Role of Pax8 and thyroid hormones in metabolic homeostasis

Programa de Doctorado en Biotecnología, Ingeniería y Tecnología QuímicaLínea de Investigación: Fundamentos y Aplicaciones de las Células TroncalesClave Programa: DBICódigo Línea: 17Two independent studies indicated that PAX8 could play a role in glucose homeostasis and pancreatic islet physiology. Pax8 expression was reported in murine islets during gestation while a genome-wide study associated a polymorphism nearby PAX8 with type 2 diabetes. In addition, Pax8 is necessary for the synthesis of thyroid hormones, which are known to exert profound effects in whole body metabolism. Herein, we show that PAX8 protects pancreatic islets from apoptosis and have identified a detrimental polymorphism that may be associated with gestational diabetes as well as gestational thyroid dysfunction. However, Pax8 +/- female mice did not develop glucose intolerance during pregnancy and displayed normal circulating T4 levels at 6 months old. In contrast to females, Pax8 +/- male mice showed decreased T4 levels in blood. However, these mice were 8 months old; suggesting that the reduction in circulating T4 levels in Pax8 +/- animals may be developed with advancing age. Interestingly, untreated Pax8 +/- male mice showed glucose intolerance and insulin resistance, while T4 supplementation rescued the phenotype. Therefore, our results indicate that the alterations observed in glucose homeostasis are due to reduced T4 levels in circulation. Noteworthy, insulin signaling was preferentially impaired in the liver of Pax8 +/- male mice, where ROS production was found significantly increased. We next sought to investigate whether T4 supplementation could enhance glucose homeostasis in healthy wild type animals. Remarkably, T4 treatment was found to enhance glucose clearance and increase insulin expression in pancreatic islets. Basal circulating insulin concentration was also higher in T4-treated mice, which could be explained by an upregulation of glucokinase expression in pancreatic islets from these animals. Importantly, T4 supplementation also increased circulating insulin levels and insulin content in pancreatic islets in a model of experimental autoimmune diabetes. In addition, T4 supplementation improved the survival rate of mice treated with streptozotocin. Therefore, our results indicate that reduced circulating T4 levels are associated with impaired glucose homeostasis whereas T4 supplementation enhances glucose clearance and blunts the onset of experimental autoimmune diabetes.Universidad Pablo de Olavide de Sevilla. Departamento de Fisiología, Anatomía y Biología CelularPostprin

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

Metabolic reprogramming by Acly inhibition using SB-204990 alters glucoregulation and modulates molecular mechanisms associated with aging

19 Páginas.-- 7 FigurasATP-citrate lyase is a central integrator of cellular metabolism in the interface of protein, carbohydrate, and lipid metabolism. The physiological consequences as well as the molecular mechanisms orchestrating the response to long-term pharmacologically induced Acly inhibition are unknown. We report here that the Acly inhibitor SB-204990 improves metabolic health and physical strength in wild-type mice when fed with a high-fat diet, while in mice fed with healthy diet results in metabolic imbalance and moderated insulin resistance. By applying a multiomic approach using untargeted metabolomics, transcriptomics, and proteomics, we determined that, in vivo, SB-204990 plays a role in the regulation of molecular mechanisms associated with aging, such as energy metabolism, mitochondrial function, mTOR signaling, and folate cycle, while global alterations on histone acetylation are absent. Our findings indicate a mechanism for regulating molecular pathways of aging that prevents the development of metabolic abnormalities associated with unhealthy dieting. This strategy might be explored for devising therapeutic approaches to prevent metabolic diseases.This work was funded by grants from the Ministerio de Economía y Competitividad, Instituto de Salud Carlos III, co-funded by Fondos FEDER (PI15/00134, PI18/01590, CPII19/00023 to A.M.M.) and the Ministerio de Ciencia e Innovación (PID2021-123965OB-100 to A.M.M.). A.M.M. is funded by the Junta de Andalucía P20_00480, the Spanish Society of Diabetes, and CSIC 202220I059. M.S.K. is funded by the Nordea Foundation (#02-2017-1749), the Novo Nordisk Foundation (#NNF17OC0027812), the Neye Foundation, the Lundbeck Foundation (#R324-2019-1492), the Ministry of Higher Education and Science of Denmark (#0238-00003B). V.C.G. is funded by the Instituto de Salud Carlos III (CP19/00046), co-funded by FEDER. F.M. is funded by the CIBERDEM of the Instituto de Salud Carlos III. A.M.M. is the guarantor of this work and, as such, had full access to all the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis. We acknowledge the support of the group of basic research on diabetes of the Spanish Society of Diabetes.Peer reviewe

