RESUMO
Context: The 12-lipoxygenase (12-LO) pathway produces proinflammatory metabolites, and its activation is implicated in islet inflammation associated with type 1 and type 2 diabetes (T2D). Objectives: We aimed to test the efficacy of ML355, a highly selective, small molecule inhibitor of 12-LO, for the preservation of islet function. Design: Human islets from nondiabetic donors were incubated with a mixture of tumor necrosis factor α , interluekin-1ß, and interferon-γ to model islet inflammation. Cytokine-treated islets and human islets from T2D donors were incubated in the presence and absence of ML355. Setting: In vitro study. Participants: Human islets from organ donors aged >20 years of both sexes and any race were used. T2D status was defined from either medical history or most recent hemoglobin A1c value >6.5%. Intervention: Glucose stimulation. Main Outcome Measures: Static and dynamic insulin secretion and oxygen consumption rate (OCR). Results: ML355 prevented the reduction of insulin secretion and OCR in cytokine-treated human islets and improved both parameters in human islets from T2D donors. Conclusions: ML355 was efficacious in improving human islet function after cytokine treatment and in T2D islets in vitro. The study suggests that the blockade of the 12-LO pathway may serve as a target for both form of diabetes and provides the basis for further study of this small molecule inhibitor in vivo.
Assuntos
Diabetes Mellitus Tipo 2/metabolismo , Glucose/farmacologia , Insulina/metabolismo , Ilhotas Pancreáticas/efeitos dos fármacos , Inibidores de Lipoxigenase/farmacologia , Consumo de Oxigênio/efeitos dos fármacos , Sulfonamidas/farmacologia , Adulto , Estudos de Casos e Controles , Feminino , Humanos , Técnicas In Vitro , Inflamação , Secreção de Insulina , Interferon gama/farmacologia , Interleucina-1beta/farmacologia , Masculino , Pessoa de Meia-Idade , Fator de Necrose Tumoral alfa/farmacologia , Adulto JovemRESUMO
Redox stress related loss of beta cell function is a feature of diabetes. Exposure of beta cells and islets to inflammatory mediators elevates reactive oxygen species (ROS) and beta cell dysfunction. Direct molecular manipulation of NADPH oxidase-1 (NOX-1) has identified a key role for NOX-1 in cytokine-induced beta cell dysfunction. Plasmid driven elevation of NOX-1 resulted in elevated ROS, loss of glucose-stimulated-insulin-secretion and increased apoptosis. These outcomes on beta cell function are analogous to cytokine treatment. In contrast, reduction of NOX-1 expression, by shRNA, conferred protection to beta cells and islets from the damaging effects of inflammatory cytokines. Collectively, these data support the therapeutic potential for NOX-1 inhibition in diabetes.
Assuntos
Apoptose , Citocinas/imunologia , Inflamação/imunologia , Células Secretoras de Insulina/imunologia , Células Secretoras de Insulina/patologia , NADH NADPH Oxirredutases/imunologia , Animais , Linhagem Celular , Diabetes Mellitus/genética , Diabetes Mellitus/imunologia , Diabetes Mellitus/metabolismo , Diabetes Mellitus/patologia , Glucose/metabolismo , Inflamação/genética , Inflamação/metabolismo , Inflamação/patologia , Insulina/metabolismo , Células Secretoras de Insulina/citologia , Células Secretoras de Insulina/metabolismo , Camundongos , NADH NADPH Oxirredutases/genética , NADH NADPH Oxirredutases/metabolismo , NADPH Oxidase 1 , Estresse Oxidativo , Interferência de RNA , RNA Interferente Pequeno/genética , Espécies Reativas de Oxigênio/metabolismo , Regulação para CimaRESUMO
Islet inflammation contributes to beta cell demise in both type 1 and type 2 diabetes. 12-Lipoxygenase (12-LO, gene expressed as ALOX12 in humans and 12-Lo in rodents in this manuscript) produces proinflammatory metabolites such as 12(S)-hydroxyeicosatetraenoic acids through dioxygenation of polyunsaturated fatty acids. 12-LO was first implicated in diabetes when the increase in 12-Lo expression and 12(S)-hydroxyeicosatetraenoic acid was noted in rodent models of diabetes. Subsequently, germline 12-Lo (-/-) was shown to prevent the development of hyperglycemia in mouse models of type 1 diabetes and in high-fat fed mice. More recently, beta cell-specific 12-Lo (-/-) was shown to protect mice against hyperglycaemia after streptozotocin and a high-fat diet. In humans, 12-LO expression is increased in pancreatic islets of autoantibody-positive, type 1 diabetic and type 2 diabetic organ donors. Interestingly, the high expression of ALOX12 is associated with the alteration in first-phase glucose-stimulated insulin secretion in human type 2 diabetic islets. To further clarify the role of islet 12-LO in diabetes and to validate 12-LO as a therapeutic target of diabetes, we have studied selective pharmacological inhibitors for 12-LO. The compounds we have identified show promise: they protect beta cell lines and human islets from apoptosis and preserve insulin secretion when challenged by proinflammatory cytokine mixture. Currently studies are underway to test the compounds in mouse models of diabetes. This review summarises a presentation given at the 'Islet inflammation in type 2 diabetes' symposium at the 2015 annual meeting of the EASD. It is accompanied two other mini-reviews on topics from this symposium (by Simone Baltrusch, DOI: 10.1007/s00125-016-3891-x and Marc Donath, DOI: 10.1007/s00125-016-3873-z ) and a commentary by the Session Chair, Piero Marchetti (DOI: 10.1007/s00125-016-3875-x ).
