Endocrine disruptors in plastics alter ß-cell physiology and increase the risk of diabetes mellitus.

Plastic pollution breaks a planetary boundary threatening wildlife and humans through its physical and chemical effects. Of the latter, the release of endocrine disrupting chemicals (EDCs) has consequences on the prevalence of human diseases related to the endocrine system. Bisphenols (BPs) and phthalates are two groups of EDCs commonly found in plastics that migrate into the environment and make low-dose human exposure ubiquitous. Here we review epidemiological, animal, and cellular studies linking exposure to BPs and phthalates to altered glucose regulation, with emphasis on the role of pancreatic ß-cells. Epidemiological studies indicate that exposure to BPs and phthalates is associated with diabetes mellitus. Studies in animal models indicate that treatment with doses within the range of human exposure decreases insulin sensitivity and glucose tolerance, induces dyslipidemia, and modifies functional ß-cell mass and serum levels of insulin, leptin, and adiponectin. These studies reveal that disruption of ß-cell physiology by EDCs plays a key role in impairing glucose homeostasis by altering the mechanisms used by ß-cells to adapt to metabolic stress such as chronic nutrient excess. Studies at the cellular level demonstrate that BPs and phthalates modify the same biochemical pathways involved in adaptation to chronic excess fuel. These include changes in insulin biosynthesis and secretion, electrical activity, expression of key genes, and mitochondrial function. The data summarized here indicate that BPs and phthalates are important risk factors for diabetes mellitus and support a global effort to decrease plastic pollution and human exposure to EDCs.

In Vitro Assays to Identify Metabolism-Disrupting Chemicals with Diabetogenic Activity in a Human Pancreatic ß-Cell Model.

There is a need to develop identification tests for Metabolism Disrupting Chemicals (MDCs) with diabetogenic activity. Here we used the human EndoC-ßH1 ß-cell line, the rat ß-cell line INS-1E and dispersed mouse islet cells to assess the effects of endocrine disruptors on cell viability and glucose-stimulated insulin secretion (GSIS). We tested six chemicals at concentrations within human exposure (from 0.1 pM to 1 µM). Bisphenol-A (BPA) and tributyltin (TBT) were used as controls while four other chemicals, namely perfluorooctanoic acid (PFOA), triphenylphosphate (TPP), triclosan (TCS) and dichlorodiphenyldichloroethylene (DDE), were used as "unknowns". Regarding cell viability, BPA and TBT increased cell death as previously observed. Their mode of action involved the activation of estrogen receptors and PPARγ, respectively. ROS production was a consistent key event in BPA-and TBT-treated cells. None of the other MDCs tested modified viability or ROS production. Concerning GSIS, TBT increased insulin secretion while BPA produced no effects. PFOA decreased GSIS, suggesting that this chemical could be a "new" diabetogenic agent. Our results indicate that the EndoC-ßH1 cell line is a suitable human ß-cell model for testing diabetogenic MDCs. Optimization of the test methods proposed here could be incorporated into a set of protocols for the identification of MDCs.

G protein-coupled estrogen receptor activation by bisphenol-A disrupts the protection from apoptosis conferred by the estrogen receptors ERα and ERß in pancreatic beta cells.

17ß-estradiol protects pancreatic ß-cells from apoptosis via the estrogen receptors ERα, ERß and GPER. Conversely, the endocrine disruptor bisphenol-A (BPA), which exerts multiple effects in this cell type via the same estrogen receptors, increased basal apoptosis. The molecular-initiated events that trigger these opposite actions have yet to be identified. We demonstrated that combined genetic downregulation and pharmacological blockade of each estrogen receptor increased apoptosis to a different extent. The increase in apoptosis induced by BPA was diminished by the pharmacological blockade or the genetic silencing of GPER, and it was partially reproduced by the GPER agonist G1. BPA and G1-induced apoptosis were abolished upon pharmacological inhibition, silencing of ERα and ERß, or in dispersed islet cells from ERß knockout (BERKO) mice. However, the ERα and ERß agonists PPT and DPN, respectively, had no effect on beta cell viability. To exert their biological actions, ERα and ERß form homodimers and heterodimers. Molecular dynamics simulations together with proximity ligand assays and coimmunoprecipitation experiments indicated that the interaction of BPA with ERα and ERß as well as GPER activation by G1 decreased ERαß heterodimers. We propose that ERαß heterodimers play an antiapoptotic role in beta cells and that BPA- and G1-induced decreases in ERαß heterodimers lead to beta cell apoptosis. Unveiling how different estrogenic chemicals affect the crosstalk among estrogen receptors should help to identify diabetogenic endocrine disruptors.

