Implicación de los astrocitos hipotalámicos en las alteraciones metabólicas y en los cambios de composición corporal inducidos por malnutrición en los periodos neonatal y adulto: nuevas acciones de la leptina y la ghrelina

Tesis doctoral inédita leída en la Universidad Autónoma de Madrid. Facultad de Medicina, Departamento de Pediatría. Fecha de lectura: 15 de Diciembre de 201

Emerging role of glial cells in the control of body weight

Glia are the most abundant cell type in the brain and are indispensible for the normal execution of neuronal actions. They protect neurons from noxious insults and modulate synaptic transmission through affectation of synaptic inputs, release of glial transmitters and uptake of neurotransmitters from the synaptic cleft. They also transport nutrients and other circulating factors into the brain thus controlling the energy sources and signals reaching neurons. Moreover, glia express receptors for metabolic hormones, such as leptin and insulin, and can be activated in response to increased weight gain and dietary challenges. However, chronic glial activation can be detrimental to neurons, with hypothalamic astrocyte activation or gliosis suggested to be involved in the perpetuation of obesity and the onset of secondary complications. It is now accepted that glia may be a very important participant in metabolic control and a possible therapeutical target. Here we briefly review this rapidly advancing fiel

Leptin in early life: A key factor for the development of the adult metabolic profile

Leptin levels during the perinatal period are important for the development of metabolic systems involved in energy homeostasis. In rodents, there is a postnatal leptin surge, with circulating leptin levels increasing around postnatal day (PND) 5 and peaking between PND 9 and PND 10. At this time circulating leptin acts as an important trophic factor for the development of hypothalamic circuits that control energy homeostasis and food seeking and reward behaviors. Blunting the postnatal leptin surge results in long-term leptin insensitivity and increased susceptibility to diet-induced obesity during adulthood. Pharmacologically increased leptin levels in the postnatal period also have long-term effects on metabolism. Nevertheless, this effect is controversial as postnatal hyperleptinemia is reported to both increase and decrease the predisposition to obesity in adulthood. The different effects reported in the literature could be explained by the different moments at which this hormone was administered, suggesting that modifications of the neonatal leptin surge at specific time points could selectively affect the development of central and peripheral systems that are undergoing modifications at this moment resulting in different metabolic and behavioral outcomes. In addition, maternal nutrition and the hormonal environment during pregnancy and lactation may also modulate the offspring’s response to postnatal modifications in leptin levels. This review highlights the importance of leptin levels during the perinatal period in the development of metabolic systems that control energy homeostasis and how modifications of these levels may induce long-lasting and potentially irreversible effects on metabolis

PAX4 preserves endoplasmic reticulum integrity preventing beta cell degeneration in a mouse model of type 1 diabetes mellitus

[Aims/hypothesis]: A strategy to enhance pancreatic islet functional beta cell mass (BCM) while restraining inflammation, through the manipulation of molecular and cellular targets, would provide a means to counteract the deteriorating glycaemic control associated with diabetes mellitus. The aims of the current study were to investigate the therapeutic potential of such a target, the islet-enriched and diabetes-linked transcription factor paired box 4 (PAX4), to restrain experimental autoimmune diabetes (EAD) in the RIP-B7.1 mouse model background and to characterise putative cellular mechanisms associated with preserved BCM. [Methods]: Two groups of RIP-B7.1 mice were genetically engineered to: (1) conditionally express either PAX4 (BPTL) or its diabetes-linked mutant variant R129W (mutBPTL) using doxycycline (DOX); and (2) constitutively express luciferase in beta cells through the use of RIP. Mice were treated or not with DOX, and EAD was induced by immunisation with a murine preproinsulin II cDNA expression plasmid. The development of hyperglycaemia was monitored for up to 4 weeks following immunisation and alterations in the BCM were assessed weekly by non-invasive in vivo bioluminescence intensity (BLI). In parallel, BCM, islet cell proliferation and apoptosis were evaluated by immunocytochemistry. Alterations in PAX4- and PAX4R129W-mediated islet gene expression were investigated by microarray profiling. PAX4 preservation of endoplasmic reticulum (ER) homeostasis was assessed using thapsigargin, electron microscopy and intracellular calcium measurements. [Results]: PAX4 overexpression blunted EAD, whereas the diabetes-linked mutant variant PAX4R129W did not convey protection. PAX4-expressing islets exhibited reduced insulitis and decreased beta cell apoptosis, correlating with diminished DNA damage and increased islet cell proliferation. Microarray profiling revealed that PAX4 but not PAX4R129W targeted expression of genes implicated in cell cycle and ER homeostasis. Consistent with the latter, islets overexpressing PAX4 were protected against thapsigargin-mediated ER-stress-related apoptosis. Luminal swelling associated with ER stress induced by thapsigargin was rescued in PAX4-overexpressing beta cells, correlating with preserved cytosolic calcium oscillations in response to glucose. In contrast, RNA interference mediated repression of PAX4-sensitised MIN6 cells to thapsigargin cell death. [Conclusions/interpretation]: The coordinated regulation of distinct cellular pathways particularly related to ER homeostasis by PAX4 not achieved by the mutant variant PAX4R129W alleviates beta cell degeneration and protects against diabetes mellitus. The raw data for the RNA microarray described herein are accessible in the Gene Expression Omnibus database under accession number GSE62846