Assuntos
Inflamação/metabolismo , Células Secretoras de Insulina/metabolismo , Células Secretoras de Insulina/patologia , Animais , Araquidonato 12-Lipoxigenase/metabolismo , Diabetes Mellitus Tipo 1/imunologia , Diabetes Mellitus Tipo 1/metabolismo , Diabetes Mellitus Tipo 1/patologia , Diabetes Mellitus Tipo 2/imunologia , Diabetes Mellitus Tipo 2/metabolismo , Diabetes Mellitus Tipo 2/patologia , Humanos , Ácidos Hidroxieicosatetraenoicos/metabolismo , Inflamação/imunologia , Inflamação/patologia , Células Secretoras de Insulina/imunologia , LipídeosRESUMO
Pathology driving ß-cell loss in diabetes is poorly defined. Chronic subclinical inflammation is associated with ß-cell dysfunction. Acute in vitro exposure of islets and ß-cells to an inflammatory cytokine cocktail (IL-1ß/TNF-α/IFN-γ) results in loss of cell function and viability. The contribution of each cytokine alone or in combination has been evaluated in homogeneous mouse ß-cell lines and primary mouse islets. Cytokine cooperation is required for ß-cell apoptosis with the most potent combinations including IL-1ß. Single cytokine exposure did not induce ß-cell apoptosis. Expression of endogenous interleukin-12 in ß-cells correlated with inflammatory cytokine combinations that induced ß-cell apoptosis. Uncoupling of the IL-12 axis by a block of IL-12 production, inhibition of IL-12 receptor/ligand interaction or disruption of IL-12 receptor signaling conferred protection to ß-cells from apoptosis induced by inflammatory cytokine stimulation. Signaling through STAT4 is indicated since disruption of IL-12 concomitantly reduced inflammatory cytokine stimulation of endogenous IFN-γ expression. Primary mouse islets isolated from mice deficient in STAT4 show resistance to inflammatory-cytokine-induced cell death when compared to islets isolated from wild type mice. Collectively, the data identify IL-12 as an important mediator of inflammation induced ß-cell apoptosis. Modulation of IL-12/STAT4 signaling may be a valuable therapeutic strategy to preserve islet/ß-cell viability in established diabetes.
Assuntos
Citocinas/fisiologia , Mediadores da Inflamação/fisiologia , Interleucina-12/metabolismo , Ilhotas Pancreáticas/fisiopatologia , Fator de Transcrição STAT4/metabolismo , Animais , Apoptose , Linhagem Celular , Células Cultivadas , Quimiocina CCL2/genética , Interleucina-12/genética , Camundongos , Transdução de SinaisRESUMO
AIMS/HYPOTHESIS: Upregulation of the reactive oxygen species (ROS)-producing enzyme NADPH oxidase (NOX)-1 in islets and beta cells follows acute exposure to inflammatory cytokines and is concomitant with beta cell dysfunction. NOX-1 is a candidate mediator of inflammation-induced beta cell dysfunction. This study aimed to determine whether selective inhibition of NADPH oxidase-1 presents a new strategy to preserve beta cell function. METHODS: Induced beta cell dysfunction was studied in primary human donor islets, isolated mouse islets and murine beta cell lines. Islets and beta cells were stimulated with inflammatory cytokines (TNF-α, IL-1ß, IFN-γ). NOX-1 activity was blocked by the selective inhibitor ML171. RESULTS: Cytokine induction of intracellular ROS was reduced 80% with 1 µmol/l ML171 in murine beta cell lines (p < 0.01). Cytokine-induced apoptosis, measured by caspase-3 activation or quantified fluorescence microscopy, was prevented in islets and beta cell lines up to 100% with ML171 in a concentration-dependent manner (p < 0.05). Functionally, glucose-stimulated insulin secretion was abolished by cytokine exposure but preserved by ML171 in isolated mouse islets and murine beta cell lines. A feed-forward regulation of NOX-1 in islets and beta cell lines was disrupted by ML171. CONCLUSIONS/INTERPRETATION: Stimulation of NOX-1 activity is a major component of inflammatory cytokine-induced beta cell dysfunction. Significant protection of beta cells is conferred with selective inhibition of NOX-1. Suppression of NOX-1 activity may present a new therapeutic strategy to preserve and protect beta cell function in diabetes.
