RESUMO
Nitric oxide (NO) plays a dual role in regulating DNA damage response (DDR) signaling in pancreatic ß-cells. As a genotoxic agent, NO activates two types of DDR signaling; however, when produced at micromolar levels by the inducible isoform of NO synthase, NO inhibits DDR signaling and DDR-induced apoptosis in a ß-cell-selective manner. DDR signaling inhibition by NO correlates with mitochondrial oxidative metabolism inhibition and decreases in ATP and NAD+. Unlike most cell types, ß-cells do not compensate for impaired mitochondrial oxidation by increasing glycolytic flux, and this metabolic inflexibility leads to a decrease in ATP and NAD+. Here, we used multiple analytical approaches to determine changes in intermediary metabolites in ß-cells and non-ß-cells treated with NO or complex I inhibitor rotenone. In addition to ATP and NAD+, glycolytic and tricarboxylic acid cycle intermediates as well as NADPH are significantly decreased in ß-cells treated with NO or rotenone. Consistent with glucose-6-phosphate residing at the metabolic branchpoint for glycolysis and the pentose phosphate pathway (NADPH), we show that mitochondrial oxidation inhibitors limit glucose uptake in a ß-cell-selective manner. Our findings indicate that the ß-cell-selective inhibition of DDR signaling by NO is associated with a decrease in ATP to levels that fall significantly below the KM for ATP of glucokinase (glucose uptake) and suggest that this action places the ß-cell in a state of suspended animation where it is metabolically inert until NO is removed, and metabolic function can be restored.
Assuntos
NAD , Óxido Nítrico , Óxido Nítrico/metabolismo , NADP/metabolismo , NAD/metabolismo , Rotenona/farmacologia , Dano ao DNA , Trifosfato de Adenosina/metabolismo , Glucose/metabolismoRESUMO
Inhibition of the bromodomain and extraterminal domain (BET) protein family is a potential strategy to prevent and treat diabetes; however, the clinical use of BET bromodomain inhibitors (BETis) is associated with adverse effects. Here, we explore a strategy for targeting BETis to ß cells by exploiting the high-zinc (Zn2+) concentration in ß cells relative to other cell types. We report the synthesis of a novel, Zn2+-chelating derivative of the pan-BETi (+)-JQ1, (+)-JQ1-DPA, in which (+)-JQ1 was conjugated to dipicolyl amine (DPA). As controls, we synthesized (+)-JQ1-DBA, a non-Zn2+-chelating derivative, and (-)-JQ1-DPA, an inactive enantiomer that chelates Zn2+. Molecular modeling and biophysical assays showed that (+)-JQ1-DPA and (+)-JQ1-DBA retain potent binding to BET bromodomains in vitro. Cellular assays demonstrated (+)-JQ1-DPA attenuated NF-ĸB target gene expression in ß cells stimulated with the proinflammatory cytokine interleukin 1ß. To assess ß-cell selectivity, we isolated islets from a mouse model that expresses green fluorescent protein in insulin-positive ß cells and mTomato in insulin-negative cells (non-ß cells). Surprisingly, Zn2+ chelation did not confer ß-cell selectivity as (+)-JQ1-DPA was equally effective in both ß and α cells; however, (+)-JQ1-DPA was less effective in macrophages, a nonendocrine islet cell type. Intriguingly, the non-Zn2+-chelating derivative (+)-JQ1-DBA displayed the opposite selectivity, with greater effect in macrophages compared with (+)-JQ1-DPA, suggesting potential as a macrophage-targeting molecule. These findings suggest that Zn2+-chelating small molecules confer endocrine cell selectivity rather than ß-cell selectivity in pancreatic islets and provide valuable insights and techniques to assess Zn2+ chelation as an approach to selectively target small molecules to pancreatic ß cells.NEW & NOTEWORTHY Inhibition of BET bromodomains is a novel potential strategy to prevent and treat diabetes mellitus. However, BET inhibitors have negative side effects. We synthesized a BET inhibitor expected to exploit the high zinc concentration in ß cells to accumulate in ß cells. We show our inhibitor targeted pancreatic endocrine cells; however, it was less effective in immune cells. A control inhibitor showed the opposite effect. These findings help us understand how to target specific cells in diabetes treatment.
