Estudi del canal de glicerol aqp7 i de l'adipoquina zag en l'àmbit de l'obesitat, la diabetis tipus 2 i la síndrome metabòlica. Dues proteïnes implicades en la lipòlisi del teixit adipós

L'obesitat incrementa les possibilitats de patir altres malalties com la diabetis mellitus tipus 2 (DM2), la dislipèmia, la hipertensió, entre d'altres. Per tal de poder avançar en noves dianes terapèutiques que millorin aquestes malalties és necessari conèixer millor la biologia del teixit adipós i els seus components. El teixit adipós blanc, a més a més d'emmagatzemar la major part de les reserves energètiques, és capaç de tenir activitat metabòlica i endocrina. Cal tenir en compte que un adequat metabolisme lipídic en l'adipòcit és clau també per aconseguir un equilibri en l'homeòstasi del metabolisme glucídic en l'ésser humà. En aquest treball hem analitzat el paper de dues proteïnes implicades en la lipòlisi del teixit adipós, d'una banda el canal de glicerol aquaporina 7 (AQP7) i per altra l'adipoquina glicoproteïna Zinc-α 2 (ZAG), en el context de l'obesitat, la síndrome metabòlica i la DM2.Així, en aquesta tesi doctoral, hem pogut arribar a les següents conclusions:1. L'expressió del canal de glicerol AQP7, en teixit adipós, es troba disminuïda en pacients afectes d'obesitat mòrbida.2. La DM2 no sembla afectar l'expressió d'aquesta aquagliceroporina en el teixit adipós.3. Ambdues aquagliceroporines, AQP7 (al teixit adipós) i AQP9 (al teixit hepàtic) mantenen un estreta relació en pacients obesos mòrbids.4. La glicoproteïna Zinc-α 2 (ZAG) sembla tenir un paper rellevant en l'homeòstasi del teixit adipós amb un fort lligam amb altres adipoquines, especialment l'adiponectina.Obesity increases the chances to suffer from other diseases such as type 2 diabetes mellitus (T2DM), hyperlipidemia, hypertension, and others. In order to get into new therapeutic targets to improve these conditions, a best knowledge of the biology of adipose tissue and its components is needed. In addition to storing the most of energy reserves, white adipose tissue is able to have metabolic and endocrine activity. It is necessary to consider that an appropriate lipid metabolism in the adipocyte is also essential to achieve a balance in the homeostasis of glucose metabolism in humans. In this study we have examined the role of two proteins involved in adipose tissue lipolysis, first glycerol channel aquaporin 7 (AQP7) and on the other hand adipokine Zinc-α 2 glycoprotein (ZAG), in the context of obesity, metabolic syndrome and T2DM. Thus, in this thesis, we have reached the following conclusions: 1. The glycerol's channel AQP7 expression, in adipose tissue, is decreased in patients with morbid obesity. 2. The DM2 does not appear to affect the expression of this aquaglyceroporin in adipose tissue. 3. Both aquaglyceroporins, AQP7 (in adipose tissue) and AQP9 (in liver tissue) have a close relationship in morbidly obese patients. 4. The Zinc-α 2 glycoprotein (ZAG) seems to play an important role in the homeostasis of adipose tissue with a strong bond with other adipokines, particularly adiponectin

Circulating pyruvate is a potent prognostic marker for critical COVID-19 outcomes

