo el mismo régimen de administración (1-44d). Otro grupo se trató con IGF-II una vez inducido el daño por MPTP (22-44 días). Para investigar el papel de IGF-II en la alteración conductual inducida por MPTP/p, se evaluó el rendimiento motor. Se realizó inmunotinción para marcadores dopaminérgicos y astrogliosis. Las células tratadas con MPP+ mostraron menos mitocondrias (EM) y con pérdida parcial/total de crestas, alteraciones de membrana y forma hinchada. IGF-II mantuvo el número de mitocondrias con morfología similar al control. La disminución de OCR tras la administración de MPP+ (30%) se recuperó con IGF-II. SPHK está implicada en este mecanismo, como indica su inhibición (MPP++IGF-II+MPA-08 6 veces > LDH vs MPP++IGF-II). En animales, IGF-II recuperó el efecto de MPTP sobre los marcadores DA-NSP y sobre el rendimiento motor. IGF-II contrarresta el aumento del estrés oxidativo y la disfunción mitocondrial inducida por la neurotoxina, el deterioro conductual y la degeneración de DA-NSP. SPHK estaría involucrada en este mecanismo.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech

Rich oleocanthal and oleacein extra virgin olive oil and inflammatory and antioxidant status in people with obesity and prediabetes. The APRIL study: A randomised, controlled crossover study

Background: Oleocanthal and oleacein are olive oil phenolic compounds with well known anti inflammatory and anti-oxidant properties. The main evidence, however, is provided by experimental studies. Few human studies have examined the health benefits of olive oils rich in these biophenols. Our aim was to assess the health properties of rich oleocanthal and oleacein extra virgin olive oil (EVOO), compared to those of common olive oil (OO), in people with prediabetes and obesity. Methods: Randomised, double-blind, crossover trial done in people aged 40e65 years with obesity (BMI 30e40 kg/m2 ) and prediabetes (HbA1c 5.7e6.4%). The intervention consisted in substituting for 1 month the oil used for food, both raw and cooked, by EVOO or OO. No changes in diet or physical activity were recommended. The primary outcome was the inflammatory status. Secondary outcomes were the oxidative status, body weight, glucose handling and lipid profile. An ANCOVA model adjusted for age, sex and treatment administration sequence was used for the statistical analysis. Results: A total of 91 patients were enrolled (33 men and 58 women) and finished the trial. A decrease in interferon-g was observed after EVOO treatment, reaching inter-treatment differences (P ¼ 0.041). Total antioxidant status increased and lipid and organic peroxides decreased after EVOO treatment, the changes reaching significance compared to OO treatment (P < 0.05). Decreases in weight, BMI and blood glucose (p < 0.05) were found after treatment with EVOO and not with OO. Conclusions: Treatment with EVOO rich in oleocanthal and oleacein differentially improved oxidative and inflammatory status in people with obesity and prediabetes.Funding for open access charge: Universidad de Málaga/CBU

Rich oleocanthal and oleacein extra virgin olive oil and inflammatory and antioxidant status in people with obesity and prediabetes. The APRIL study: A randomised, controlled crossover study

10 Páginas.-- 2 Figuras.-- 6 TablasOleocanthal and oleacein are olive oil phenolic compounds with well known anti-inflammatory and anti-oxidant properties. The main evidence, however, is provided by experimental studies. Few human studies have examined the health benefits of olive oils rich in these biophenols. Our aim was to assess the health properties of rich oleocanthal and oleacein extra virgin olive oil (EVOO), compared to those of common olive oil (OO), in people with prediabetes and obesity.his study was funded by Consejeria de Salud y Familias, Junta de Andalucia (PI-0247-2016) and Instituto de Salud Carlos III, Ministerio de Sanidad, Gobierno de España, (PI17/01004). FJBS, GRM and REB belong to the regional “Nicolás Monardes” research program from Consejería de Salud, Junta de Andalucía, Spain (C-0070-2012, C-0060-2012 and C-0030-2016). IRG holds a Río Hortega contract from Instituto de Salud Carlos III (CM20/00225) cofunded by European Social Fund 2014–2020, EU “The ESF invests in your future”. CIBERDEM is an initiative of the Instituto de Salud Carlos III, EU. Funding for Open Access charge: Universidad de Málaga/CBUA.Peer reviewe

Differences in the neovascular potential of thymus versus subcutaneous adipose-derived stem cells from patients with myocardial ischemia.