Assuntos
Quelantes , Células Secretoras de Insulina , Zinco , Animais , Zinco/química , Zinco/farmacologia , Zinco/metabolismo , Quelantes/farmacologia , Células Secretoras de Insulina/efeitos dos fármacos , Células Secretoras de Insulina/metabolismo , Camundongos , Fatores de Transcrição/metabolismo , Fatores de Transcrição/antagonistas & inibidores , Triazóis/farmacologia , Triazóis/química , Humanos , Masculino , Azepinas/farmacologia , Azepinas/química , Células Secretoras de Glucagon/efeitos dos fármacos , Células Secretoras de Glucagon/metabolismo , Camundongos Endogâmicos C57BL , Proteínas que Contêm Bromodomínio , Proteínas NuclearesRESUMO
We have shown that nitric oxide limits ataxia-telangiectasia mutated signaling by inhibiting mitochondrial oxidative metabolism in a ß-cell selective manner. In this study, we examined the actions of nitric oxide on a second DNA damage response transducer kinase, ataxia-telangiectasia and Rad3-related protein (ATR). In ß-cells and non-ß-cells, nitric oxide activates ATR signaling by inhibiting ribonucleotide reductase; however, when produced at inducible nitric oxide synthase-derived (low micromolar) levels, nitric oxide impairs ATR signaling in a ß-cell selective manner. The inhibitory actions of nitric oxide are associated with impaired mitochondrial oxidative metabolism and lack of glycolytic compensation that result in a decrease in ß-cell ATP. Like nitric oxide, inhibitors of mitochondrial respiration reduce ATP levels and limit ATR signaling in a ß-cell selective manner. When non-ß-cells are forced to utilize mitochondrial oxidative metabolism for ATP generation, their response is more like ß-cells, as nitric oxide and inhibitors of mitochondrial respiration attenuate ATR signaling. These studies support a dual role for nitric oxide in regulating ATR signaling. Nitric oxide activates ATR in all cell types examined by inhibiting ribonucleotide reductase, and in a ß-cell selective manner, inducible nitric oxide synthase-derived levels of nitric oxide limit ATR signaling by attenuating mitochondrial oxidative metabolism and depleting ATP.
Assuntos
Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Dano ao DNA , Células Secretoras de Insulina/metabolismo , Mitocôndrias/metabolismo , Óxido Nítrico Sintase Tipo II/metabolismo , Óxido Nítrico/farmacologia , Animais , Células Cultivadas , Células Secretoras de Insulina/efeitos dos fármacos , Camundongos , Mitocôndrias/efeitos dos fármacos , Ratos , Transdução de SinaisRESUMO
Viral infection is an environmental trigger that has been suggested to initiate pancreatic ß-cell damage, leading to the development of autoimmune diabetes. Viruses potently activate the immune system and can damage ß cells by either directly infecting them or stimulating the production of secondary effector molecules (such as proinflammatory cytokines) during bystander activation. However, how and where ß cells recognize viruses is unclear, and the antiviral responses that are initiated following virus recognition are incompletely understood. In this study, we show that the ß-cell response to dsRNA, a viral replication intermediate known to activate antiviral responses, is determined by the cellular location of sensing (intracellular versus extracellular) and differs from the cellular response to cytokine treatment. Using biochemical and immunological methods, we show that ß cells selectively respond to intracellular dsRNA by expressing type I interferons (IFNs) and inducing apoptosis, but that they do not respond to extracellular dsRNA. These responses differ from the activities of cytokines on ß cells, which are mediated by inducible nitric oxide synthase expression and ß-cell production of nitric oxide. These findings provide evidence that the antiviral activities of type I IFN production and apoptosis are elicited in ß cells via the recognition of intracellular viral replication intermediates and that ß cells lack the capacity to respond to extracellular viral intermediates known to activate innate immune responses.