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

Mellado-Gil, José Manuel et al.[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.This work was funded by grants from the Consejeria de Salud, Fundacion Publica Andaluza Progreso y Salud, Junta de Andalucia (PI-0727-2010 to BRG and PI-0085-2013 to PIL), Consejeria de Economia, Innovacion y Ciencia (P10.CTS.6359 to BRG), Ministerio de Ciencia e Innovacion (BFU2013-42789-P to IQ) and the Ministerio de Economia y Competidividad, Instituto de Salud Carlos III co-funded by Fondos FEDER (PI10/00871 and PI13/00593 to BRG). NC-V is supported by a JDRF subsidy (17-2013-372 to BRG.). AM-M is a recipient of a Miguel Servet grant (CP14/00105) from the Instituto de Salud Carlos III co-funded by Fondos FEDER and EF-M is a recipient of a Juan de la Cierva Fellowship. PM is supported by Swiss National Science Foundation grant 310030-141162, and the European Union grant IMIDIA, C2008-T7. BOB is supported by grants from the Lee Kong Chian School of Medicine, Nanyang Technological University, Singapore, Republic of Singapore.Peer Reviewe

Long non-coding RNAs as key modulators of pancreatic β-cell mass and function

Numerous studies have sought to decipher the genetic and other mechanisms contributing to β-cell loss and dysfunction in diabetes mellitus. However, we have yet to fully understand the etiology of the disease or to develop satisfactory treatments. Since the majority of diabetes susceptibility loci are mapped to non-coding regions within the genome, understanding the functions of non-coding RNAs in β-cell biology might provide crucial insights into the pathogenesis of type 1 (T1D) and type 2 (T2D) diabetes. During the past decade, numerous studies have indicated that long non-coding RNAs play important roles in the maintenance of β-cell mass and function. Indeed, lncRNAs have been shown to be involved in controlling β-cell proliferation during development and/or β-cell compensation in response to hyperglycaemia. LncRNAs such as TUG-1 and MEG3 play a role in both β-cell apoptosis and function, while others sensitize β-cells to apoptosis in response to stress signals. In addition, several long non-coding RNAs have been shown to regulate the expression of β-cell-enriched transcription factors in cis or in trans. In this review, we provide an overview of the roles of lncRNAs in maintaining β-function and mass, and discuss their relevance in the development of diabetes.Published versionGAR was supported by a Wellcome Trust Investigator Award (212625/Z/18/Z), MRC Programme grants (MR/R022259/1, MR/J0003042/1, MR/L020149/1) and by Diabetes UK (BDA/11/0004210, BDA/15/0005275, BDA 16/0005485) project grants. This project has received funding from the European Union’s Horizon 2020 research and innovation programme via the Innovative Medicines Initiative 2 Joint Undertaking under grant agreement No 115881 (RHAPSODY). This Joint Undertaking receives support from the European Union’s Horizon 2020 research and innovation programme and EFPIA