A simple high efficiency intra-islet transduction protocol using lentiviral vectors

Successful normalization of blood glucose in patients transplanted with pancreatic islets isolated from cadaveric donors established the proof-of-concept that Type 1 Diabetes Mellitus is a curable disease. Nonetheless, major caveats to the widespread use of this cell therapy approach have been the shortage of islets combined with the low viability and functional rates subsequent to transplantation. Gene therapy targeted to enhance survival and performance prior to transplantation could offer a feasible approach to circumvent these issues and sustain a durable functional β-cell mass in vivo. However, efficient and safe delivery of nucleic acids to intact islet remains a challenging task. Here we describe a simple and easy-to-use lentiviral transduction protocol that allows the transduction of approximately 80 % of mouse and human islet cells while preserving islet architecture, metabolic function and glucose-dependent stimulation of insulin secretion. Our protocol will facilitate to fully determine the potential of gene expression modulation of therapeutically promising targets in entire pancreatic islets for xenotransplantation purposes

Ghrelin regulates glucose and glutamate transporters in hypothalamic astrocytes

Hypothalamic astrocytes can respond to metabolic signals, such as leptin and insulin, to modulate adjacent neuronal circuits and systemic metabolism. Ghrelin regulates appetite, adiposity and glucose metabolism, but little is known regarding the response of astrocytes to this orexigenic hormone. We have used both in vivo and in vitro approaches to demonstrate that acylated ghrelin (acyl-ghrelin) rapidly stimulates glutamate transporter expression and glutamate uptake by astrocytes. Moreover, acyl-ghrelin rapidly reduces glucose transporter (GLUT) 2 levels and glucose uptake by these glial cells. Glutamine synthetase and lactate dehydrogenase decrease, while glycogen phosphorylase and lactate transporters increase in response to acyl-ghrelin, suggesting a change in glutamate and glucose metabolism, as well as glycogen storage by astrocytes. These effects are partially mediated through ghrelin receptor 1A (GHSR-1A) as astrocytes do not respond equally to desacyl-ghrelin, an isoform that does not activate GHSR-1A. Moreover, primary astrocyte cultures from GHSR-1A knock-out mice do not change glutamate transporter or GLUT2 levels in response to acyl-ghrelin. Our results indicate that acyl-ghrelin may mediate part of its metabolic actions through modulation of hypothalamic astrocytes and that this effect could involve astrocyte mediated changes in local glucose and glutamate metabolism that alter the signals/nutrients reaching neighboring neurons.This work was funded by grants from Fondo de Investigación Sanitaria (PI100747; PI1302195), Ministerio de Ciencia e Innovación (BFU2011–27492; BFU2014-51836-C2-2-R) and Fondos FEDER, Centro de Investigación Biomédica en Red Fisiopatología de Obesidad y Nutrición (CIBEROBN), Instituto de Salud Carlos III, and Fundación de Endocrinología y Nutrición. SD was funded by the Swedish Research Council (Vetenskapsrådet grant 2012– 1758), Läkarutbildningsavtalet Göteborg grant at Sahlgrenska Hospital (ALFGBG-138741, The European Union Seventh Framework Programme under Grant Agreement 607310, Nudge-it