Assuntos
Células Secretoras de Insulina/virologia , RNA de Cadeia Dupla/metabolismo , Animais , Apoptose/efeitos dos fármacos , Caspases/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Dano ao DNA , Ativação Enzimática/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos dos fármacos , Inflamação/patologia , Interferon Tipo I/metabolismo , Masculino , Óxido Nítrico Sintase Tipo II/metabolismo , Poli I-C/farmacologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos Sprague-Dawley , Transdução de Sinais/efeitos dos fármacos , Receptor 3 Toll-Like/metabolismoRESUMO
Oxidative stress is thought to promote pancreatic ß-cell dysfunction and contribute to both type 1 and type 2 diabetes. Reactive oxygen species (ROS), such as superoxide and hydrogen peroxide, are mediators of oxidative stress that arise largely from electron leakage during oxidative phosphorylation. Reports that ß-cells express low levels of antioxidant enzymes, including catalase and GSH peroxidases, have supported a model in which ß-cells are ill-equipped to detoxify ROS. This hypothesis seems at odds with the essential role of ß-cells in the control of metabolic homeostasis and organismal survival through exquisite coupling of oxidative phosphorylation, a prominent ROS-producing pathway, to insulin secretion. Using glucose oxidase to deliver H2O2 continuously over time and Amplex Red to measure extracellular H2O2 concentration, we found here that ß-cells can remove micromolar levels of this oxidant. This detoxification pathway utilizes the peroxiredoxin/thioredoxin antioxidant system, as selective chemical inhibition or siRNA-mediated depletion of thioredoxin reductase sensitized ß-cells to continuously generated H2O2 In contrast, when delivered as a bolus, H2O2 induced the DNA damage response, depleted cellular energy stores, and decreased ß-cell viability independently of thioredoxin reductase inhibition. These findings show that ß-cells have the capacity to detoxify micromolar levels of H2O2 through a thioredoxin reductase-dependent mechanism and are not as sensitive to oxidative damage as previously thought.
Assuntos
Peróxido de Hidrogênio/metabolismo , Células Secretoras de Insulina/metabolismo , Peroxirredoxinas/metabolismo , Tiorredoxinas/metabolismo , Animais , Sobrevivência Celular , Dano ao DNA , Secreção de Insulina , Masculino , Oxirredução , Ratos , Ratos Sprague-Dawley , Tiorredoxina Dissulfeto Redutase/metabolismoRESUMO
Both reactive nitrogen and oxygen species (RNS and ROS), such as nitric oxide, peroxynitrite, and hydrogen peroxide, have been implicated as mediators of pancreatic ß-cell damage during the pathogenesis of autoimmune diabetes. While ß-cells are thought to be vulnerable to oxidative damage due to reportedly low levels of antioxidant enzymes, such as catalase and glutathione peroxidase, we have shown that they use thioredoxin reductase to detoxify hydrogen peroxide. Thioredoxin reductase is an enzyme that participates in the peroxiredoxin antioxidant cycle. Peroxiredoxins are expressed in ß-cells and, when overexpressed, protect against oxidative stress, but the endogenous roles of peroxiredoxins in the protection of ß-cells from oxidative damage are unclear. Here, using either glucose oxidase or menadione to continuously deliver hydrogen peroxide, or the combination of dipropylenetriamine NONOate and menadione to continuously deliver peroxynitrite, we tested the hypothesis that ß-cells use peroxiredoxins to detoxify both of these reactive species. Either pharmacological peroxiredoxin inhibition with conoidin A or specific depletion of cytoplasmic peroxiredoxin 1 (Prdx1) using siRNAs sensitizes INS 832/13 cells and rat islets to DNA damage and death induced by hydrogen peroxide or peroxynitrite. Interestingly, depletion of peroxiredoxin 2 (Prdx2) had no effect. Together, these results suggest that ß-cells use cytoplasmic Prdx1 as a primary defense mechanism against both ROS and RNS.
Assuntos
Dano ao DNA , Peróxido de Hidrogênio/toxicidade , Células Secretoras de Insulina/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , Peroxirredoxinas/metabolismo , Ácido Peroxinitroso/toxicidade , Animais , Morte Celular , Linhagem Celular Tumoral , Citoplasma/enzimologia , Citoproteção , Inibidores Enzimáticos/farmacologia , Células Secretoras de Insulina/enzimologia , Células Secretoras de Insulina/patologia , Masculino , Peroxirredoxinas/antagonistas & inibidores , Peroxirredoxinas/genética , Quinoxalinas/farmacologia , Interferência de RNA , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Ratos Sprague-Dawley , Transdução de Sinais , Tiorredoxina Redutase 1/metabolismoRESUMO
Endogenous nitric oxide (NO) generated by inducible NO synthase (iNOS) promotes glioblastoma cell proliferation and invasion and also plays a key role in glioblastoma resistance to chemotherapy and radiotherapy. Non-ionizing photodynamic therapy (PDT) has anti-tumor advantages over conventional glioblastoma therapies. Our previous studies revealed that glioblastoma U87 cells up-regulate iNOS after a photodynamic challenge and that the resulting NO not only increases resistance to apoptosis but renders surviving cells more proliferative and invasive. These findings were largely based on the effects of inhibiting iNOS activity and scavenging NO. Demonstrating now that iNOS expression in photostressed U87 cells is mediated by NF-κB, we hypothesized that (i) recognition of acetylated lysine (acK) on NF-κB p65/RelA by bromodomain and extra-terminal (BET) protein Brd4 is crucial; and (ii) by suppressing iNOS expression, a BET inhibitor (JQ1) would attenuate the negative effects of photostress. The following evidence was obtained. (i) Like iNOS, Brd4 protein and p65-acK levels increased severalfold in photostressed cells. (ii) JQ1 at minimally toxic concentrations had no effect on Brd4 or p65-acK up-regulation after PDT but strongly suppressed iNOS, survivin, and Bcl-xL up-regulation, along with the growth and invasion spurt of PDT-surviving cells. (iii) JQ1 inhibition of NO production in photostressed cells closely paralleled that of growth/invasion inhibition. (iv) Finally, at 1% the concentration of iNOS inhibitor 1400W, JQ1 reduced post-PDT cell aggressiveness to a far greater extent. This is the first evidence for BET inhibitor targeting of iNOS expression in cancer cells and how such targeting can markedly improve therapeutic efficacy.