Roles of extracellular vesicles associated non-coding RNAs in Diabetes Mellitus

Extracellular vesicles (EVs), especially exosomes (50 to 150 nm), have been shown to play important roles in a wide range of physiological and pathological processes, including metabolic diseases such as Diabetes Mellitus (DM). In the last decade, several studies have demonstrated how EVs are involved in cell-to-cell communication. EVs are enriched in proteins, mRNAs and non-coding RNAs (miRNAs, long non-coding RNAs and circRNAS, among others) which are transferred to recipient cells and may have a profound impact in either their survival or functionality. Several studies have pointed out the contribution of exosomal miRNAs, such as miR-l42-3p and miR-26, in the development of Type 1 and Type 2 DM (T1DM and T2DM), respectively. In addition, some miRNA families such as miR-let7 and miR-29 found in exosomes have been associated with both types of diabetes, suggesting that they share common etiological features. The knowledge about the role of exosomal long non-coding RNAs in this group of diseases is more immature, but the exosomal lncRNA MALAT1 has been found to be elevated in the plasma of individuals with T2DM, while more than 169 lncRNAs were reported to be differentially expressed between healthy donors and people with T1DM. Here, we review the current knowledge about exosomal non-coding RNAs in DM and discuss their potential as novel biomarkers and possible therapeutic targets.The authors were/are funded by grants from Consejería de Salud, Fundación Pública Andaluza Progreso y Salud, Junta de Andalucía (PI-0727-2010, December 2010 to BG, PI-0085-2013),; Consejería de Economía, Innovación y Ciencia, Junta de Andalucía (P10-CTS-6359 to BG); Ministerio de Economía y Competitividad, Instituto de Salud Carlos III, cofounded by Fondos FEDER (PI10/00871, January 2011 and PI13/00593, January 2014 to BG); Ministerio de Economía y Competitividad, Plan Nacional(BFU2017-83588-P, January 2018 and PID2021-123083NB-I00, sept 2022 to BG); JuvenileDiabetes Research Foundation JDRF (17-2013-372, August 2013 and 2-SRA-2019-837-S-B, August2019 to BG); Special thanks to ALUSVI (Asociación Lucha y Sonríe por la Vida, Pilas), and the Fundacion DiabetesCERO for their unconditional financial support. LL-N is also funded by Procedimiento de selección de personal investigador Doctor con cargo a las ayudas concedidas mediante la Línea de Ayudas para Captación, Incorporación y Movilidad de Capital Humano de I+D+i (20804)

The role of Pax8 in Pancreatic Islet Physiology

Motivation: Currently, there are 347 million patients suffering Diabetes Mellitus (DM), raising the obvious need for more effective DM treatments. In this sense, the study of mechanisms that would allow the regeneration and protection of pancreatic insulin producing cells (β-cells) represents a promising strategy to generate novel therapies to treat DM. Recently, a genome wide analysis showed that a specific polymorphism in the transcription factor Pax8 is associated with increased incidence of type II DM (1). In parallel, Riecket et al. determined that this gene was robustly induced in murine pancreatic islets during pregnancy, a situation of high metabolic demand (2). Interestingly, Pax8 is crucial for thyroid development and thyroxine (T4) production. Remarkably, a low concentration of circulating T4 is associated with gestational DM, which suggests that T4 may be required for the adaptation of pancreatic islets to pregnancy (3). These evidences indicate that alterations in Pax8 expression may be involved in the development of DM.Methods: In order to asess the physioloigcal role of Pax8 in islet physiology, isolated islets were transduced using lentiviral vectors harbouring GFP (MOCK) or Pax8. A microarray was performed to determine the genetic networks affected by Pax8 overexpressionin pancreatic islets. In addition, apoptosis was measured using a Cell death kit and immunohistochemical studies were conducted in order to determine the transduction efficiency (GFP), islets’ morphology (insulin/glucagon) and apoptosis (cleaved caspase 3). In parallel, oral glucose tolerance tests (OGTT) and insulin tolerance tests (ITT) were performed in non-pregnant and pregnant Pax8 heterozygous and wild type mice to determine the role of Pax8 in glucose homeostasis during pregnancy.Results: Microarray data revealed that the transcriptional profile of pancreatic islets overexpressing Pax8 is associated with diabetogenesis and that interferon signalling is dramatically modulated. Moreover, our results indicate that Pax8 overexpression protects β-cells from apoptosis. The results for in vivo studies cannot be shown since they are under consideration for a patent. Conclusions: Our results indicate that Pax8 protects pancreatic islets from apoptosis in vitro, suggesting that Pax8 may modulate genetic pathways involved in the maintainance of islets integrity.

