RESUMO
When the liver is injured, hepatocyte numbers decrease, while cell size, nuclear size and ploidy increase. The expansion of non-parenchymal cells such as cholangiocytes, myofibroblasts, progenitors and inflammatory cells also indicate chronic liver damage, tissue remodeling and disease progression. In this protocol, we describe a simple high-throughput approach for calculating changes in the cellular composition of the liver that are associated with injury, chronic disease and cancer. We show how information extracted from two-dimensional (2D) tissue sections can be used to quantify and calibrate hepatocyte nuclear ploidy within a sample and enable the user to locate specific ploidy subsets within the liver in situ. Our method requires access to fixed/frozen liver material, basic immunocytochemistry reagents and any standard high-content imaging platform. It serves as a powerful alternative to standard flow cytometry techniques, which require disruption of freshly collected tissue, loss of spatial information and potential disaggregation bias.
Assuntos
Núcleo Celular/metabolismo , Hepatócitos/metabolismo , Ensaios de Triagem em Larga Escala/métodos , Ploidias , Animais , Automação , Calibragem , Análise de Dados , Feminino , Citometria de Fluxo , Fluorescência , Processamento de Imagem Assistida por Computador , Fígado/metabolismo , Camundongos Endogâmicos C57BLRESUMO
AIMS/HYPOTHESIS: Non-alcoholic fatty liver disease (NAFLD) is frequently associated with type 2 diabetes mellitus. Progression of NAFLD is mediated, among other things, by activation of inflammatory pathways. In the present study, the role of the proinflammatory cytokine LIGHT (TNFSF14) was explored in NAFLD and type 2 diabetes mellitus in mice deficient for the cytokine. METHODS: Light-deficient (Light-/-) mice and WT controls were fed a regular chow diet (RCD) or a high-fat high-cholesterol diet (HFHCD) for 16 weeks. The expression of LIGHT and its receptors, herpes virus entry mediator (HVEM) and lymphotoxin ß receptor (LTßR), was investigated in both dietary regimens. Glucose tolerance, insulin sensitivity, non-alcoholic fatty liver (NAFL), systemic and tissue inflammation, and metabolic gene expression were explored in Light-/- and WT mice fed an RCD and an HFHCD. The effect of Light deficiency was also evaluated in hepatic tissue and in inflammation in HFHCD-fed Irs2+/- mice with impaired insulin signalling. RESULTS: Light deficiency did not have an effect on metabolism, in NAFL or in tissue and systemic inflammation, in RCD-fed WT mice. HVEM and LTßR were markedly increased in livers of HFHCD-fed WT mice compared with RCD-fed WT controls. In WT mice under HFHCD, Light deficiency improved glucose tolerance and insulin sensitivity. Non-alcoholic fatty liver disease activity (NAS) score, hepatic CD3+ T lymphocytes and F4/80+ macrophages were decreased in HFHCD-fed Light-/- mice compared with HFHCD-fed WT controls. Consistent with a potential role of adipose tissue in hepatic homeostasis, Light-/- mice exhibited augmented anti-inflammatory F4/80+CD206+ adipose tissue macrophages and reduced proinflammatory F4/80+CD11c+ adipose tissue macrophages. Moreover, adipose tissue explants from Light-/- mice showed diminished secretion of monocyte chemoattractant protein 1 (MCP1), TNF-α and IL-17 cytokines. Circulating Light-/- leucocytes consistently displayed augmented levels of the patrolling Ly6Clow monocytes, decreased Th9 T cell subset and diminished plasma TNF-α and IL-6 levels. Similarly, Light deficiency in Irs2+/- mice, which display impaired insulin signalling, also reduced NAFL as well as systemic and adipose tissue inflammation. Analysis of hepatic gene expression in Light-/- mouse livers showed reduced levels of Zbtb16, the transcription factor essential for natural killer T (NKT) cell function, and two genes related to NAFLD and fibrosis, Klf6 and Tlr4. CONCLUSIONS/INTERPRETATION: These results indicate that Light deficiency in HFHCD improves hepatic glucose tolerance, and reduces hepatic inflammation and NAFL. This is accompanied by decreased systemic inflammation and adipose tissue cytokine secretion and by changes in the expression of key genes such as Klf6 and Tlr4 involved in NAFLD. These results suggest that therapies to block LIGHT-dependent signalling might be useful to restore hepatic homeostasis and to restrain NAFLD.