Assuntos
Óxido Nítrico Sintase Tipo II/metabolismo , Fotoquimioterapia/métodos , Proteínas/metabolismo , Apoptose/efeitos dos fármacos , Azepinas , Proteínas de Ciclo Celular , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Glioblastoma/tratamento farmacológico , Glioblastoma/metabolismo , Humanos , NF-kappa B , Óxido Nítrico/antagonistas & inibidores , Óxido Nítrico/metabolismo , Óxido Nítrico Sintase/metabolismo , Óxido Nítrico Sintase Tipo II/genética , Proteínas Nucleares/metabolismo , Protoporfirinas/metabolismo , Fatores de Transcrição/metabolismo , Triazóis , Regulação para Cima/efeitos dos fármacosRESUMO
Fluorescent protein reporter genes are widely used to identify and sort murine pancreatic ß-cells. In this study, we compared use of the MIP-GFP transgene, which exhibits aberrant expression of human growth hormone (hGH), with a newly derived Ins2Apple allele that lacks hGH expression on the expression of sex-specific genes. ß-Cells from MIP-GFP transgenic mice exhibit changes in the expression of 7,733 genes, or greater than half of their transcriptome, compared with ß-cells from Ins2Apple/+ mice. To determine how these differences might affect a typical differential gene expression study, we analyzed the effect of sex on gene expression using both reporter lines. Six hundred fifty-seven differentially expressed genes were identified between male and female ß-cells containing the Ins2Apple allele. Female ß-cells exhibit higher expression of Xist, Tmed9, Arpc3, Eml2, and several islet-enriched transcription factors, including Nkx2-2 and Hnf4a, whereas male ß-cells exhibited a generally higher expression of genes involved in cell cycle regulation. In marked contrast, the same male vs. female comparison of ß-cells containing the MIP-GFP transgene revealed only 115 differentially expressed genes, and comparison of the 2 lists of differentially expressed genes revealed only 17 that were common to both analyses. These results indicate that 1) male and female ß-cells differ in their expression of key transcription factors and cell cycle regulators and 2) the MIP-GFP transgene may attenuate sex-specific differences that distinguish male and female ß-cells, thereby impairing the identification of sex-specific variations.
Assuntos
Proteínas de Fluorescência Verde/genética , Hormônio do Crescimento Humano/genética , Células Secretoras de Insulina/metabolismo , Insulina/genética , Animais , Feminino , Expressão Gênica , Genes Reporter/genética , Proteínas de Fluorescência Verde/metabolismo , Proteína Homeobox Nkx-2.2 , Proteínas de Homeodomínio , Hormônio do Crescimento Humano/metabolismo , Humanos , Masculino , Camundongos , Camundongos Transgênicos , Proteínas Nucleares , Regiões Promotoras Genéticas , RNA Mensageiro/metabolismo , Fatores Sexuais , Fatores de Transcrição , TransgenesRESUMO
Pancreatic ß-cells are essential for survival, being the only cell type capable of insulin secretion. While they are believed to be vulnerable to damage by inflammatory cytokines such as interleukin-1 beta (IL-1ß) and interferon-gamma, we have recently identified physiological roles for cytokine signaling in rodent ß-cells that include the stimulation of antiviral and antimicrobial gene expression and the inhibition of viral replication. In this study, we examine cytokine-stimulated changes in gene expression in human islets using single-cell RNA sequencing. Surprisingly, the global responses of human islets to cytokine exposure were remarkably blunted compared to our previous observations in the mouse. The small population of human islet cells that were cytokine responsive exhibited increased expression of IL-1ß-stimulated antiviral guanylate-binding proteins, just like in the mouse. Most human islet cells were not responsive to cytokines, and this lack of responsiveness was associated with high expression of genes encoding ribosomal proteins. We further correlated the expression levels of RPL5 with stress response genes, and when expressed at high levels, RPL5 is predictive of failure to respond to cytokines in all endocrine cells. We postulate that donor causes of death and isolation methodologies may contribute to stress of the islet preparation. Our findings indicate that activation of stress responses in human islets limits cytokine-stimulated gene expression, and we urge caution in the evaluation of studies that have examined cytokine-stimulated gene expression in human islets without evaluation of stress-related gene expression.