Physiology of pancreatic ß-cells: Ion channels and molecular mechanisms implicated in stimulus-secretion coupling.

The human and mouse islet of Langerhans is an endocrine organ composed of five different cells types; insulin-secreting ß-cells, glucagon-producing α-cells, somatostatin-producing δ-cells, pancreatic polypeptide-secreting PP cells and ɛ-cells that secretes ghrelin. The most important cells are the pancreatic ß-cells that comprise around 45-50% of human islets and 75-80% in the mouse. Pancreatic ß-cells secrete insulin at high glucose concentration, thereby finely regulating glycaemia by the hypoglycaemic effects of this hormone. Different ion channels are implicated in the stimulus-secretion coupling of insulin. An increase in the intracellular ATP concentration leads to closure KATP channels, depolarizing the cell and opening voltage-gated calcium channels. The increase of intracellular calcium concentration induced by calcium entry through voltage-gated calcium channels promotes insulin secretion. Here, we briefly describe the diversity of ion channels present in pancreatic ß-cells and the different mechanisms that are responsible to induce insulin secretion in human and mouse cells. Moreover, we described the pathophysiology due to alterations in the physiology of the main ion channels present in pancreatic ß-cell and its implication to predispose metabolic disorders as type 2 diabetes mellitus.

Bisphenol-S and Bisphenol-F alter mouse pancreatic ß-cell ion channel expression and activity and insulin release through an estrogen receptor ERß mediated pathway.

Bisphenol-S (BPS) and Bisphenol-F (BPF) are current Bisphenol-A (BPA) substitutes. Here we used pancreatic ß-cells from wild type (WT) and estrogen receptor ß (ERß) knockout (BERKO) mice to investigate the effects of BPS and BPF on insulin secretion, and the expression and activity of ion channels involved in ß-cell function. BPS or BPF rapidly increased insulin release and diminished ATP-sensitive K+ (KATP) channel activity. Similarly, 48 h treatment with BPS or BPF enhanced insulin release and decreased the expression of several ion channel subunits in ß-cells from WT mice, yet no effects were observed in cells from BERKO mice. PaPE-1, a ligand designed to preferentially trigger extranuclear-initiated ER pathways, mimicked the effects of bisphenols, suggesting the involvement of extranuclear-initiated ERß pathways. Molecular dynamics simulations indicated differences in ERß ligand-binding domain dimer stabilization and solvation free energy among different bisphenols and PaPE-1. Our data suggest a mode of action involving ERß whose activation alters three key cellular events in ß-cell, namely ion channel expression and activity, and insulin release. These results may help to improve the hazard identification of bisphenols.

Transcriptomic Analysis Reveals the Wound Healing Activity of Mussel Myticin C.

Myticin C is the most studied antimicrobial peptide in the marine mussel Mytilusgalloprovincialis. Although it is constitutively expressed in mussel hemocytes and displays antibacterial, antiviral, and chemotactic functions, recent work has suggested that this molecule is mainly activated after tissue injury. Therefore, the main objective of this work was to characterize the hemocytes' transcriptomic response after a myticin C treatment, in order to understand the molecular changes induced by this cytokine-like molecule. The transcriptome analysis revealed the modulation of genes related to cellular movement, such as myosin, transgelin, and calponin-like proteins, in agreement with results of functional assays, where an implication of myticin C in the in vitro activation of hemocytes and migration was evidenced. This was also observed in vivo after a tissue injury, when hemocytes, with high concentrations of myticin C, migrated to the damaged area to heal the wound. All these properties allowed us to think about the biotechnological application of these molecules as wound healers. Human keratinocytes and larvae zebrafish models were used to confirm this hypothesis. Accelerated regeneration after a wound or tail fin amputation was observed after treatment with the myticin C peptide, supporting the chemotactic and healing activity of myticin C.

