Altered surface expression of insulin-degrading enzyme on monocytes and lymphocytes from COVID-19 patients both at diagnosis and after hospital discharge

Although the COVID-19 disease has developed into a worldwide pandemic, its pathophysiology remains to be fully understood. Insulin-degrading enzyme (IDE), a zinc-metalloprotease with a high affinity for insulin, has been found in the interactomes of multiple SARS-CoV-2 proteins. However, the relevance of IDE in the innate and adaptative immune responses elicited by circulating peripheral blood mononuclear cells is unknown. Here, we show that IDE is highly expressed on the surface of circulating monocytes, T-cells (both CD4+ and CD4−), and, to a lower extent, in B-cells from healthy controls. Notably, IDE’s surface expression was upregulated on monocytes from COVID-19 patients at diagnosis, and it was increased in more severe patients. However, IDE’s surface expression was downregulated (relative to healthy controls) 3 months after hospital discharge in all the studied immune subsets, with this effect being more pronounced in males than in females, and thus it was sex-dependent. Additionally, IDE levels in monocytes, CD4+ T-cells, and CD4− T-cells were inversely correlated with circulating insulin levels in COVID-19 patients (both at diagnosis and after hospital discharge). Of note, high glucose and insulin levels downregulated IDE surface expression by ~30% in the monocytes isolated from healthy donors, without affecting its expression in CD4+ T-cells and CD4− T-cells. In conclusion, our studies reveal the sex- and metabolism-dependent regulation of IDE in monocytes, suggesting that its regulation might be important for the recruitment of immune cells to the site of infection, as well as for glucometabolic control, in COVID-19 patients.This work was funded by the European Commission–NextGenerationEU (Regulation EU 2020/2094), through CSIC’s Global Health Platform (PTI Salud Global) and Junta de Castilla y León (Proyectos COVID 07.04.467B04.74011.0) to D.B. The project leading to these results had received funding from the “La Caixa” Foundation under agreement LCF/PR/PR18/51130007 to G.P. and grant PID2019-110496RB-C22 funded by MCIN/AEI/10.13039/501100011033 to G.P. This research was funded by the Programa Estratégico Instituto de Biología y Genética Molecular (IBGM), Junta de León and the European Social Fund (ORDER EDU/574/2018)

Fine mapping and conditional analysis identify a new mutation in the autoimmunity susceptibility gene BLK that leads to reduced half-life of the BLK protein

OBJECTIVES: To perform fine mapping of the autoimmunity susceptibility gene BLK and identify functional variants involved in systemic lupus erythematosus (SLE). METHODS: Genotyping of 1163 European SLE patients and 1482 controls and imputation were performed covering the BLK gene with 158 single-nucleotide polymorphisms. Logistic regression analysis was done using PLINK and conditional analyses using GENABEL's test score. Transfections of BLK constructs on HEK293 cells containing the novel mutation or the wild type form were analysed for their effect on protein half-life using a protein stability assay, cycloheximide and western blot. CHiP-qPCR for detection of nuclear factor κ B (NFkB) binding. RESULTS: Fine mapping of BLK identified two independent genetic effects with functional consequences: one represented by two tightly linked associated haplotype blocks significantly enriched for NFκB-binding sites and numerous putative regulatory variants whose risk alleles correlated with low BLK mRNA levels. Binding of NFkBp50 and p65 to an associated 1.2 Kb haplotype segment was confirmed. A second independent genetic effect was represented by an Ala71Thr, low-frequency missense substitution with an OR=2.31 (95% CI 1.38 to 3.86). The 71Thr decreased BLK protein half-life. CONCLUSIONS: These results show that rare and common regulatory variants in BLK are involved in disease susceptibility and both, albeit independently, lead to reduced levels of BLK protein

Assessing the Impact of SARS-CoV-2 Lineages and Mutations on Patient Survival

Objectives: More than two years into the COVID-19 pandemic, SARS-CoV-2 still remains a global public health problem. Successive waves of infection have produced new SARS-CoV-2 variants with new mutations for which the impact on COVID-19 severity and patient survival is uncertain. Methods: A total of 764 SARS-CoV-2 genomes, sequenced from COVID-19 patients, hospitalized from 19th February 2020 to 30 April 2021, along with their clinical data, were used for survival analysis. Results: A significant association of B.1.1.7, the alpha lineage, with patient mortality (log hazard ratio (LHR) = 0.51, C.I. = [0.14,0.88]) was found upon adjustment by all the covariates known to affect COVID-19 prognosis. Moreover, survival analysis of mutations in the SARS-CoV-2 genome revealed 27 of them were significantly associated with higher mortality of patients. Most of these mutations were located in the genes coding for the S, ORF8, and N proteins. Conclusions: This study illustrates how a combination of genomic and clinical data can provide solid evidence for the impact of viral lineage on patient survival.This work was supported by Spanish Ministry of Science and Innovation (grant PID2020- 117979RB-I00), the Instituto de Salud Carlos III (ISCIII), co-funded with European Regional Development Funds (ERDF) (grant IMP/00019), and has also been funded by Consejería de Salud y Familias, Junta de Andalucía (grants COVID-0012-2020 and PS-2020-342) and the postdoctoral contract of Carlos Loucera (PAIDI2020- DOC_00350), co-funded by the European Social Fund (FSE) 2014-2020. ELIXIR-CONVERGE—H2020 (871075).Peer reviewe