Assuntos
Citocinas , Ilhotas Pancreáticas , Análise de Célula Única , Humanos , Análise de Célula Única/métodos , Ilhotas Pancreáticas/metabolismo , Ilhotas Pancreáticas/efeitos dos fármacos , Citocinas/metabolismo , Citocinas/genética , Células Secretoras de Insulina/metabolismo , Células Secretoras de Insulina/efeitos dos fármacos , Análise de Sequência de RNA , Estresse Fisiológico/efeitos dos fármacos , Interleucina-1beta/metabolismo , Proteínas Ribossômicas/genética , Proteínas Ribossômicas/metabolismo , Masculino , Camundongos , Animais , RNA-Seq , Feminino , Pessoa de Meia-Idade , Análise da Expressão Gênica de Célula ÚnicaRESUMO
Reactive oxygen species (ROS), such as hydrogen peroxide, are formed when molecular oxygen obtains additional electrons, increasing its reactivity. While low concentrations of hydrogen peroxide are necessary for regulation of normal cellular signaling events, high concentrations can be toxic. To maintain this balance between beneficial and deleterious concentrations of hydrogen peroxide, cells utilize antioxidants. Our recent work supports a primary role for peroxiredoxin, thioredoxin, and thioredoxin reductase as the oxidant defense pathway used by insulin-producing pancreatic ß-cells. These three players work in an antioxidant cycle based on disulfide exchange, with oxidized targets ultimately being reduced using electrons provided by NADPH. Peroxiredoxins also participate in hydrogen peroxide-based signaling through disulfide exchange with redox-regulated target proteins. This chapter will describe the catalytic mechanisms of thioredoxin, thioredoxin reductase, and peroxiredoxin and provide an in-depth look at the roles these enzymes play in antioxidant defense pathways of insulin-secreting ß-cells. Finally, we will evaluate the physiological relevance of peroxiredoxin-mediated hydrogen peroxide signaling as a regulator of ß-cell function.
Assuntos
Antioxidantes , Insulinas , Humanos , Oxidantes , Peróxido de Hidrogênio/metabolismo , Peroxirredoxinas/metabolismo , Tiorredoxina Dissulfeto Redutase/química , Tiorredoxina Dissulfeto Redutase/metabolismo , Estresse Oxidativo/fisiologia , Tiorredoxinas/metabolismo , Insulinas/metabolismoRESUMO
While exposure to inflammatory cytokines is thought to contribute to pancreatic ß-cell damage during diabetes, primarily because cytokine-induced nitric oxide impairs ß-cell function and causes cell death with prolonged exposure, we hypothesize that there is a physiological role for cytokine signaling that protects ß-cells from a number of environmental stresses. This hypothesis is derived from the knowledge that ß-cells are essential for survival even though they have a limited capacity to replicate, yet they are exposed to high cytokine levels during infection as most of the pancreatic blood flow is directed to islets. Here, mouse islets were subjected to single-cell RNA sequencing following 18-h cytokine exposure. Treatment with IL-1ß and IFN-γ stimulates expression of inducible nitric oxide synthase (iNOS) mRNA and antiviral and immune-associated genes as well as repression of islet identity factors in a subset of ß- and non-ß-endocrine cells in a nitric oxide-independent manner. Nitric oxide-dependent expression of genes encoding heat shock proteins was observed in both ß- and non-ß-endocrine cells. Interestingly, cells with high expression of heat shock proteins failed to increase antiviral and immune-associated gene expression, suggesting that nitric oxide may be an internal "off switch" to prevent the negative effects of prolonged cytokine signaling in islet endocrine cells. We found no evidence for pro-apoptotic gene expression following 18-h cytokine exposure. Our findings suggest that the primary functions of cytokines and nitric oxide are to protect islet endocrine cells from damage, and only when regulation of cytokine signaling is lost does irreversible damage occur.