Assuntos
Citoproteção/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Células Secretoras de Insulina/efeitos dos fármacos , NADH NADPH Oxirredutases/antagonistas & inibidores , Fenotiazinas/farmacologia , Animais , Células Cultivadas , Humanos , Inflamação/prevenção & controle , Células Secretoras de Insulina/fisiologia , Interferon gama/efeitos adversos , Interleucina-1beta/efeitos adversos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , NADPH Oxidase 1 , Ratos , Fator de Necrose Tumoral alfa/efeitos adversosRESUMO
AIMS/HYPOTHESIS: Islet inflammation leads to loss of functional pancreatic beta cell mass. Increasing evidence suggests that activation of 12-lipoxygenase leads to inflammatory beta cell loss. This study evaluates new specific small-molecule inhibitors of 12-lipoxygenase for protecting rodent and human beta cells from inflammatory damage. METHODS: Mouse beta cell lines and mouse and human islets were treated with inflammatory cytokines IL-1ß, TNFα and IFNγ in the absence or presence of novel selective 12-lipoxygenase inhibitors. Glucose-stimulated insulin secretion (GSIS), gene expression, cell survival and 12-S-hydroxyeicosatetraenoic acid (12-S-HETE) levels were evaluated using established methods. Pharmacokinetic analysis was performed with the lead inhibitor in CD1 mice. RESULTS: Inflammatory cytokines led to the loss of human beta cell function, elevated cell death, increased inflammatory gene expression and upregulation of 12-lipoxygenase expression and activity (measured by 12-S-HETE generation). Two 12-lipoxygenase inhibitors, Compounds 5 and 9, produced a concentration-dependent reduction of stimulated 12-S-HETE levels. GSIS was preserved in the presence of the 12-lipoxygenase inhibitors. 12-Lipoxygenase inhibition preserved survival of primary mouse and human islets. When administered orally, Compound 5 reduced plasma 12-S-HETE in CD1 mice. Compounds 5 and 9 preserved the function and survival of human donor islets exposed to inflammatory cytokines. CONCLUSIONS/INTERPRETATION: Selective inhibition of 12-lipoxygenase activity confers protection to beta cells during exposure to inflammatory cytokines. These concept validation studies identify 12-lipoxygenase as a promising target in the prevention of loss of functional beta cells in diabetes.
Assuntos
Araquidonato 12-Lipoxigenase/metabolismo , Inibidores Enzimáticos/farmacologia , Células Secretoras de Insulina/efeitos dos fármacos , Células Secretoras de Insulina/metabolismo , Ácido 12-Hidroxi-5,8,10,14-Eicosatetraenoico/metabolismo , Animais , Apoptose/efeitos dos fármacos , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Ensaio de Imunoadsorção Enzimática , Humanos , Técnicas In Vitro , Interleucina-1beta/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Reação em Cadeia da Polimerase em Tempo Real , Fator de Necrose Tumoral alfa/metabolismoRESUMO
Human lipoxygenases (LOXs) are a family of iron-containing enzymes which catalyze the oxidation of polyunsaturated fatty acids to provide the corresponding bioactive hydroxyeicosatetraenoic acid (HETE) metabolites. These eicosanoid signaling molecules are involved in a number of physiologic responses such as platelet aggregation, inflammation, and cell proliferation. Our group has taken a particular interest in platelet-type 12-(S)-LOX (12-LOX) because of its demonstrated role in skin diseases, diabetes, platelet hemostasis, thrombosis, and cancer. Herein, we report the identification and medicinal chemistry optimization of a 4-((2-hydroxy-3-methoxybenzyl)amino)benzenesulfonamide-based scaffold. Top compounds, exemplified by 35 and 36, display nM potency against 12-LOX, excellent selectivity over related lipoxygenases and cyclooxygenases, and possess favorable ADME properties. In addition, both compounds inhibit PAR-4 induced aggregation and calcium mobilization in human platelets and reduce 12-HETE in ß-cells.