Base de datos de abejas ibéricas

Las abejas son un grupo extremadamente diverso con más de 1000 especies descritas en la península ibérica. Además, son excelentes polinizadores y aportan numerosos servicios ecosistémicos fundamentales para la mayoría de ecosistemas terrestres. Debido a los diversos cambios ambientales inducidos por el ser humano, existen evidencias del declive de algunas de sus poblaciones para ciertas especies. Sin embargo, conocemos muy poco del estado de conservación de la mayoría de especies y de muchas de ellas ignoramos cuál es su distribución en la península ibérica. En este trabajo presentamos un esfuerzo colaborativo para crear una base de datos de ocurrencias de abejas que abarca la península ibérica e islas Baleares que permitirá resolver cuestiones como la distribución de las diferentes especies, preferencia de hábitat, fenología o tendencias históricas. En su versión actual, esta base de datos contiene un total de 87 684 registros de 923 especies recolectados entre 1830 y 2022, de los cuales un 87% presentan información georreferenciada. Para cada registro se incluye información relativa a la localidad de muestreo (89%), identificador y colector de la especie (64%), fecha de captura (54%) y planta donde se recolectó (20%). Creemos que esta base de datos es el punto de partida para conocer y conservar mejor la biodiversidad de abejas en la península ibérica e Islas Baleares. Se puede acceder a estos datos a través del siguiente enlace permanente: https://doi.org/10.5281/zenodo.6354502ABSTRACT: Bees are a diverse group with more than 1000 species known from the Iberian Peninsula. They have increasingly received special attention due to their important role as pollinators and providers of ecosystem services. In addition, various rapid human-induced environmental changes are leading to the decline of some of its populations. However, we know very little about the conservation status of most species and for many species, we hardly know their true distributions across the Iberian Peninsula. Here, we present a collaborative effort to collate and curate a database of Iberian bee occurrences to answer questions about their distribution, habitat preference, phenology, or historical trends. In total we have accumulated 87 684 records from the Iberian Peninsula and the Balearic Islands of 923 different species with 87% of georeferenced records collected between 1830 and 2022. In addition, each record has associated information such as the sampling location (89%), collector and person who identified the species (64%), date of the capture (54%) and plant species where the bees were captured (20%). We believe that this database is the starting point to better understand and conserve bee biodiversity in the Iberian Peninsula. It can be accessed at: https://doi.org/10.5281/zenodo.6354502Esta base de datos se ha realizado con la ayuda de los proyectos EUCLIPO (Fundação para a Ciência e a Tecnologia, LISBOA-01-0145-FEDER-028360/EUCLIPO) y SAFEGUARD (ref. 101003476 H2020 -SFS-2019-2).info:eu-repo/semantics/publishedVersio

Loss of insulin-degrading enzyme function leads to impaired glucagon signalling and enhanced mitochondrial respiration in hepatocytes