Assuntos
Glicemia/análise , Diabetes Mellitus Tipo 2/metabolismo , Fígado Gorduroso/genética , Fígado/patologia , Membro 14 da Superfamília de Ligantes de Fatores de Necrose Tumoral/genética , Membro 14 da Superfamília de Ligantes de Fatores de Necrose Tumoral/metabolismo , Tecido Adiposo/metabolismo , Animais , Peso Corporal , Constrição Patológica/metabolismo , Citocinas/metabolismo , Progressão da Doença , Feminino , Homeostase , Inflamação/metabolismo , Fator 6 Semelhante a Kruppel/metabolismo , Fígado/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Tamanho do Órgão , Transdução de Sinais , Receptor 4 Toll-Like/metabolismoRESUMO
Involvement of IRS2 in the proliferative effects of IGF-I of follicular thyroid cells has been described, but there are no evidences for in vivo participation of IRS2. This study aimed to analyse the in vivo relevance of IRS2 in the proliferation and apoptosis of thyroid cells by immunocytochemical studies for PCNA, Ki67, and active-caspase-3 in thyroid cells of IRS2 knockout (IRS2-KO) mice, jointly to TUNEL assay. Thyroid hormones were lower in IRS2-KO mice than in their wild-type (WT) counterparts. Increases in the area, perimeter and diameter of thyroid follicles of IRS2-KO mice were observed, which also showed increased proliferation rate of follicular cells and decreased percentage of apoptotic cells that was more evident in the central than in the marginal region of the gland. Sex-related differences were also found, since the follicular epithelium height was higher in male than in female mice. The percentage of proliferating cells showed significant changes in male but not in female mice, and apoptotic cells were more abundant in female than in male IRS2-KO animals, without significant differences between WT-animals. Therefore, our results suggest that IRS2 could be involved in the maintenance of thyroid cells population and in the normal physiology of the thyroid gland.
Assuntos
Proteínas Substratos do Receptor de Insulina/metabolismo , Glândula Tireoide/metabolismo , Animais , Apoptose/fisiologia , Proliferação de Células/fisiologia , Feminino , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Transdução de Sinais/fisiologiaRESUMO
Insulin provides important information to tissues about feeding behavior and energy status. Defective insulin signaling is associated with ageing, tissue dysfunction, and impaired wound healing. In the liver, insulin resistance leads to chronic damage and fibrosis, but it is unclear how tissue-repair mechanisms integrate insulin signals to coordinate an appropriate injury response or how they are affected by insulin resistance. In this study, we demonstrate that insulin resistance impairs local cellular crosstalk between the fibrotic stroma and bipotent adult liver progenitor cells (LPCs), whose paracrine interactions promote epithelial repair and tissue remodeling. Using insulin-resistant mice deficient for insulin receptor substrate 2 (Irs2), we highlight dramatic impairment of proregenerative fibroblast growth factor 7 (Fgf7) signaling between stromal niche cells and LPCs during chronic injury. We provide a detailed account of the role played by IRS2 in promoting Fgf7 ligand and receptor (Fgfr2-IIIb) expression by the two cell compartments, and we describe an insulin/IRS2-dependent feed-forward loop capable of sustaining hepatic re-epithelialization by driving FGFR2-IIIb expression. Finally, we shed light on the regulation of IRS2 and FGF7 within the fibrotic stroma and show-using a human coculture system-that IRS2 silencing shifts the equilibrium away from paracrine epithelial repair in favor of fibrogenesis. Hence, we offer a compelling insight into the contribution of insulin resistance to the pathogenesis of chronic liver disease and propose IRS2 as a positive regulator of communication between cell types and the transition between phases of stromal to epithelial repair.