Assuntos
Araquidonato 12-Lipoxigenase/metabolismo , Derivados de Benzeno/síntese química , Inibidores de Lipoxigenase/síntese química , Sulfonamidas/síntese química , Animais , Derivados de Benzeno/química , Derivados de Benzeno/farmacologia , Disponibilidade Biológica , Plaquetas/efeitos dos fármacos , Plaquetas/metabolismo , Cálcio/metabolismo , Humanos , Ilhotas Pancreáticas/efeitos dos fármacos , Ilhotas Pancreáticas/enzimologia , Inibidores de Lipoxigenase/química , Inibidores de Lipoxigenase/farmacologia , Camundongos , Agregação Plaquetária/efeitos dos fármacos , Relação Estrutura-Atividade , Sulfonamidas/química , Sulfonamidas/farmacologiaRESUMO
Predictions of diabetes prevalence over the next decades warrant the aggressive discovery of new approaches to stop or reverse loss of functional beta cell mass. Beta cells are recognized to have a relatively high sensitivity to reactive oxygen species (ROS) and become dysfunctional under oxidative stress conditions. New discoveries have identified NADPH oxidases in beta cells as contributors to elevated cellular ROS. Reviewed are recent reports that evidence a role for NADPH oxidase-1 (NOX-1) in beta cell dysfunction. NOX-1 is stimulated by inflammatory cytokines that are elevated in diabetes. First, regulation of cytokine-stimulated NOX-1 expression has been linked to inflammatory lipid mediators derived from 12-lipoxygenase activity. For the first time in beta cells these data integrate distinct pathways associated with beta cell dysfunction. Second, regulation of NOX-1 in beta cells involves feed-forward control linked to elevated ROS and Src-kinase activation. This potentially results in unbridled ROS generation and identifies candidate targets for pharmacologic intervention. Third, consideration is provided of new, first-in-class, selective inhibitors of NOX-1. These compounds could have an important role in assessing a disruption of NOX-1/ROS signaling as a new approach to preserve and protect beta cell mass in diabetes.
RESUMO
AIMS: Type 1 diabetes (T1D) is characterized by autoimmune depletion of insulin-producing pancreatic beta cells. We showed previously that deletion of the 12/15-lipoxygenase enzyme (12/15-LO, Alox15 gene) in NOD mice leads to nearly 100 percent protection from T1D. In this study, we test the hypothesis that cytokines involved in the IL-12/12/15-LO axis affect both macrophage and islet function, which contributes to the development of T1D. METHODS: 12/15-LO expression was clarified in immune cells by qRT-PCR, and timing of expression was tested in islets using qRT-PCR and Western blotting. Expression of key proinflammatory cytokines and pancreatic transcription factors was studied in NOD and NOD-Alox15(null) macrophages and islets using qRT-PCR. The two mouse strains were also assessed for the ability of splenocytes to transfer diabetes in an adoptive transfer model, and beta cell mass. RESULTS: 12/15-LO is expressed in macrophages, but not B and T cells of NOD mice. In macrophages, 12/15-LO deletion leads to decreased proinflammatory cytokine mRNA and protein levels. Furthermore, splenocytes from NOD-Alox15(null) mice are unable to transfer diabetes in an adoptive transfer model. In islets, expression of 12/15-LO in NOD mice peaks at a crucial time during insulitis development. The absence of 12/15-LO results in maintenance of islet health with respect to measurements of islet-specific transcription factors, markers of islet health, proinflammatory cytokines, and beta cell mass. CONCLUSIONS: These results suggest that 12/15-LO affects islet and macrophage function, causing inflammation, and leading to autoimmunity and reduced beta cell mass.
Assuntos
Araquidonato 12-Lipoxigenase/genética , Araquidonato 15-Lipoxigenase/genética , Diabetes Mellitus Tipo 1/genética , Macrófagos/enzimologia , Oxigenases/genética , Animais , Araquidonato 12-Lipoxigenase/metabolismo , Araquidonato 15-Lipoxigenase/metabolismo , Diabetes Mellitus Tipo 1/terapia , Humanos , Insulina/metabolismo , Células Secretoras de Insulina/citologia , Células Secretoras de Insulina/metabolismo , Interleucina-12/metabolismo , Ilhotas Pancreáticas/citologia , Ilhotas Pancreáticas/enzimologia , Ilhotas Pancreáticas/metabolismo , Macrófagos/citologia , Macrófagos/metabolismo , Camundongos , Camundongos Endogâmicos NOD/genéticaRESUMO
Elevated cellular reactive species, which can be produced by diabetic serum conditions such as elevated inflammatory cytokines, lipotoxicity or glucotoxicity contribute to islet beta cell dysfunction and cell death. Cellular pathways that result in beta cell oxidative stress are poorly resolved. In this study, stimulation of human donor islets, primary mouse islets or homogeneous beta cell lines with a cocktail of inflammatory cytokines (TNFα, IL-1ß, and INFγ) significantly induced NADPH oxidase-1 (NOX-1) gene expression (p<0.05). This pro-inflammatory cytokine cocktail concomitantly induced loss of islet glucose stimulated insulin response (p<0.05), elevated expression of MCP-1 (p<0.01), increased cellular reactive oxygen species (ROS) and induced cell death. Inhibitors of NADPH oxidase, apocynin and diphenyleneiodonium, and a dual selective NOX1/4 inhibitor, blocked ROS generation (p<0.01) and induction of MCP-1 (p<0.05) by pro-inflammatory cytokines in beta cells. It has previously been reported that pro-inflammatory cytokine stimulation induces 12-lipoxygenase (12-LO) expression in human islets. 12-Hydroxyeicosatetraenoic acid (12-HETE), a product of 12-LO activity, stimulated NOX-1 expression in human islets (p<0.05). A novel selective inhibitor of 12-LO blocked induction of NOX-1, production of ROS and pro-caspase 3 cleavage by pro-inflammatory cytokines in INS-1 beta cells (p<0.01). Inhibition was not seen with a structurally related but inactive analog. Importantly, islets from human type 2 diabetic donors have an elevated expression of NOX-1 (p<0.05). This study describes an integrated pathway in beta cells that links beta cell dysfunction induced by pro-inflammatory cytokines with 12-lipoxygenase and NADPH oxidase (NOX-1) activation. Inhibitors of this pathway may provide a new therapeutic strategy to preserve beta cell mass in diabetes.