Rag1 immunodeficiency-induced early aging and senescence in zebrafish are dependent on chronic inflammation and oxidative stress.

In mammals, recombination activating gene 1 (RAG1) plays a crucial role in adaptive immunity, generating a vast range of immunoglobulins. Rag1-/- zebrafish (Danio rerio) are viable and reach adulthood without obvious signs of infectious disease in standard nonsterile conditions, suggesting that innate immunity could be enhanced to compensate for the lack of adaptive immunity. By using microarray analysis, we confirmed that the expression of immunity- and apoptosis-related genes was increased in the rag1-/- fish. This tool also allows us to notice alterations of the DNA repair and cell cycle mechanisms in rag1-/- zebrafish. Several senescence and aging markers were analyzed. In addition to the lower lifespan of rag1-/- zebrafish compared to their wild-type (wt) siblings, rag1-/- showed a higher incidence of cell cycle arrest and apoptosis, a greater amount of phosphorylated histone H2AX, oxidative stress and decline of the antioxidant mechanisms, an upregulated expression and activity of senescence-related genes and senescence-associated ß-galactosidase, respectively, diminished telomere length, and abnormal self-renewal and repair capacities in the retina and liver. Metabolomic analysis also demonstrated clear differences between wt and rag1-/- fish, as was the deficiency of the antioxidant metabolite l-acetylcarnitine (ALCAR) in rag1-/- fish. Therefore, Rag1 activity does not seem to be limited to V(D)J recombination but is also involved in senescence and aging. Furthermore, we confirmed the senolytic effect of ABT-263, a known senolytic compound and, for the first time, the potential in vivo senolytic activity of the antioxidant agent ALCAR, suggesting that this metabolite is essential to avoid premature aging.

Antiviral Activity of a Turbot (Scophthalmus maximus) NK-Lysin Peptide by Inhibition of Low-pH Virus-Induced Membrane Fusion.

Global health is under attack by increasingly-frequent pandemics of viral origin. Antimicrobial peptides are a valuable tool to combat pathogenic microorganisms. Previous studies from our group have shown that the membrane-lytic region of turbot (Scophthalmus maximus) NK-lysine short peptide (Nkl71⁻100) exerts an anti-protozoal activity, probably due to membrane rupture. In addition, NK-lysine protein is highly expressed in zebrafish in response to viral infections. In this work several biophysical methods, such as vesicle aggregation, leakage and fluorescence anisotropy, are employed to investigate the interaction of Nkl71⁻100 with different glycerophospholipid vesicles. At acidic pH, Nkl71⁻100 preferably interacts with phosphatidylserine (PS), disrupts PS membranes, and allows the content leakage from vesicles. Furthermore, Nkl71⁻100 exerts strong antiviral activity against spring viremia of carp virus (SVCV) by inhibiting not only the binding of viral particles to host cells, but also the fusion of virus and cell membranes, which requires a low pH context. Such antiviral activity seems to be related to the important role that PS plays in these steps of the replication cycle of SVCV, a feature that is shared by other families of virus-comprising members with health and veterinary relevance. Consequently, Nkl71⁻100 is shown as a promising broad-spectrum antiviral candidate.

Plasma proteomic analysis of zebrafish following spring viremia of carp virus infection.

To better understand spring viremia of carp virus (SVCV) pathogenesis in zebrafish proteomic analysis was used to examine the plasma protein profile in SVCV-infected zebrafish. A total of 3062 proteins were identified. Of those 137, 63 and 31 proteins were enriched in blood samples harvested at 1, 2 and 5 days post SVCV infection, respectively. These altered host proteins were classified based on their biological function: 23 proteins under the response to stimulus term were identified. Interestingly, at the top of the up-regulated proteins during SVCV infection were the proteins of the vitellogenin family (Vtg) and the grass carp reovirus-induced gene (Gig) proteins. Real-time RT-PCR evaluation of samples from internal organs verified that SVCV infection induced vtg and gig2 gene expression already at day 1 post-infection. Western blot analysis revealed the presence of Vtg protein only in blood of SVCV-infected fish. This is the first proteomic study to reveal the involvement of Vtg proteins in adult fish response to viral challenge. It also highlights the role of Gig proteins as important factors in antiviral response in fish. This work provides valuable relevant insight into virus-host interaction and the identification of molecular markers of fish response to virus.