COVID-19; Cuantificación fluorométrica; PiruvatoCOVID-19; Quantificació fluoromètrica; PiruvatCOVID-19; Fluorometric quantification; PyruvateBackground: Coronavirus-19 (COVID-19) disease is driven by an unchecked immune response to the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus which alters host mitochondrial-associated mechanisms. Compromised mitochondrial health results in abnormal reprogramming of glucose metabolism, which can disrupt extracellular signalling. We hypothesized that examining mitochondrial energy-related signalling metabolites implicated in host immune response to SARS-CoV-2 infection would provide potential biomarkers for predicting the risk of severe COVID-19 illness. Methods: We used a semi-targeted serum metabolomics approach in 273 patients with different severity grades of COVID-19 recruited at the acute phase of the infection to determine the relative abundance of tricarboxylic acid (Krebs) cycle-related metabolites with known extracellular signaling properties (pyruvate, lactate, succinate and α-ketoglutarate). Abundance levels of energy-related metabolites were evaluated in a validation cohort (n=398) using quantitative fluorimetric assays. Results: Increased levels of four energy-related metabolites (pyruvate, lactate, a-ketoglutarate and succinate) were found in critically ill COVID-19 patients using semi-targeted and targeted approaches (p<0.05). The combined strategy proposed herein enabled us to establish that circulating pyruvate levels (p<0.001) together with body mass index (p=0.025), C-reactive protein (p=0.039), D-Dimer (p<0.001) and creatinine (p=0.043) levels, are independent predictors of critical COVID-19. Furthermore, classification and regression tree (CART) analysis provided a cut-off value of pyruvate in serum (24.54 µM; p<0.001) as an early criterion to accurately classify patients with critical outcomes. Conclusion: Our findings support the link between COVID-19 pathogenesis and immunometabolic dysregulation, and show that fluorometric quantification of circulating pyruvate is a cost-effective clinical decision support tool to improve patient stratification and prognosis prediction.This work has been developed in the framework of the COVIDOMICS’ project supported by Direcció General de Recerca i Innovació en Salut (DGRIS), Departament de Salut, Generalitat de Catalunya (PoC-6-17 and PoC1-5). The research was also funded by the Programa de Suport als Grups de Recerca AGAUR (2017SGR948), the SPANISH AIDS Research Network [RD16/0025/0006]-ISCIII-FEDER (Spain) and the Centro de Investigación Biomédica en Red de Enfermedades Infecciosas-ISCIII [CB21/13/00020], Madrid, Spain. LR is supported by the Instituto de Salud Carlos III (ISCIII) under grant agreement “CD20/00105” through the program “Contratos Sara Borrell”. FV is supported by grants from the Programa de Intensificación de Investigadores (INT20/00031)-ISCIII and by “Premi a la Trajectòria Investigadora dels Hospitals de l’ICS 2018”. AR is supported by a grant from IISPV through the project “2019/IISPV/05” (Boosting Young Talent), by GeSIDA through the “III Premio para Jóvenes Investigadores 2019” and by the Instituto de Salud Carlos III (ISCIII) under grant agreement “CP19/00146” through the Miguel Servet Program. This study was also supported by grants SAF2015–65019-R and RTI2018–093919-B-100 (to SF-V.) funded by MCIN/AEI and by “ERFD A way of making Europe”; PI19/01337 to FV, PI20/00095 to VC.-M, PI20/00326 to AR and PI20/00338 to JV funded by ISCIII, cofinanced by the European Regional Development Fund (ERDF), and from Fundación Bancaria Caixa d’Estalvis i Pensions de Barcelona (HR20-00051 to SF-V). The Spanish Biomedical Research Center in Diabetes and Associated Metabolic Disorders (CIBERDEM) (CB07708/0012) is an initiative of the Instituto de Salud Carlos III. SF-V acknowledges support from the Miguel Servet tenure-track program (CP10/00438 and CPII16/00008) from the Fondo de Investigación Sanitaria, cofinanced by the ERDF. VC-M acknowledges support from the Ramón y Cajal program (RYC2019-026490-I) from the Spanish Ministry of Science and Innovation, cofinanced by the ERDF. The work was also supported by Consejeria de Salud y Familia (COVID-0005-2020), Consejeria de Transformacion Economica, Industria, Conocimiento y Universidades Junta de Andalucia (CV20-85418to ER-M) and Instituto de Salud Carlos III (ISCIII) under grant agreement CP19/00159 to AGV “a way to make Europe”. ER-M was supported by the Spanish Research Council (CSIC). The funders have no roles in study design, data collection, data analysis, interpretation or the writing of this research

Arterial Stiffness Is Increased in Patients With Type 1 Diabetes Without Cardiovascular Disease

Altres ajuts: Financial support was provided by the Associació Catalana de Diabetis (Beca Gonçal Lloveras 2008); the Fundació la Marató de TV3-2008 (project no. 081410); FIS PS09/01360, Instituto de Salud Carlos III, Ministerio de Sanidad y Consumo, Spain; and by an intensification grant to J.M.G.-C. (Instituto de Salud Carlos III, Ministerio de Sanidad y Consumo, Spain)

Arterial Stiffness Is Increased in Patients With Type 1 Diabetes Without Cardiovascular Disease

Altres ajuts: Financial support was provided by the Associació Catalana de Diabetis (Beca Gonçal Lloveras 2008); the Fundació la Marató de TV3-2008 (project no. 081410); FIS PS09/01360, Instituto de Salud Carlos III, Ministerio de Sanidad y Consumo, Spain; and by an intensification grant to J.M.G.-C. (Instituto de Salud Carlos III, Ministerio de Sanidad y Consumo, Spain)