Trabajo presentado en el 58th Annual Meeting European Association for the Study of Diabetes, celebrado en Estocolmo (Suecia) del 19 al 23 de septiembre de 2022.[Background and aims]: Insulin-degrading enzyme (IDE) is a ubiquitous metalloprotease that degrades insulin and glucagon among other substrates. By decades, its main function has been attributed to hepatic insulin clearance, a process that regulates availability of insulin levels. Recent studies indicate a more important role of this protein in insulin sensitivity and glucose homeostasis. Much less attention has been dedicated to its role on regulating other hormones action and sensitivity, particularly glucagon and its control in hepatic mitochondrial function. In this work we aim to elucidate the effect of IDE on glucagon signalling and its impact on energy metabolism in hepatocytes. Material and methods: Liver specific IDE-KO (L-IDE-KO) mice were obtained by breeding homozygous mice for a floxed Ide allele with albumin-Cre mice. L-IDE-KO and WT livers were used to obtain tissue extracts and primary hepatocytes for culture. The mouse hepatocyte cell line (AML12) was transduced with an shRNA-Ide by means of a lentiviral vector and obtaining a stable line (AML12-shRNA-Ide). L-IDE-KO primary hepatocytes and AML12-shRNA-Ide with their respective controls were stimulated with glucagon and the signalling pathway was analysed by western blot and ELISA. Mitochondrial function and energy metabolism of AML12-shRNA-Ide and control cells were assessed by Seahorse XFe24 Analyzer with a Mito Stress Assay. [Results]: Liver extracts and primary hepatocytes from L-IDE-KO mice, compared to WT, showed decreased expression of glucagon receptor (~60%), CREB protein (~40%), and diminished phosphorylation of CREB (~50%) upon glucagon stimulation. Ide gene expression and IDE protein levels were reduced by ~50% in AML12-shRNA-Ide cells. At basal state, glucagon receptor, FoxO1 and CREB protein were significantly lower in AML12-shRNA-Ide cells than in control cells, (~40%, ~75% and ~75%, respectively). Glucagon stimulation resulted in less (~30%) cAMP levels and changes in the kinetic of glucagon-mediated phosphorylation of CREB and other PKA substrates in AML12-shRNA-Ide. Seahorse analyses showed that both oxygen consumption and extracelluar acidification rates increased 2-fold in AML12-shRNA-Ide with a 2-fold increment of mitochondrial and glycolytic ATP production. [Conclusion]: Reduced IDE expression in mouse hepatocytes has a deleterious effect on glucagon signalling, affecting this intracellular pathway in parallel with a shift to a more energetic phenotype. These findings suggest that IDE is necessary for a correct glucagon signalling and energy regulation in hepatocytes

Unraveling the role of the insulin-degrading enzyme (IDE) on hepatic insulin and glucagon signaling

Trabajo presentado en el 11th Ciberdem Annual Meeting celebrado en modalidad online, del 3 al 5 de noviembre de 2020.IDE is a protease that degrades insulin and glucagon. Genetic Ide polymorphisms are linked to increased risk of developing T2DM. Reduced IDE levels associates with lower insulin clearance in T2DM patients. However, molecular mechanisms linking IDE with the pathophysiology of T2DM are poorly defined. To investigate the role of IDE on T2DM, we fed mice with a standard diet (SD) or high-fat diet (HFD) and examined the impact of loss- versus -gain of IDE function on insulin and glucagon action in hepatocytes. L-IDE-KO mice fed a SD exhibited insulin resistance and glucose intolerance with no change in hepatic insulin clearance. Insulin resistance was associated with increased FoxO1 activation, reduced insulin receptor (IR) protein levels, and reduced phosphorylation of CEACAM1, which promotes receptor-mediated insulin uptake to be degraded. L-IDE-KO mice exacerbates hyperinsulinemia and insulin resistance in the setting of HFD-induced obesity, in parallel to an increase in pancreatic beta-cell function without altering insulin clearance. Insulin resistance was associated with increased FoxO1 activation and ~2-fold increase of GLUT2 protein levels. Conversely, gain of IDE function (adenoviral delivery) improves glucose tolerance and insulin sensitivity, in parallel to a reciprocal ~2-fold reduction in hepatic GLUT2 protein levels. Furthermore, in response to insulin, IDE co-immunoprecipitates with the IR. On the other hand, hepatic IDE depletion did not alter circulating glucagon levels in mice fed SD or HFD. Interestingly, L-IDE-KO mice showed reduced CREB protein levels. Likewise, depletion of Ide in AML12 cells significantly reduced CREB protein levels, in parallel with increased glycogen synthase protein levels. In conclusion, loss of IDE function aggravates insulin resistance and glucose intolerance, whereas gain of IDE function exerts beneficial effects on glucose tolerance and insulin sensitivity in the setting of obesity, providing a strong rationale for developing pharmacological compounds targeting IDE activation for T2DM treatment

Targeting Insulin-Degrading Enzyme in Insulin Clearance

© 2021 by the authors.Hepatic insulin clearance, a physiological process that in response to nutritional cues clears ~50–80% of circulating insulin, is emerging as an important factor in our understanding of the pathogenesis of type 2 diabetes mellitus (T2DM). Insulin-degrading enzyme (IDE) is a highly conserved Zn2+-metalloprotease that degrades insulin and several other intermediate-size peptides. Both, insulin clearance and IDE activity are reduced in diabetic patients, albeit the cause-effect relationship in humans remains unproven. Because historically IDE has been proposed as the main enzyme involved in insulin degradation, efforts in the development of IDE inhibitors as therapeutics in diabetic patients has attracted attention during the last decades. In this review, we retrace the path from Mirsky’s seminal discovery of IDE to the present, highlighting the pros and cons of the development of IDE inhibitors as a pharmacological approach to treating diabetic patients.This research was funded by the Ministerio de Economía, Industria y Competitividad, grant number SAF2016-77871-C2-2-R to G.P.; Ministerio de Ciencia e Innovación PID2019-110496RB-C22 to G.P.; European Foundation for the Study of Diabetes (European Diabetes Research Programme on New Targets for Type 2 Diabetes supported by MSD-2017) to G.P.; European Foundation for the Study of Diabetes (EFSD) -Novo Nordisk to B.M.; and the US National Institutes of Health (GM115617) to M.A.L. The project leading to these results has received funding from “La Caixa” Foundation, under agreement LCF/PR/PR18/51130007 to G.P. C.M.G.-C. was supported by a fellowship from the Junta de Castilla y León and the European Social Fund (ORDER EDU/574/2018). B.M. was supported by a “Rising Star” fellowship (EFSD-Novo Nordisk)