Chromatin immunoprecipitation and high throughput sequencing of SVCV-infected zebrafish reveals novel epigenetic histone methylation patterns involved in antiviral immune response.

Chromatin immunoprecipitation (ChIP) and high throughput sequencing (ChIP-seq) have been used to assess histone methylation (epigenetic modification) dynamics within the internal organs of zebrafish after spring viremia of carp virus (SVCV) infection. Our results show H3K4me3 up-methylation in gene promoters associated with innate immune response during the first 5 days after SVCV infection. Gene Ontology (GO) enrichment analysis confirmed up-methylation in 218 genes in the "immune system process" category. In particular, the promoters of interferon (ifn), interferon stimulated genes (isg), Toll-like receptors (tlr) and c-reactive protein (crp) multi gene sets were marked with the permissive H3K4 methylation. Higher histone 3 methylation was associated with higher transcription levels of the corresponding genes. Therefore, the evidence presented here suggests that transcriptional regulation at the promoter level of key immune genes of the interferon signaling pathway and c-reactive proteins genes can be modulated by epigenetic modification of histones. This study emphasizes the importance of epigenetic control in the response of zebrafish to SVCV infection.

Beta-glucan enhances the response to SVCV infection in zebrafish.

The antiviral effects of beta-glucan, an immunostimulatory agent were studied in zebrafish both in vitro and in vivo. Here we show that zebrafish ZF4 cells as well as whole fish primed with yeast ß-glucan zymosan exhibited increased cytokine expression and elevated response to spring viremia of carp virus (SVCV) infection. In vitro, previous treatment of ß-glucan enhanced ZF4 cell viability against SVCV infection which is associated to the activation of interferon signaling pathway and inflammatory cytokines gene expression. In vivo, the SVCV-infected fish primed with ß-glucan had a higher survival rate (≈73%) than the control SVCV-infected group (≈33%). Additionally, up-regulation of the expression of a set of genes involved in innate immune response was detected in zebrafish intraperitoneally injected of ß-glucan: il1b, il6, il8, il10 and tnfa transcripts showed increased expression that appear to be rapid (2 days) but not long-lived (less than 2 weeks). The present study is, to our knowledge, the first to combine cell culture and in vivo approaches to describe host response to ß-glucan stimulation and viral infection in zebrafish.

Neutralization of viral infectivity by zebrafish c-reactive protein isoforms.

This work explores the unexpected in vivo and in vitro anti-viral functions of the seven c-reactive protein (crp1-7) genes of zebrafish (Danio rerio). First results showed heterogeneous crp1-7 transcript levels in healthy wild-type zebrafish tissues and organs and how those levels heterogeneously changed not only after bacterial but also after viral infections, including those in adaptive immunity-deficient rag1-/- mutants. As shown by microarray hybridization and proteomic techniques, crp2/CRP2 and crp5/CRP5 transcripts/proteins were among the most modulated during in vivo viral infection situations including the highest responses in the absence of adaptive immunity. In contrast crp1/CRP1/and crp7/CRP7 very often remained unmodulated. All evidences suggested that zebrafish crp2-6/CRP2-6 may have in vivo anti-viral activities in addition to their well known anti-bacterial and/or physiological functions in mammalians. Confirming those expectations, in vitro neutralization and in vivo protection against spring viremia carp virus (SVCV) infections were demonstrated by crp2-6/CRP2-6 using crp1-7 transfected and/or CRP1-7-enriched supernatant-treated fish cells and crp2-5-injected one-cell stage embryo eggs, respectively. All these findings discovered a crp1-7/CRP1-7 primitive anti-viral functional diversity.These findings may help to study similar functions on the one-gene-coded human CRP, which is widely used as a clinical biomarker for bacterial infections, tissue inflammation and coronary heart diseases.