Assuntos
Araquidonato 12-Lipoxigenase/metabolismo , Células Secretoras de Insulina/metabolismo , NADH NADPH Oxirredutases/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Acetofenonas/farmacologia , Animais , Apoptose/efeitos dos fármacos , Caspase 3/metabolismo , Quimiocina CCL2/biossíntese , Diabetes Mellitus/metabolismo , Diabetes Mellitus/patologia , Ácido Eicosapentaenoico/análogos & derivados , Ácido Eicosapentaenoico/antagonistas & inibidores , Ácido Eicosapentaenoico/farmacologia , Ativação Enzimática , Humanos , Células Secretoras de Insulina/patologia , Interferon gama/farmacologia , Interleucina-1beta/farmacologia , Camundongos , NADH NADPH Oxirredutases/antagonistas & inibidores , NADPH Oxidase 1 , Oniocompostos/farmacologia , Espécies Reativas de Oxigênio/metabolismo , Fator de Necrose Tumoral alfa/farmacologiaRESUMO
Type 1 diabetes mellitus results from the autoimmune and inflammatory destruction of insulin-producing islet ß cells, rendering individuals devoid of insulin production. Recent studies suggest that combination therapies consisting of anti-inflammatory agents and islet growth-promoting factors have the potential to cause sustained recovery of ß cell mass, leading to amelioration or reversal of type 1 diabetes in mouse models. In this study, we hypothesized that the combination of the anti-inflammatory agent lisofylline (LSF) with an active peptide fragment of islet neogenesis associated protein (INGAP peptide) would lead to remission of type 1 diabetes in the non-obese diabetic (NOD) mouse. We treated groups of spontaneously diabetic NOD mice with combinations of LSF, INGAP peptide, or control saline parenterally for up to 6 weeks. Our results demonstrate that the mice receiving combined treatment with LSF and INGAP peptide exhibited partial remission of diabetes with increased plasma insulin levels. Histologic assessment of pancreata in mice receiving combined therapy revealed the presence of islet insulin staining, increased ß cell replication, and evidence of Pdx1-positivity in ductal cells. By contrast, diabetic animals showed severe insulitis with no detectible insulin or Pdx1 staining. We conclude that the novel combination treatment with LSF and INGAP peptide has the potential to ameliorate hyperglycemia in the setting of established type 1 diabetes via the recovery of endogenous ß cells and warrant further studies.
RESUMO
We report the discovery of novel small molecule inhibitors of platelet-type 12-human lipoxygenase, which display nanomolar activity against the purified enzyme, using a quantitative high-throughput screen (qHTS) on a library of 153607 compounds. These compounds also exhibit excellent specificity, >50-fold selectivity vs the paralogues, 5-human lipoxygenase, reticulocyte 15-human lipoxygenase type-1, and epithelial 15-human lipoxygenase type-2, and >100-fold selectivity vs ovine cyclooxygenase-1 and human cyclooxygenase-2. Kinetic experiments indicate this chemotype is a noncompetitive inhibitor that does not reduce the active site iron. Moreover, chiral HPLC separation of two of the racemic lead molecules revealed a strong preference for the (-)-enantiomers (IC(50) of 0.43 ± 0.04 and 0.38 ± 0.05 µM) compared to the (+)-enantiomers (IC(50) of >25 µM for both), indicating a fine degree of selectivity in the active site due to chiral geometry. In addition, these compounds demonstrate efficacy in cellular models, which underscores their relevance to disease modification.