Assuntos
Doença Hepática Crônica Induzida por Substâncias e Drogas/metabolismo , Fator 7 de Crescimento de Fibroblastos/metabolismo , Proteínas Substratos do Receptor de Insulina/metabolismo , Animais , Modelos Animais de Doenças , Células Epiteliais/metabolismo , Fator 7 de Crescimento de Fibroblastos/fisiologia , Glucose/metabolismo , Humanos , Insulina/metabolismo , Proteínas Substratos do Receptor de Insulina/genética , Proteínas Substratos do Receptor de Insulina/fisiologia , Resistência à Insulina/fisiologia , Fígado/metabolismo , Camundongos , Receptores de Fatores de Crescimento de Fibroblastos/metabolismo , Transdução de Sinais/fisiologia , Células-Tronco/metabolismo , Células-Tronco/fisiologiaRESUMO
Insulin is one of the standard components used to culture primary neurospheres. Although it stimulates growth of different types of cells, the effects of insulin on adult neural stem cells (NSCs) have not been well characterized. Here, we reveal that insulin stimulates proliferation, but not survival or self-renewal, of adult NSCs. This effect is mediated by insulin receptor substrate 2 (IRS2) and subsequent activation of the protein kinase B (or Akt), leading to increased activity of the G1-phase cyclin-dependent kinase 4 (Cdk4) and cell cycle progression. Neurospheres isolated from Irs2-deficient mice are reduced in size and fail to expand in culture and this impaired proliferation is rescued by introduction of a constitutively active Cdk4 (Cdk4R24C/R24C ). More interestingly, activation of the IRS2/Akt/Cdk4 signaling pathway by insulin is also necessary for the generation in vitro of neurons and oligodendrocytes from NSCs. Furthermore, the IRS2/Cdk4 pathway is also required for neuritogenesis, an aspect of neuronal maturation that has not been previously linked to regulation of the cell cycle. Differentiation of NSCs usually follows exit from the cell cycle due to increased levels of CDK-inhibitors which prevent activation of CDKs. In contrast, our data indicate that IRS2-mediated Cdk4 activity in response to a mitogen such as insulin promotes terminal differentiation of adult NSCs. Stem Cells 2017;35:2403-2416.
Assuntos
Diferenciação Celular/efeitos dos fármacos , Quinase 4 Dependente de Ciclina/metabolismo , Insulina/farmacologia , Animais , Ciclo Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Fase G1/efeitos dos fármacos , Proteínas Substratos do Receptor de Insulina/metabolismo , Camundongos , Células-Tronco Neurais/citologia , Células-Tronco Neurais/efeitos dos fármacos , Neurogênese/efeitos dos fármacos , Fosforilação/efeitos dos fármacosRESUMO
Gap junctions are present in many cell types throughout the animal kingdom and allow fast intercellular electrical and chemical communication between neighboring cells. Connexin-36 (Cx36), the major neuronal gap junction protein, synchronizes cellular activity in the brain, but also in other organs. Here we identify a sex-specific role for Cx36 within the hypothalamic-pituitary-gonadal (HPG) axis at the level of the anterior pituitary gland (AP). We show that Cx36 is expressed in gonadotropes of the AP sustaining their synchronous activity. Cx36 ablation affects the entire downstream HPG axis in females, but not in males. We demonstrate that Cx36-mediated coupling between gonadotropes in the AP supports gonadotropin-releasing hormone-induced secretion of luteinizing hormone. Furthermore, we provide evidence for negative feedback regulation of Cx36 expression in the AP by estradiol. We thus, conclude that hormonally-controlled plasticity of gap junction communication at the level of the AP constitutes an additional mechanism affecting female reproduction.