Adipose tissue-derived multipotent mesenchymal cells (ASCs) participate in the formation of blood vessels under hypoxic conditions. It is probable that the susceptibility of ASCs to the influence of age and aging-associated pathologies compromises their therapeutic effectiveness depending on the adipose tissue (AT) depot. Our aim was to examine the neovascular potential under hypoxic conditions of ASCs-derived from thymic (thymASCs) and subcutaneous (subASCs) AT from 39 subjects with and without type 2 diabetes mellitus (T2DM) and of different ages who were undergoing coronary bypass surgery (CBS). We confirmed a significant decrease in the percentage of CD34+ CD31- CD45- subASCs in the cell yield of subASCs and in the survival of cultured endothelial cells in the medium conditioned by the hypox-subASCs with increasing patient age, which was not observed in thymASCs. While the length of the tubules generated by hypox-subASCs tended to correlate negatively with patient age, tubule formation capacity of the hypoxic thymASCs increased significantly. Compared with subASCs, thymASCs from subjects over age 65 and without T2DM showed higher cell yield, tubule formation capacity, VEGF secretion levels and the ability to promote endothelial cell survival in their conditioned medium. Deterioration in subASCs neovascular potential relative to thymASCs derived from these subjects was accompanied by higher expression levels of NOX4 mRNA and fibrotic proteins. Our results indicate that thymASCs from patients over age 65 and without T2DM have a higher angiogenic potential than those from the other patient groups, suggesting they may be a good candidate for angiogenic therapy in subjects undergoing CBS.Ministry of Economy and Knowledge, Grant/Award Number: CTS‐7895/2011; This workwas co‐funded by the European Unionthrough the European Regional DevelopmentFund (FEDER) and supported by grants fromthe Ministry of Economy and Competitiveness,Institute of Health Carlos III, Grant/AwardNumber: PI15/01114, PI13/02628 and PI12/0235

miR-20b, miR-296, and Let-7f Expression in Human Adipose Tissue is Related to Obesity and Type 2 Diabetes.

This study aimed to analyze the potential association of different microRNA (miRNA) molecules with both type 2 diabetes (T2D) and obesity and determine their target genes. Quantitative PCR was used to analyze the miR-20b, miR-296, and Let-7f levels in human visceral and subcutaneous adipose tissues (ATs) in relation to obesity and T2D, miRTarBase 4.0 was used for validation of target genes, and the Protein Analysis Through Evolutionary Relationships (PANTHER) Classification System and the Database for Annotation, Visualization and Integrated Discovery (DAVID) were used to annotate the biological processes of the predicted targets. In AT, miR-20b, miR-296, and Let-7f levels were significantly different between normoglycemic subjects and those with T2D. In visceral adipose tissue, miRNA levels were higher in normoglycemic/obesity samples than in T2D/obesity samples. miR-20b-miR-296 and Let-7f target genes that showed significant differences in both ATs in relation to obesity and T2D were CDKN1A, CX3CL1, HIF1A, PPP2R1B, STAT3, and VEGFA. These genes are known to be principally involved in the vascular endothelial growth factor (VEGF) and WNT pathways. This study provides experimental evidence of the possible correlation between AT miR-20b-miR-296-Let-7f with obesity and T2D, which might involve vascular endothelial growth factor and WNT-dependent pathways that are regulated by six different genes, suggesting a novel signaling pathway that could be important for understanding the mechanisms underlying the AT dysfunction associated with obesity and T2D.This work was cofunded by the European Union through the European Regional Development Fund (FEDER) and was supported by grants from the Ministry of Economy and Competitiveness, Institute of Health Carlos III (PI15/01114, PI13/02628; PI12/02355), Ministry of Economy and Knowledge (PI‐CTS‐08181/2011; CTS‐7895/2011), and Consejeria de Salud, Junta de Andalucia (PI‐0092‐2017). RE is under a contract of a “Nicolas Monarde” program from the Servicio Andaluz de Salud