Complicaciones nefrourológicas en pacientes sometidos a resección pulmonar según la utilización o no de sondaje vesical

Introducción: La implementación del protocolo ERAS (Enhanced Recovery After Surgery) en cirugía torácica ha implicado un cambio en el manejo perioperatorio de los pacientes. Una de las nuevas recomendaciones es evitar la colocación sistémica del catéter urinario en cirugía pulmonar. Existe poca evidencia en la bibliografía sobre las complicaciones nefrourológicas (URC) postoperatorias. Por ello, diseñamos un estudio con el fin de evaluar la incidencia de URC en la población sometida a resección pulmonar por videotoracoscopia. Material y Métodos: Realizamos un estudio longitudinal y prospectivo en la Unidad de Reanimación Postanestésica (URPA) en un hospital de tercer nivel durante el periodo comprendido entre abril 2019 y julio del 2020. Se recogieron variables perioperatorias así como la presencia de URC. Resultados: De los 62 pacientes (82%) ingresaron en URPA sin catéter urinario, 5 (8%) presentaron URC.  El 60% (3 de 5) de los pacientes con complicaciones presentaban volúmenes vesicales estimados por ecografía altos (>300 ml) a su ingreso en URPA y el 80% (4 de 5) a las 4 horas. Estas complicaciones no implicaron un deterioro significativo de la función renal durante el ingreso. Conclusiones: La recomendación de evitar el sondaje urinario en cirugía de resección pulmonar parece una práctica segura, aunque existe una incidencia no despreciable de URC en el postoperatorio inmediato. Sería muy interesante disponer de herramientas que permitan una detección y monitorización de los pacientes con riesgo incrementado para favorecer la detección precoz de complicaciones

Evaluation of cytokines as robust diagnostic biomarkers for COVID-19 detection

Producción CientíficaAntigen tests or polymerase chain reaction (PCR) amplification are currently COVID-19 diagnostic tools. However, developing complementary diagnosis tools is mandatory. Thus, we performed a plasma cytokine array in COVID-19 patients to identify novel diagnostic biomarkers. A discovery–validation study in two independent prospective cohorts was performed. The discovery cohort included 136 COVID-19 and non-COVID-19 patients recruited consecutively from 24 March to 11 April 2020. Forty-five cytokines’ quantification by the MAGPIX system (Luminex Corp., Austin, TX, USA) was performed in plasma samples. The validation cohort included 117 patients recruited consecutively from 15 to 25 April 2020 for validating results by ELISA. COVID-19 patients showed different levels of multiple cytokines compared to non-COVID-19 patients. A single chemokine, IP-10, accurately identified COVID-19 patients who required hospital admission (AUC: 0.962; 95%CI (0.933–0.992); p < 0.001)). The results were validated in an independent cohort by multivariable analysis (OR: 25.573; 95%CI (8.127–80.469); p < 0.001) and AUROC (AUC: 0.900; 95%CI (0.846–0.954); p < 0.001). Moreover, showing IP-10 plasma levels over 173.35 pg/mL identified COVID-19 with higher sensitivity (86.20%) than the first SARS-CoV-2 PCR. Our discover–validation study identified IP-10 as a robust biomarker in clinical practice for COVID-19 diagnosis at hospital. Therefore, IP-10 could be used as a complementary tool in clinical practice, especially in emergency departments.Instituto de Salud Carlos III (grant COV20/00491)Consejo Superior de Investigaciones científicas (grant CSIC-COV19-016/202020E155)Junta de Castilla y León (project COVID 07.04.467B04.74011.0)IBGM excellence programme (grant CLU-2029-02

Maximal respiratory pressure reference equations in healthy adults and cut-off points for defining respiratory muscle weakness