Assuntos
Citocinas , Óxido Nítrico , Camundongos , Animais , Citocinas/genética , Óxido Nítrico/metabolismo , Óxido Nítrico Sintase/genética , Interferon gama/farmacologia , Antivirais , Proteínas de Choque TérmicoRESUMO
Reactive oxygen species (ROS) have been implicated as mediators of pancreatic ß-cell damage. While ß-cells are thought to be vulnerable to oxidative damage, we have shown, using inhibitors and acute depletion, that thioredoxin reductase, thioredoxin, and peroxiredoxins are the primary mediators of antioxidant defense in ß-cells. However, the role of this antioxidant cycle in maintaining redox homeostasis and ß-cell survival in vivo remains unclear. Here, we generated mice with a ß-cell specific knockout of thioredoxin reductase 1 (Txnrd1fl/fl; Ins1Cre/+ , ßKO). Despite blunted glucose-stimulated insulin secretion, knockout mice maintain normal whole-body glucose homeostasis. Unlike pancreatic islets with acute Txnrd1 inhibition, ßKO islets do not demonstrate increased sensitivity to ROS. RNA-sequencing analysis revealed that Txnrd1-deficient ß-cells have increased expression of nuclear factor erythroid 2-related factor 2 (Nrf2)-regulated genes, and altered expression of genes involved in heme and glutathione metabolism, suggesting an adaptive response. Txnrd1-deficient ß-cells also have decreased expression of factors controlling ß-cell function and identity which may explain the mild functional impairment. Together, these results suggest that Txnrd1-knockout ß-cells compensate for loss of this essential antioxidant pathway by increasing expression of Nrf2-regulated antioxidant genes, allowing for protection from excess ROS at the expense of normal ß-cell function and identity.
Assuntos
Antioxidantes , Fator 2 Relacionado a NF-E2 , Camundongos , Animais , Antioxidantes/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Fator 2 Relacionado a NF-E2/genética , Oxirredução , Camundongos Knockout , Glucose , Homeostase/genéticaRESUMO
Oxidative stress is hypothesized to play a role in pancreatic ß-cell damage, potentially contributing to ß-cell dysfunction and death in both type 1 and type 2 diabetes. Oxidative stress arises when naturally occurring reactive oxygen species (ROS) are produced at levels that overwhelm the antioxidant capacity of the cell. ROS, including superoxide and hydrogen peroxide, are primarily produced by electron leak during mitochondrial oxidative metabolism. Additionally, peroxynitrite, an oxidant generated by the reaction of superoxide and nitric oxide, may also cause ß-cell damage during autoimmune destruction of these cells. ß-cells are thought to be susceptible to oxidative damage based on reports that they express low levels of antioxidant enzymes compared to other tissues. Furthermore, markers of oxidative damage are observed in islets from diabetic rodent models and human patients. However, recent studies have demonstrated high expression of various isoforms of peroxiredoxins, thioredoxin, and thioredoxin reductase in ß-cells and have provided experimental evidence supporting a role for these enzymes in promoting ß-cell function and survival in response to a variety of oxidative stressors. This mini-review will focus on the mechanism by which thioredoxins and peroxiredoxins detoxify ROS and on the protective roles of these enzymes in ß-cells. Additionally, we speculate about the role of this antioxidant system in promoting insulin secretion.
Assuntos
Antioxidantes/farmacologia , Diabetes Mellitus Tipo 1/prevenção & controle , Diabetes Mellitus Tipo 2/prevenção & controle , Células Secretoras de Insulina/efeitos dos fármacos , Estresse Oxidativo , Peroxirredoxinas/farmacologia , Tiorredoxinas/farmacologia , Diabetes Mellitus Tipo 1/metabolismo , Diabetes Mellitus Tipo 1/patologia , Diabetes Mellitus Tipo 2/metabolismo , Diabetes Mellitus Tipo 2/patologia , Humanos , Células Secretoras de Insulina/patologiaRESUMO
Exposure to proinflammatory cytokines is believed to contribute to pancreatic ß-cell damage during diabetes development. Although some cytokine-mediated changes in islet gene expression are known, the heterogeneity of the response is not well-understood. After 6-h treatment with IL-1ß and IFN-γ alone or together, mouse islets were subjected to single-cell RNA sequencing. Treatment with both cytokines together led to expression of inducible nitric oxide synthase mRNA (Nos2) and antiviral and immune-associated genes in a subset of ß-cells. Interestingly, IL-1ß alone activated antiviral genes. Subsets of δ- and α-cells expressed Nos2 and exhibited similar gene expression changes as ß-cells, including increased expression of antiviral genes and repression of identity genes. Finally, cytokine responsiveness was inversely correlated with expression of genes encoding heat shock proteins. Our findings show that all islet endocrine cell types respond to cytokines, IL-1ß induces the expression of protective genes, and cellular stress gene expression is associated with inhibition of cytokine signaling.