Assuntos
Araquidonato 12-Lipoxigenase/efeitos dos fármacos , Inibidores de Lipoxigenase/farmacologia , Ácido 12-Hidroxi-5,8,10,14-Eicosatetraenoico/antagonistas & inibidores , Animais , Plaquetas/enzimologia , Humanos , Ilhotas Pancreáticas/efeitos dos fármacos , Cinética , Inibidores de Lipoxigenase/síntese química , Inibidores de Lipoxigenase/farmacocinética , Camundongos , Ovinos , Estereoisomerismo , Relação Estrutura-AtividadeRESUMO
The 12/15-lipoxygenase enzymes react with fatty acids producing active lipid metabolites that are involved in a number of significant disease states. The latter include type 1 and type 2 diabetes (and associated complications), cardiovascular disease, hypertension, renal disease, and the neurological conditions Alzheimer's disease and Parkinson's disease. A number of elegant studies over the last thirty years have contributed to unraveling the role that lipoxygenases play in chronic inflammation. The development of animal models with targeted gene deletions has led to a better understanding of the role that lipoxygenases play in various conditions. Selective inhibitors of the different lipoxygenase isoforms are an active area of investigation, and will be both an important research tool and a promising therapeutic target for treating a wide spectrum of human diseases.
Assuntos
Araquidonato 12-Lipoxigenase/metabolismo , Araquidonato 15-Lipoxigenase/metabolismo , Tecido Adiposo/enzimologia , Tecido Adiposo/patologia , Animais , Vasos Sanguíneos/enzimologia , Vasos Sanguíneos/patologia , Vasos Sanguíneos/fisiologia , Vasos Sanguíneos/fisiopatologia , Doença , Humanos , Rim/enzimologia , Rim/patologia , Rim/fisiologia , Rim/fisiopatologiaRESUMO
Islet neogenesis associated protein (INGAP) stimulates experimental pancreatic islet growth, as evidenced by elevated markers of beta cell mass, in rodents, dogs and primates. Previous analyses of mice that have a transgenic expression of INGAP targeted to the exocrine pancreas have indicated additional biological activity attributed to INGAP. In this study we report on mice with a targeted expression of INGAP to the islet beta cell. The beta cell transgenic mice (IP-INGAP) showed enhanced normalization of blood glucose during IPGTT. Further, IP-INGAP mice had a significant delay in development of hyperglycemia following a diabetogenic dose of streptozotocin. INGAP conferred beta cell protection and enhanced islet function. Analysis of oxidative stress genes in IP-INGAP mice revealed a decrease in islet expression of the NADPH oxidase, NOX1, in both basal state and in response to pro-inflammatory cytokine stimulation. These data are consistent with a pleiotropic role for INGAP and reveal new pathways to target in the discovery of improved diabetic therapies.
Assuntos
Diabetes Mellitus Experimental/induzido quimicamente , Glucose/metabolismo , Hiperglicemia/induzido quimicamente , Células Secretoras de Insulina/metabolismo , Proteínas/metabolismo , Animais , Glicemia/metabolismo , Caspase 3/metabolismo , Diabetes Mellitus Experimental/metabolismo , Regulação da Expressão Gênica , Hiperglicemia/metabolismo , Insulina/metabolismo , Secreção de Insulina , Ilhotas Pancreáticas/efeitos dos fármacos , Ilhotas Pancreáticas/metabolismo , Masculino , Camundongos , Camundongos Transgênicos , NADH NADPH Oxirredutases/genética , NADPH Oxidase 1 , Proteínas Associadas a Pancreatite , Proteínas Recombinantes/metabolismo , Estreptozocina , Transcrição GênicaRESUMO
Building on the elaborate research studies that have helped map out key decision points in the process of pancreas development, reprogramming of pluripotent embryonic stem cells or induced pluripotent stem cells offers the possibility of overcoming restrictions on tissue supply associated with transplantation of donor islets. In a healthy pancreas, the beta-cell mass can exhibit significant plasticity, as reflected in the normal adaptive response in beta-cell mass to offset the metabolic challenge associated with pregnancy and obesity. In this article, alternative strategies and potential sources of pancreatic stem cells are considered.