[Abstract] Introduction: Maximal inspiratory and expiratory pressures (PImax/PEmax) reference equations obtained in healthy people are needed to correctly interpret respiratory muscle strength. Currently, no clear cut-off points defining respiratory muscle weakness are available. We aimed to establish sex-specific reference equations for PImax/PEmax in a large sample of healthy adults and to objectively determine cut-off points for respiratory muscle weakness. Methods: A multicentre cross-sectional study was conducted across 14 Spanish centres. Healthy non-smoking volunteers aged 18-80 years stratified by sex and age were recruited. PImax/PEmax were assessed using uniform methodology according to international standards. Multiple linear regressions were used to obtain reference equations. Cut-off points for respiratory muscle weakness were established by using T-scores. Results: The final sample consisted of 610 subjects (314 females; 48 [standard deviation, SD: 17] years). Reference equations for PImax/PEmax included body mass index and a squared term of the age as independent variables for both sexes (p<0.01). Cut-off points for respiratory muscle weakness based on T-scores ≥2.5 SD below the peak mean value achieved at a young age were: 62 and 83cmH2O for PImax and 81 and 109cmH2O for PEmax in females and males, respectively. Conclusion: These reference values, based on the largest dataset collected in a European population to date using uniform methodology, help identify cut-off points for respiratory muscle weakness in females and males. These data will help to better identify the presence of respiratory muscle weakness and to determine indications for interventions to improve respiratory muscle function

Association between vitamin D receptor rs731236 (Taq1) polymorphism and risk for restless legs syndrome in the Spanish caucasian population

Varios trabajos recientes sugieren un posible papel de la deficiencia de vitamina D en la etiología o el síndrome de las piernas inquietas (RLS). Hemos analizado la posible relación de 2 polimorfismos de un solo nucleótido (SNP) en el receptor de la vitamina D3 (GEN VDR) con el riesgo de SPI. Hemos estudiado la variante alélica genotipo y frecuencias de VDR rs2228570 y rs731236 VDR SNPs en 205 RLS pacientes y 445 controles sanos mediante un ensayo TaqMan. Las frecuencias de los rs731236AAgenotype y la variante alélica rs731236un SPI fue significativamente inferior en los pacientes que en los controles (P<0,005 y 0,01, respectivamente). El síndrome de las piernas inquietas pacientes portadoras de la variante alélica rs731236G había una edad temprana en el inicio, y los portadores del genotipo GG731236rs tuvieron mayor severidad de RLS, aunque estos datos desaparecieron después de los análisis multivariados. Ninguno de los SNPs estudiados estaba relacionada con la positividad de la historia familiar de SPI. Estos resultados sugieren una modesta, pero significativa asociación entre rs731236 VDR SNP y el riesgo de síndrome de piernas inquietas.Several recent works suggest a possible role of vitamin D deficiency in the etiology or restless legs syndrome (RLS). We analyzed the possible relationship of 2 common single nucleotide polymorphisms (SNPs) in the vitamin D3 receptor (VDR) gene with the risk for RLS. We studied the genotype and allelic variant frequencies of VDR rs2228570 and VDR rs731236 SNPs in 205 RLS patients and 445 healthy controls using a TaqMan essay. The frequencies of the rs731236AAgenotype and the allelic variant rs731236A were significantly lower in RLS patients than in controls (P<0.005 and<0.01, respectively). Restless legs syndrome patients carrying the allelic variant rs731236G had an earlier age at onset, and those carrying the rs731236GG genotype had higher severity of RLS, although these data disappeared after multivariate analyses. None of the SNPs studied was related with the positivity of family history of RLS. These results suggest a modest, but significant association between VDR rs731236 SNP and the risk for RLS.• Instituto de Salud Carlos III, Madrid, Fondo de Investigación Sanitaria: Ayudas PI12/00241, PI12/00324, y RETICS RD12/0013/0002 • Junta de Extremadura: GR15026 y PRIS10016 • Ministerio de Ciencia e Innovación: Ayudas SAF2006-10126 (2006–2009) y SAF2010-22329-C02-01 (2011-2013) • Parciamente financiado con Fondos FEDERpeerReviewe