Primary Cilia in Pancreatic ß- and ¿-Cells: Time to Revisit the Role of Insulin-Degrading Enzyme

The primary cilium is a narrow organelle located at the surface of the cell in contact with the extracellular environment. Once underappreciated, now is thought to efficiently sense external environmental cues and mediate cell-to-cell communication, because many receptors, ion channels, and signaling molecules are highly or differentially expressed in primary cilium. Rare genetic disorders that affect cilia integrity and function, such as Bardet-Biedl syndrome and Alström syndrome, have awoken interest in studying the biology of cilium. In this review, we discuss recent evidence suggesting emerging roles of primary cilium and cilia-mediated signaling pathways in the regulation of pancreatic β- and α-cell functions, and its implications in regulating glucose homeostasisThe project leading to this Review has received funding from “La Caixa” Foundation, under agreement LCF/PR/PR18/51130007 to GP; Grants PID2019-110496RB-C21 and PID2019-110496RB-C22 funded by MCIN/AEI/10.13039/501100011033 to IC-C and GP, respectively; European Foundation for the Study of Diabetes Rising Star Fellowship to BM supported by EFSD-Novo Nordisk; This research was funded by the Programa Estratégico Instituto de Biología y Genética Molecular (IBGM), Junta de Castilla y León (CCVC8485). CG-C and EC-Á were supported by a fellowship from the Junta de Castilla y León and the European Social Fund (ORDER EDU/574/2018 and ORDER EDU/556/2019, respectively)

Hepatic insulin-degrading enzyme regulates glucose and insulin homeostasis in diet-induced obese mice

© 2020 The Authors.The insulin-degrading enzyme (IDE) is a metalloendopeptidase with a high affinity for insulin. Human genetic polymorphisms in Ide have been linked to increased risk for T2DM. In mice, hepatic Ide ablation causes glucose intolerance and insulin resistance when mice are fed a regular diet. [Objective]: These studies were undertaken to further investigate its regulatory role in glucose homeostasis and insulin sensitivity in diet-induced obesity. [Methods]: To this end, we have compared the metabolic effects of loss versus gain of IDE function in mice fed a high-fat diet (HFD). [Results]: We demonstrate that loss of IDE function in liver (L-IDE-KO mouse) exacerbates hyperinsulinemia and insulin resistance without changes in insulin clearance but in parallel to an increase in pancreatic β-cell function. Insulin resistance was associated with increased FoxO1 activation and a ~2-fold increase of GLUT2 protein levels in the liver of HFD-fed mice in response to an intraperitoneal injection of insulin. Conversely, gain of IDE function (adenoviral delivery) improves glucose tolerance and insulin sensitivity, in parallel to a reciprocal ~2-fold reduction in hepatic GLUT2 protein levels. Furthermore, in response to insulin, IDE co-immunoprecipitates with the insulin receptor in liver lysates of mice with adenoviral-mediated liver overexpression of IDE. [Conclusions]: We conclude that IDE regulates hepatic insulin action and whole-body glucose metabolism in diet-induced obesity via insulin receptor levels.This work was supported by grants from the Ministerio de Economía, Industria y Competitividad: SAF2016-77871-C2-1-R to IC; SAF2016-77871-C2-2-R to GP; This work was supported by grants from the Ministerio de Ciencia e Innovación PID2019-110496RB-C21 to IC; PID2019-110496RB-C22 to GP. European Foundation for the Study of Diabetes (European Diabetes Research Programme on New Targets for Type 2 Diabetes supported by MSD-2017) to IC and GP. The project leading to these results has received funding from “la Caixa” Foundation, under agreement LCF/PR/PR18/51130007 to GP. This work was suppoted by grant from NIH GM115617 to ML

Insulin-Degrading Enzyme (IDE) Regulates Glucagon Signaling and Mitochondrial Respiration in Hepatocytes

Trabajo presentado en las 83rd Scientific Sessions American Diabetes Association, celebradas en San Diego (Estados Unidos) del 23 al 26 de junio de 2023