Zinc-α2-Glycoprotein Modulates AKT-Dependent Insulin Signaling in Human Adipocytes by Activation of the PP2A Phosphatase

<div><p>Objective</p><p>Evidence from mouse models suggests that zinc-α2-glycoprotein (ZAG) is a novel anti-obesity adipokine. In humans, however, data are controversial and its physiological role in adipose tissue (AT) remains unknown. Here we explored the molecular mechanisms by which ZAG regulates carbohydrate metabolism in human adipocytes.</p><p>Methods</p><p>ZAG action on glucose uptake and insulin action was analyzed. β1 and β2-adrenoreceptor (AR) antagonists and siRNA targeting PP2A phosphatase were used to examine the mechanisms by which ZAG modulates insulin sensitivity. Plasma levels of ZAG were measured in a lean patient cohort stratified for HOMA-IR.</p><p>Results</p><p>ZAG treatment increased basal glucose uptake, correlating with an increase in <i>GLUT</i> expression, but induced insulin resistance in adipocytes. Pretreatment of adipocytes with propranolol and a specific β1-AR antagonist demonstrated that ZAG effects on basal glucose uptake and GLUT4 expression are mediated via β1-AR, whereas inhibition of insulin action is dependent on β2-AR activation. ZAG treatment correlated with an increase in PP2A activity. Silencing of the PP2A catalytic subunit abrogated the negative effect of ZAG on insulin-stimulated AKT phosphorylation and glucose uptake but not on GLUT4 expression and basal glucose uptake. ZAG circulating levels were unchanged in a lean patient cohort stratified for HOMA-IR. Neither glucose nor insulin was associated with plasma ZAG.</p><p>Conclusions</p><p>ZAG inhibits insulin-induced glucose uptake in human adipocytes by impairing insulin signaling at the level of AKT in a β2-AR- and PP2A-dependent manner.</p></div

ZAG activation of PP2A phosphatase impairs insulin-stimulated glucose uptake and AKT phosphorylation in SGBS adipocytes.

<p><b>(A)</b> Differentiated human adipose cells were cultured with 25 μg/ml ZAG for 24 hours and PP2A activity was measured. <b>(B)</b> PP2A activity was measured in mature adipocytes pre-treated with 1 μM propranolol or 300 nM CPG20712A (CPG) prior to culture with ZAG. Data are presented as mean±SEM (n = 3). *, P < 0.01 vs control. (<b>C</b>) Adipose cells were transfected with 100 nM siRNA against the α-catalytic subunit of PP2A (PP2A-Cα) or RISC-free (control cells) and cultured with or without 25 μg/ml ZAG for 24 hours prior to stimulation with 100 nM insulin (Ins) for 30 minutes. Glucose uptake was measured by incorporation of labelled 2-deoxyglucose into the cells during the last 10 minutes of culture. Results from 3 independent experiments performed in triplicate are expressed as pmol glc/mg prot/10 min. Right panels represent percentage of stimulation produced by insulin over control cells (no insulin, without or with ZAG, respectively). *, P < 0.01. <b>(D)</b> PP2A-Cα protein expression was analyzed by western blot in control and transfected cells. A representative experiment is shown together with densitometric analysis (3 independent experiments). *, P < 0.01. <b>(E)</b> GLUT4 mRNA levels were analyzed by qPCR in siRNA control and PP2A-Cα-silenced adipocytes in the absence or presence of ZAG. Data are presented as mean±SEM (n = 3). *, P < 0.01 vs control. <b>(F)</b> Cells transfected as in D were cultured or not with 25 μg/ml ZAG for 24 hours prior to stimulation with 100 nM insulin (Ins) for 15 minutes. Cell lysates were analyzed by western blotting using antibodies against phosphorylated and total Akt (Ser473). A representative experiment is shown together with densitometric analysis of phosphorylated <i>vs</i> total proteins (3 independent experiments). *, P < 0.01.</p

Clinical, anthropometric, and analytical characteristics (units) according to insulin resistance classification.

<p>The results are given as the mean±SD. BMI: body mass index; HOMA-IR: homeostasis model assessment of insulin resistance index; DBP: Diastolic Blood Pressure; SBP: Systolic Blood Pressure. ^a and ^b indicate significant differences between the means of the different groups:</p><p>^a: P<0.05</p><p>^b: P<0.01(ANOVA or Student´s t where appropriate).</p><p>Clinical, anthropometric, and analytical characteristics (units) according to insulin resistance classification.</p