RESUMO
Single nucleotide polymorphisms near the Ink4/Arf locus have been associated with type-2 diabetes mellitus. Previous studies indicate a protective role of the locus in the carbohydrate metabolism derangement associated with ageing in wild-type mice. The present study demonstrates that the increased Ink4/Arf locus expression in 1-year-old mice, partially-deficient for the insulin receptor substrate (IRS)2 (Irs2+/-SuperInk4/Arf mice) ameliorates hepatic steatosis, inflammation and insulin resistance. Irs2+/-SuperInk4/Arf mice displayed improved glucose tolerance and insulin sensitivity compared with Irs2+/- mice which were glucose intolerant and insulin resistant compared with age-matched wild-type mice. These changes in Irs2+/- mice were accompanied by enhanced hepatic steatosis, proinflammatory macrophage phenotype, increased Ly6C(hi)-monocyte percentage, T-lymphocyte activation and MCP1 and TNF-α cytokine levels. In Irs2+/-SuperInk4/Arf mice, steatosis and inflammatory parameters were markedly reduced and similar to those of wild-type counterparts. In vivo insulin signalling also revealed reduced activation of the IRS/AKT-dependent signalling in Irs2+/- mice. This was restored upon increased locus expression in Irs2+/-SuperInk4/Arf mice which display similar activation levels as those for wild-type mice. In vivo treatment of Irs2+/-SuperInk4/Arf mice with TNF-α diminished insulin canonical IRS/AKT-signalling and enhanced the stress SAPK/JNK-phosphoSer307IRS1-pathway suggesting that cytokine levels might potentially affect glucose homeostasis through changes in these insulin-signalling pathways. Altogether, these results indicate that enhanced Ink4/Arf locus expression restores glucose homeostasis and that this is associated with diminished hepatic steatosis and inflammation in mice with insulin resistance. Therefore, pharmacological interventions targeted to modulate the Ink4/Arf locus expression could be a tentative therapeutic approach to alleviate the inflammation associated with insulin resistance.
RESUMO
Pancreatic ß-cells play a central role in type 2 diabetes (T2D) development, which is characterized by the progressive decline of the functional ß-cell mass that is associated mainly with increased ß-cell apoptosis. Thus, understanding how to enhance survival of ß-cells is key for the management of T2D. The insulin receptor substrate-2 (IRS-2) protein is pivotal in mediating the insulin/IGF signaling pathway in ß-cells. In fact, IRS-2 is critically required for ß-cell compensation in conditions of increased insulin demand and for ß-cell survival. Tungstate is a powerful antidiabetic agent that has been shown to promote ß-cell recovery in toxin-induced diabetic rodent models. In this study, we investigated whether tungstate could prevent the onset of diabetes in a scenario of dysregulated insulin/IGF signaling and massive ß-cell death. To this end, we treated mice deficient in IRS2 (Irs2(-/-)), which exhibit severe ß-cell loss, with tungstate for 3 wk. Tungstate normalized glucose tolerance in Irs2(-/-) mice in correlation with increased ß-cell mass, increased ß-cell replication, and a striking threefold reduction in ß-cell apoptosis. Islets from treated Irs2(-/-) exhibited increased phosphorylated Erk1/2. Interestingly, tungstate repressed apoptosis-related genes in Irs2(-/-) islets in vitro, and ERK1/2 blockade abolished some of these effects. Gene expression profiling showed evidence of a broad impact of tungstate on cell death pathways in islets from Irs2(-/-) mice, consistent with reduced apoptotic rates. Our results support the finding that ß-cell death can be arrested in the absence of IRS2 and that therapies aimed at reversing ß-cell mass decline are potential strategies to prevent the progression to T2D.