Assuntos
Células-Tronco Embrionárias/fisiologia , Pâncreas/embriologia , Pâncreas/fisiologia , Medicina Regenerativa/métodos , Medicina Regenerativa/tendências , Animais , Diferenciação Celular/fisiologia , Desenvolvimento Embrionário/fisiologia , Feminino , Humanos , Células Secretoras de Insulina/citologia , Células Secretoras de Insulina/fisiologia , Modelos Biológicos , Pâncreas/crescimento & desenvolvimento , GravidezRESUMO
OBJECTIVE: Islet neogenesis-associated protein (INGAP) can enhance beta-cell mass to offset progression of diabetes. Identifying how transcription factors regulate INGAP gene expression could reveal key checkpoints governing islet neogenesis. METHODS: Protein complex interactions at the INGAP promoter were detected using a beta-galactosidase reporter, these protein-DNA complexes being validated in competitive electrophoresis mobility shift assays. The relevance of the revealed promoter interactions was confirmed in small interfering RNA (siRNA) gene knockdown studies. RESULTS: Pdx-1 negatively regulates stimulation of the INGAP promoter by Pan-1/NeuroD. Independently, Pdx-1, Pan-1, and NeuroD bind to the INGAP promoter as revealed by electrophoresis mobility shift assay studies. In combination, Pdx-1 selectively displaces NeuroD from a DNA-binding complex with Pan-1 to form a non-DNA-binding unit. The importance of this interaction is shown in HIT cells that have a forced reduction of Pdx-1 expression. In siRNA/Pdx-1-depleted HIT cells, the interaction of Pan-1/NeuroD with the INGAP promoter is increased 6-fold. Furthermore, endogenous INGAP expression is detected in Pdx-1-depleted cells. CONCLUSIONS: These data reveal a dynamic interaction between Pdx-1, NeuroD, and Pan-1 for the regulation of INGAP promoter activity. Modulating molecular regulators of DNA expression may be a consideration in diabetic therapies that translate exogenous stimuli into new endogenous beta-cell mass.
Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Proteínas de Homeodomínio/metabolismo , Regiões Promotoras Genéticas/genética , Transativadores/metabolismo , Animais , Antígenos de Neoplasias/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Biomarcadores Tumorais/genética , Western Blotting , Linhagem Celular , Linhagem Celular Tumoral , Cricetinae , DNA/genética , DNA/metabolismo , Ensaio de Desvio de Mobilidade Eletroforética , Proteínas de Homeodomínio/genética , Humanos , Insulinoma/genética , Insulinoma/metabolismo , Insulinoma/patologia , Lectinas Tipo C/genética , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/patologia , Proteínas Associadas a Pancreatite , Ligação Proteica , Interferência de RNA , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fatores de Transcrição TCF/genética , Fatores de Transcrição TCF/metabolismo , Transativadores/genética , Proteína 1 Semelhante ao Fator 7 de Transcrição , TransfecçãoRESUMO
OBJECTIVE: Adult islet neogenesis is believed to recapitulate elements of pancreatic endocrine development. Identifying factors that regulate islet neogenesis-associated protein (INGAP) gene activity could provide links to pancreas development. METHODS: Predicted transcriptional regulators of INGAP were screened in an INGAP-promoter-reporter assay. Based upon their temporal expression, the occurrence of INGAP-positive cells during pancreas embryonic development were studied. RESULTS: Pancreatic transcription factors, PDX-1, Ngn3, NeuroD, and Isl-1, activated the INGAP promoter, but PAX4, PAX6, and Nkx2.2 did not. The INGAP-positive cells were present in the developing pancreatic bud of the mouse embryo. Emerging clusters of unorganized endocrine cells were INGAP positive. These cells coexpressed insulin or somatostatin, but glucagon-expressing cells remained distinct. The INGAP-positive cells were also detected in the maturing neonatal endocrine cells organized into islets. In direct contrast to the embryo, glucagon localized with most INGAP-positive cells in the postnatal endocrine cells. The INGAP-positive cells juxtaposed pancreatic duct cells. A subset of 5-bromo-2'-deoxyuridine-positive/INGAP-positive cells was detected in the neonatal pancreas. CONCLUSIONS: These data implicate INGAP and/or Reg family proteins in endocrine cell patterning during embryonic development and suggest that INGAP immunoreactivity is a key marker associated with early endocrine cells.
Assuntos
Pâncreas/embriologia , Proteínas/análise , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/farmacologia , Diferenciação Celular , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Proteína Homeobox Nkx-2.2 , Proteínas de Homeodomínio/farmacologia , Humanos , Insulina/análise , Proteínas com Homeodomínio LIM , Camundongos , Proteínas do Tecido Nervoso/farmacologia , Proteínas Nucleares , Proteínas Associadas a Pancreatite , Regiões Promotoras Genéticas/efeitos dos fármacos , Regiões Promotoras Genéticas/genética , Proteínas/genética , Transativadores/farmacologia , Fatores de TranscriçãoRESUMO
The Reg-related protein family member INGAP (islet neogenesis-associated protein) is a pleiotropic factor enhancing islet neogenesis, neurite growth, beta-cell protection, and beta-cell function. Using an antibody to the N-termini of INGAP, we have identified that immunoreactivity to INGAP localized to the pancreatic endocrine cells in mouse. INGAP- and insulin-immunoreactive cells are mutually exclusive, with INGAP-immunoreactive cells being preserved after streptozotocin-mediated destruction of beta-cells. Glucagon- and INGAP-immunoreactive cells colocalize, although respective antigen expression occurs in different intracellular locations. These data suggest that INGAP-immunoreactive cells include alpha-cells; however, detection of single INGAP-immunoreactive/glucagon-negative cells indicates that this may not be exclusive. In addition to mouse, detection of islet endocrine cells that were INGAP immunoreactive/glucagon immunoreactive/insulin negative was also observed in islets from human, monkey, and rat. These findings reveal that INGAP and/or related group 3 Reg proteins have a conserved expression in the pancreatic islet.