Assuntos
Citocinas/farmacologia , Ilhotas Pancreáticas/metabolismo , Animais , Apoptose/efeitos dos fármacos , Linhagem Celular , Citocinas/metabolismo , Expressão Gênica/genética , Perfilação da Expressão Gênica/métodos , Células Secretoras de Insulina/metabolismo , Células Secretoras de Insulina/fisiologia , Interferon gama/metabolismo , Interleucina-1beta/genética , Ilhotas Pancreáticas/fisiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Óxido Nítrico Sintase Tipo II/metabolismo , RNA Mensageiro/metabolismo , Análise de Sequência de RNA/métodos , Análise de Célula Única/métodos , Transcriptoma/genéticaRESUMO
The immune response to a chronic viral infection is uniquely tailored to balance viral control and immunopathology. The role of myeloid cells in shaping the response to chronic viral infection, however, is poorly understood. We perform single-cell RNA sequencing of myeloid cells during acute and chronic lymphocytic choriomeningitis virus (LCMV) infection to address this question. Our analysis identifies a cluster of suppressive neutrophils that is enriched in chronic infection. Furthermore, suppressive neutrophils highly express the gene encoding Proviral integration site for Moloney murine leukemia virus-1 (PIM1), a kinase known to promote mitochondrial fitness and cell survival. Pharmacological inhibition of PIM1 selectively diminishes suppressive neutrophil-mediated immunosuppression without affecting the function of monocytic myeloid-derived suppressor cells (M-MDSCs). Decreased accumulation of suppressive neutrophils leads to increased CD8 T cell function and viral control. Mechanistically, PIM kinase activity is required for maintaining fused mitochondrial networks in suppressive neutrophils, but not in M-MDSCs, and loss of PIM kinase function causes increased suppressive neutrophil apoptosis.
Assuntos
Neutrófilos/metabolismo , Proteínas Proto-Oncogênicas c-pim-1/metabolismo , Viroses/imunologia , Doença Crônica , HumanosRESUMO
A sustained increase in intracellular Ca2+ concentration (referred to hereafter as excitotoxicity), brought on by chronic metabolic stress, may contribute to pancreatic ß-cell failure. To determine the additive effects of excitotoxicity and overnutrition on ß-cell function and gene expression, we analyzed the impact of a high-fat diet (HFD) on Abcc8 knockout mice. Excitotoxicity caused ß-cells to be more susceptible to HFD-induced impairment of glucose homeostasis, and these effects were mitigated by verapamil, a Ca2+ channel blocker. Excitotoxicity, overnutrition, and the combination of both stresses caused similar but distinct alterations in the ß-cell transcriptome, including additive increases in genes associated with mitochondrial energy metabolism, fatty acid ß-oxidation, and mitochondrial biogenesis and their key regulator Ppargc1a Overnutrition worsened excitotoxicity-induced mitochondrial dysfunction, increasing metabolic inflexibility and mitochondrial damage. In addition, excitotoxicity and overnutrition, individually and together, impaired both ß-cell function and identity by reducing expression of genes important for insulin secretion, cell polarity, cell junction, cilia, cytoskeleton, vesicular trafficking, and regulation of ß-cell epigenetic and transcriptional program. Sex had an impact on all ß-cell responses, with male animals exhibiting greater metabolic stress-induced impairments than females. Together, these findings indicate that a sustained increase in intracellular Ca2+, by altering mitochondrial function and impairing ß-cell identity, augments overnutrition-induced ß-cell failure.