Corrigendum: Zebra Fish Lacking Adaptive Immunity Acquire an Antiviral Alert State Characterized by Upregulated Gene Expression of Apoptosis, Multigene Families, and Interferon-Related Genes.

[This corrects the article on p. 121 in vol. 8, PMID: 28243233.].

Zebra Fish Lacking Adaptive Immunity Acquire an Antiviral Alert State Characterized by Upregulated Gene Expression of Apoptosis, Multigene Families, and Interferon-Related Genes.

To investigate fish innate immunity, we have conducted organ and cell immune-related transcriptomic as well as immunohistologic analysis in mutant zebra fish (Danio rerio) lacking adaptive immunity (rag1-/-) at different developmental stages (egg, larvae, and adult), before and after infection with spring viremia carp virus (SVCV). The results revealed that, compared to immunocompetent zebra fish (rag1+/+ ), rag1-/- acquired increased resistance to SVCV with age, correlating with elevated transcript levels of immune genes in skin/fins and lymphoid organs (head kidney and spleen). Gene sets corresponding to apoptotic functions, immune-related multigene families, and interferon-related genes were constitutively upregulated in uninfected adult rag1-/- zebra fish. Overexpression of activated CASPASE-3 in different tissues before and after infection with SVCV further confirmed increased apoptotic function in rag1-/- zebra fish. Concurrently, staining of different tissue samples with a pan-leukocyte antibody marker showed abundant leukocyte infiltrations in SVCV-infected rag1-/- fish, coinciding with increased transcript expression of genes related to NK-cells and macrophages, suggesting that these genes played a key role in the enhanced immune response of rag1-/- zebra fish to SVCV lethal infection. Overall, we present evidence that indicates that rag1-/- zebra fish acquire an antiviral alert state while they reach adulthood in the absence of adaptive immunity. This antiviral state was characterized by (i) a more rapid response to viral infection, which resulted in increased survival, (ii) the involvement of NK-cell- and macrophage-mediated transcript responses rather than B- and/or T-cell dependent cells, and (iii) enhanced apoptosis, described here for the first time, as well as the similar modulation of multigene family/interferon-related genes previously associated to fish that survived lethal viral infections. From this and other studies, it might be concluded that some of the characteristics of mammalian trained immunity are present in lower vertebrates.

Restricted replication of viral hemorrhagic septicemia virus (VHSV) in a birnavirus-carrier cell culture.

Viral hemorrhagic septicemia virus (VHSV) and infectious pancreatic necrosis virus (IPNV) are economically important pathogens of the salmonid aquaculture industry. In previous work we demonstrated that a cell line persistently infected with IPNV (EPCIPNV) exhibited antiviral activity against superinfection with the heterologous virus VHSV. This work extends our study by analyzing the replication of VHSV in the IPNV-persistently infected cells. At early and late stages of infection VHSV RNA synthesis, as well as VHSV-induced syncytia formation, were examined in EPCIPNV cultures. During the course of VHSV infection the accumulation of VHSV RNA is inhibited in EPCIPNV cells. Typical VHSV-induced membrane fusion at the late stages of infection is also absent in the IPNV carrier cultures. VHSV binding and fusion to EPCIPNV cells did not appear to be impaired, but a potent inhibitory effect on VHSV RNA synthesis is exerted at early times of infection in the IPNV carrier culture. In conclusion, the EPCIPNV cells are considered to be a useful system to study viral interference as well to analyze the mechanisms underlying the phenomenon of superinfection exclusion.

Autophagy-inducing peptides from mammalian VSV and fish VHSV rhabdoviral G glycoproteins (G) as models for the development of new therapeutic molecules.

It has not been elucidated whether or not autophagy is induced by rhabdoviral G glycoproteins (G) in vertebrate organisms for which rhabdovirus infection is lethal. Our work provides the first evidence that both mammalian (vesicular stomatitis virus, VSV) and fish (viral hemorrhagic septicemia virus, VHSV, and spring viremia carp virus, SVCV) rhabdoviral Gs induce an autophagic antiviral program in vertebrate cell lines. The transcriptomic profiles obtained from zebrafish genetically immunized with either Gsvcv or Gvhsv suggest that autophagy is induced shortly after immunization and therefore, it may be an important component of the strong antiviral immune responses elicited by these viral proteins. Pepscan mapping of autophagy-inducing linear determinants of Gvhsv and Gvsv showed that peptides located in their fusion domains induce autophagy. Altogether these results suggest that strategies aimed at modulating autophagy could be used for the prevention and treatment of rhabdoviral infections such as rabies, which causes thousands of human deaths every year.