Targeting pancreatic expressed PAX genes for the treatment of diabetes mellitus and pancreatic neuroendocrine tumors

[Introduction]: Four members of the PAX family, PAX2, PAX4, PAX6 and PAX8 are known to be expressed in the pancreas. Accumulated evidences indicate that several pancreatic expressed PAX genes play a significant role in pancreatic development/functionality and alterations in these genes are involved in the pathogenesis of pancreatic diseases. [Areas covered]: In this review, we summarize the ongoing research related to pancreatic PAX genes in diabetes mellitus and pancreatic neuroendocrine tumors. We dissect the current knowledge at different levels; from mechanistic studies in cell lines performed to understand the molecular processes controlled by pancreatic PAX genes, to in vivo studies using rodent models that over-express or lack specific PAX genes. Finally, we describe human studies associating variants on pancreatic-expressed PAX genes with pancreatic diseases. [Expert opinion]: Based on the current literature, we propose that future interventions to treat pancreatic neuroendocrine tumors and diabetes mellitus could be developed via the modulation of PAX4 and/or PAX6 regulated pathways.This work was supported by grants from the Consejería de Salud, Fundación Pública Andaluza Progreso y Salud, Junta de Andalucía; Consejería de Economía, Innovación y Ciencia, Instituto de Salud Carlos III, Fundación Vencer el Cancer and ALUSVI.Peer Reviewe

Understanding the effects of thyroxine supplementation to explore novel venues for the treatment of type 2 diabetes mellitus

Resumen del póster presentado al XXVIII Congreso Nacional de la Sociedad Española de diabetes, celebrado en Bilbao del 20 al 22 de abril de 2016.[Introduction and objectives]: Thyroid hormone (T4) supplementation is known to increase glucose utilization in insulin-target tissues yet patients suffering from hyperthyroidism develop long term metabolic disorders that are, in certain cases, associated with type 2 diabetes mellitus (T2DM). Herein we sought to understand the underlying mechanism of this dichotomy with the goal to target thyroid hormone signalling pathways beneficial for the treatment of T2DM. [Material and methods]: Newborn C57/Bl6 mice were treated with T4 until the age of 32 weeks. Body weight was monitored monthly and physical performance was evaluated by rotarod test. Percentage of glycated haemoglobin (HbA1c), oral glucose tolerance test (OGTT), insulin tolerance test (ITT) and glucose quantifications during a 24 hours fasting test were determined to assess metabolic homeostasis. Prior to sacrifice at 32 weeks of age, mice were either injected with insulin (1.5 IU.Kg-1) or saline solution. Protein extracts were then prepared from skeletal muscles and total protein levels for AKT, GSK3β and FOXO1 as well as AKT phosphorylation, implicated in insulin signaling, were assessed by Western blot analysis. [Results]: T4-treated mice exhibited a 12-34% lower body weight during the course of the study and displayed improved physical performance. Mice exposed to exogenous T4 showed lower circulating glucose in OGTT (30%), and ITT (37%) as compared to untreated animals, suggesting enhanced glucose clearance. Consistent with the latter, the percentage of HbA1c was reduced in T4-treated mice. Total AKT, GSK3β and FOXO1 protein levels were higher in skeletal muscles of T4-treated mice as compared to untreated mice under basal conditions (e.g. non-insulin treated). Interestingly, increased AKT phosphorylation (Serine 473) was also detected in non-insulin treated T4-supplemented mice. Insulin injection induced maximal AKT phosphorylation in both control and T4-treated mice. [Conclusions]: Our data suggest that T4 supplementation enhances skeletal muscle glucose uptake under basal conditions through increased AKT phosphorylation, resulting in improved metabolic homeostasis and physical performance, while reducing body weight gain. We propose that, although hyperthyroidism is a serious disease that prevents the use of T4 in the clinic, interventions based on the modulation of T4-targets may be a promising strategy for the development of novel therapies for the treatment of T2DM.Peer reviewe