Assuntos
Cálcio/metabolismo , Células Secretoras de Insulina/metabolismo , Hipernutrição/metabolismo , Animais , Células Cultivadas , Dieta Hiperlipídica , Feminino , Regulação da Expressão Gênica , Glucose/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Mitocôndrias/fisiologia , Caracteres Sexuais , TranscriptomaRESUMO
We used mice lacking Abcc8, a key component of the ß-cell KATP-channel, to analyze the effects of a sustained elevation in the intracellular Ca2+ concentration ([Ca2+]i) on ß-cell identity and gene expression. Lineage tracing analysis revealed the conversion of ß-cells lacking Abcc8 into pancreatic polypeptide cells but not to α- or δ-cells. RNA-sequencing analysis of FACS-purified Abcc8-/- ß-cells confirmed an increase in Ppy gene expression and revealed altered expression of more than 4,200 genes, many of which are involved in Ca2+ signaling, the maintenance of ß-cell identity, and cell adhesion. The expression of S100a6 and S100a4, two highly upregulated genes, is closely correlated with membrane depolarization, suggesting their use as markers for an increase in [Ca2+]i Moreover, a bioinformatics analysis predicts that many of the dysregulated genes are regulated by common transcription factors, one of which, Ascl1, was confirmed to be directly controlled by Ca2+ influx in ß-cells. Interestingly, among the upregulated genes is Aldh1a3, a putative marker of ß-cell dedifferentiation, and other genes associated with ß-cell failure. Taken together, our results suggest that chronically elevated ß-cell [Ca2+]i in Abcc8-/- islets contributes to the alteration of ß-cell identity, islet cell numbers and morphology, and gene expression by disrupting a network of Ca2+-regulated genes.
Assuntos
Sinalização do Cálcio/genética , Polaridade Celular , Regulação da Expressão Gênica/genética , Expressão Gênica/genética , Células Secretoras de Insulina/metabolismo , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/fisiologia , Cálcio/metabolismo , Adesão Celular/genética , Proteínas de Ciclo Celular/metabolismo , Linhagem da Célula/genética , Células Secretoras de Insulina/citologia , Canais KATP/genética , Camundongos , Células Secretoras de Polipeptídeo Pancreático/fisiologia , Proteína A6 Ligante de Cálcio S100 , Proteína A4 de Ligação a Cálcio da Família S100/metabolismo , Proteínas S100/metabolismo , Receptores de Sulfonilureias/deficiênciaRESUMO
Using a transgenic mouse model to express MafA, Pdx1, and Neurog3 (3TF) in a pancreatic acinar cell- and doxycycline-dependent manner, we discovered that the outcome of transcription factor-mediated acinar to ß-like cellular reprogramming is dependent on both the magnitude of 3TF expression and on reprogramming-induced inflammation. Overly robust 3TF expression causes acinar cell necrosis, resulting in marked inflammation and acinar-to-ductal metaplasia. Generation of new ß-like cells requires limiting reprogramming-induced inflammation, either by reducing 3TF expression or by eliminating macrophages. The new ß-like cells were able to reverse streptozotocin-induced diabetes 6 days after inducing 3TF expression but failed to sustain their function after removal of the reprogramming factors.
Assuntos
Células Acinares/patologia , Reprogramação Celular , Inflamação/patologia , Células Secretoras de Insulina/patologia , Pâncreas/patologia , Células Acinares/efeitos dos fármacos , Adenoviridae/metabolismo , Alelos , Animais , Reprogramação Celular/efeitos dos fármacos , Diabetes Mellitus Experimental/patologia , Doxiciclina/farmacologia , Perfilação da Expressão Gênica , Proteínas de Homeodomínio/metabolismo , Imunidade , Células Secretoras de Insulina/efeitos dos fármacos , Macrófagos/efeitos dos fármacos , Macrófagos/patologia , Metaplasia , Camundongos Transgênicos , Tamanho do Órgão/efeitos dos fármacos , Ductos Pancreáticos/patologia , Reprodutibilidade dos Testes , Fatores de Transcrição/metabolismo , TransgenesRESUMO
The human growth hormone (hGH) minigene is frequently used in the derivation of transgenic mouse lines to enhance transgene expression. Although this minigene is present in the transgenes as a secondcistron, and thus not thought to be expressed, we found that three commonly used lines, Pdx1-Cre(Late), RIP-Cre, and MIP-GFP, each expressed significant amounts of hGH in pancreatic islets. Locally secreted hGH binds to prolactin receptors on ß cells, activates STAT5 signaling, and induces pregnancy-like changes in gene expression, thereby augmenting pancreatic ß cell mass and insulin content. In addition, islets of Pdx1-Cre(Late) mice have lower GLUT2 expression and reduced glucose-induced insulin release and are protected against the ß cell toxin streptozotocin. These findings may be important when interpreting results obtained when these and other hGH minigene-containing transgenic mice are used.