Assuntos
Antígenos de Neoplasias/metabolismo , Biomarcadores Tumorais/metabolismo , Ilhotas Pancreáticas/metabolismo , Lectinas Tipo C/metabolismo , Proteínas/metabolismo , Animais , Reações Antígeno-Anticorpo , Linhagem Celular , Reações Cruzadas , Diabetes Mellitus Experimental/metabolismo , Glucagon/metabolismo , Células Secretoras de Glucagon/metabolismo , Haplorrinos , Humanos , Imuno-Histoquímica , Insulina/metabolismo , Espaço Intracelular/metabolismo , Masculino , Camundongos , Proteínas Associadas a Pancreatite , Transporte Proteico , Ratos , Proteínas Recombinantes/metabolismo , Especificidade da EspécieRESUMO
OBJECTIVES: Diabetes is a serious health problem. It has been proposed that islet neogenesis from pancreatic progenitor cells may restore insulin secretion in diabetic mammals. Islet neogenesis- associated protein (INGAP) stimulates islet neogenesis; therefore, we hypothesized that it would stimulate islet neogenesis in dogs. METHODS: Forty nondiabetic beagle dogs were randomly divided into 4 groups. Group 1 received daily intramuscular injections of vehicle, whereas the other 3 groups received daily INGAP injections of 0.5, 1.5, or 10 mg/kg. After 30 days, pancreatic tissues were collected, and RNA and histological sections were analyzed. RESULTS: In dogs treated with 1.5 mg/kg INGAP, there was a significant (P < 0.001) increase in the percentage of insulin-positive cells (P < 0.001) and insulin gene expression. There was a trend to increased insulin-positive cells and gene expression with treatments of 0.5 and 10 mg/kg peptide. Protein gene product 9.5-positive cells were increased with treatment. CONCLUSIONS: These results indicate that INGAP stimulates cells in the pancreatic duct epithelium of healthy dogs (putative islet progenitor cells) to develop along a neuroendocrine pathway and form new islets in response to INGAP peptide. The INGAP might be an effective therapy for diabetes.
Assuntos
Antígenos de Neoplasias/farmacologia , Biomarcadores Tumorais/farmacologia , Ilhotas Pancreáticas/citologia , Ilhotas Pancreáticas/efeitos dos fármacos , Animais , Antígenos de Neoplasias/genética , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/farmacocinética , Divisão Celular/efeitos dos fármacos , Cricetinae , Diabetes Mellitus Tipo 1/tratamento farmacológico , Diabetes Mellitus Tipo 2/tratamento farmacológico , Cães , Feminino , Imunofluorescência , Injeções Intramusculares , Injeções Intravenosas , Ilhotas Pancreáticas/metabolismo , Lectinas Tipo C/genética , Masculino , Mesocricetus , Proteínas Associadas a Pancreatite , RNA Mensageiro/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/farmacocinética , Proteínas Recombinantes de Fusão/farmacologia , Regeneração/efeitos dos fármacosRESUMO
Islet neogenesis associated protein (INGAP) is a protein factor that can stimulate new islet mass from adult pancreatic progenitor cells. In models of islet neogenesis, INGAP expression is elevated in pancreatic acinar cells. Using a transgenic model to drive a sustained expression of INGAP in pancreatic acinar cells, we have identified a protection to chemical-induced hyperglycemia. A sustained expression of INGAP during development did not perturb islet development or basal blood glucose homeostasis, although beta-cell mass and pancreatic insulin content were significantly increased in the INGAP transgenic mice. When challenged with a diabetogenic dose of streptozotocin (STZ), mice carrying the INGAP transgene did not become hyperglycemic. In contrast, wild-type mice became and remained hyperglycemic, blood glucose > 550 mg/dl. The serum insulin levels and islet morphology were preserved in the transgenic mice after STZ treatment. These data suggest that the sustained expression of INGAP in the acinar pancreas confers resistance to a diabetogenic insult. The INGAP transgenic mouse provides a new model to uncover factors that are protective to diabetes onset and biomarkers to track beta-cell pathology.