Increasing versatility of the DNA vaccines through modification of the subcellular location of plasmid-encoded antigen expression in the in vivo transfected cells.

The route of administration of DNA vaccines can play a key role in the magnitude and quality of the immune response triggered after their administration. DNA vaccines containing the gene of the membrane-anchored glycoprotein (gpG) of the fish rhabdoviruses infectious haematopoietic necrosis virus (IHNV) or viral haematopoietic septicaemia virus (VHSV), perhaps the most effective DNA vaccines generated so far, confer maximum protection when injected intramuscularly in contrast to their low efficacy when injected intraperitoneally. In this work, taking as a model the DNA vaccine against VHSV, we focused on developing a more versatile DNA vaccine capable of inducing protective immunity regardless of the administration route used. For that, we designed two alternative constructs to gpG1₋507 (the wild type membrane-anchored gpG of VHSV) encoding either a soluble (gpG1₋462) or a secreted soluble (gpG(LmPle20-462)) form of the VHSV-gpG. In vivo immunisation/challenge assays showed that only gpG(LmPle20-462) (the secreted soluble form) conferred protective immunity against VHSV lethal challenge via both intramuscular and intraperitoneal injection, being this the first description of a fish viral DNA vaccine that confers protection when administered intraperitoneally. Moreover, this new DNA vaccine construct also conferred protection when administered in the presence of an oil adjuvant suggesting that DNA vaccines against rhabdoviruses could be included in the formulation of current multicomponent-intaperitoneally injectable fish vaccines formulated with an oil adjuvant. On the other hand, a strong recruitment of membrane immunoglobulin expressing B cells, mainly membrane IgT, as well as t-bet expressing T cells, at early times post-immunisation, was specifically observed in the fish immunised with the secreted soluble form of the VHSV-gpG protein; this may indicate that the subcellular location of plasmid-encoded antigen expression in the in vivo transfected cells could be an important factor in determining the ways in which DNA vaccines prime the immune response.

pH-dependent solution structure and activity of a reduced form of the host-defense peptide myticin C (Myt C) from the mussel Mytilus galloprovincialis.

Myticin C (Myt C) is a highly variable host-defense peptide (HDP) associated to the immune response in the mediterranean mussel (Mytilus galloprovincialis), which has shown to be active across species due to its strong antiviral activity against a fish rhabdovirus found in fish cells overexpressing this HDP. However, the potential antimicrobial properties of any synthetic analogue of Myt C has not yet been analysed. Thus, in this work we have synthesised the sequence of the mature peptide of Myt C variant c and analysed the structure activity relationships of its reduced (non-oxidized) form (red-MytCc). In contrast to results previously reported for oxidized isoforms of mussel myticins, red-MytCc was not active against bacteria at physiological pH and showed a moderate antiviral activity against the viral haemorrhagic septicaemia (VHS) rhabdovirus. However, its chemotactic properties remained active. Structure/function studies in neutral and acid environments by means of infrared spectroscopy indicated that the structure of red-MytCc is pH dependent, with acid media increasing its alpha-helical content. Furthermore, red-MytCc was able to efficiently aggregate artificial phospholipid membranes at low pH, as well as to inhibit the Escherichia coli growth, suggesting that this activity is attributable to its more structured form in an acidic environment. All together, these results highlight the dynamic and environmentally sensitive behavior of red-Myt C in solution, and provide important insights into Myt C structure/activity relationships and the requirements to exert its antimicrobial/immunomodulatory activities. On the other hand, the pH-dependent direct antimicrobial activity of Myt C suggests that this HDP may be a suitable template for the development of antimicrobial agents that would function selectively in specific pH environments, which are sorely needed in this "antibiotic-resistance era".