Assuntos
Hipoglicemiantes/administração & dosagem , Proteínas Substratos do Receptor de Insulina/deficiência , Proteínas Substratos do Receptor de Insulina/fisiologia , Células Secretoras de Insulina/efeitos dos fármacos , Compostos de Tungstênio/administração & dosagem , Animais , Apoptose/efeitos dos fármacos , Apoptose/genética , Sobrevivência Celular/efeitos dos fármacos , Diabetes Mellitus Tipo 2/prevenção & controle , Regulação para Baixo/efeitos dos fármacos , Intolerância à Glucose/tratamento farmacológico , Células Secretoras de Insulina/fisiologia , Fígado/metabolismo , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Sistema de Sinalização das MAP Quinases/fisiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fosforilação/efeitos dos fármacos , Transdução de SinaisRESUMO
BACKGROUND: Neuroblastma cell lines contain a side-population of cells which express stemness markers. These stem-like cells may represent the potential underlying mechanism for resistance to conventional therapy and recurrence of neuroblastoma in patients. METHODOLOGY/PRINCIPAL FINDINGS: To develop novel strategies for targeting the side-population of neurobastomas, we analyzed the effects of 13-cis-retinoic acid (RA) combined with the proteasome inhibitor MG132. The short-term action of the treatment was compared with effects after a 5-day recovery period during which both chemicals were withdrawn. RA induced growth arrest and differentiation of SH-SY5Y and SK-N-BE(2) neuroblastoma cell lines. Inhibition of the proteasome caused apoptosis in both cell lines, thus, revealing the critical role of this pathway in the regulated degradation of proteins involved in neuroblastoma proliferation and survival. The combination of RA with MG132 induced apoptosis in a dose-dependent manner, in addition to promoting G2/M arrest in treated cultures. Interestingly, expression of stem cell markers such as Nestin, Sox2, and Oct4 were reduced after the recovery period of combined treatment as compared with untreated cells or treated cells with either compound alone. Consistent with this, neurosphere formation was significantly impaired by the combined treatment of RA and MG132. CONCLUSIONS: Given that stem-like cells are associated with resistant to conventional therapy and are thought to be responsible for relapse, our results suggest that dual therapy of RA and proteasome inhibitor might be beneficial for targeting the side-population of cells associated residual disease in high-risk neuroblastoma.
Assuntos
Leupeptinas/farmacologia , Células-Tronco Neoplásicas/efeitos dos fármacos , Inibidores de Proteassoma/farmacologia , Tretinoína/farmacologia , Apoptose/efeitos dos fármacos , Western Blotting , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Relação Dose-Resposta a Droga , Sinergismo Farmacológico , Citometria de Fluxo , Pontos de Checagem da Fase G2 do Ciclo Celular/efeitos dos fármacos , Humanos , Microscopia Confocal , Células-Tronco Neoplásicas/metabolismo , Células-Tronco Neoplásicas/patologia , Nestina/metabolismo , Neuroblastoma/metabolismo , Neuroblastoma/patologia , Fator 3 de Transcrição de Octâmero/metabolismo , Fatores de Transcrição SOXB1/metabolismo , Células da Side Population/efeitos dos fármacos , Células da Side Population/metabolismo , Células da Side Population/patologia , Fatores de TempoRESUMO
Insulin receptor substrate (IRS) proteins are key mediators of insulin and insulin-like growth factor (IGF) signalling. In mice, deletion of Irs1 is associated with profound growth retardation and increased longevity whereas Irs2-deficiency causes diabetes and female infertility. Clinical studies suggest that diabetes and obesity diminish male fertility. However, the role of IRS proteins in male reproduction is unknown. We observed that testis weight is reduced by 45% in Irs2-deficient mice as compared with control males. The weight of these organs in Irs1(-/-) males was similar to controls; however, since Irs1-deficient mice are 50% smaller, testis weight:body weight was increased in this model. Neonatal Irs2(-/-) mice also exhibited reduced testicular size, suggesting that impairments in this model occur during development. Histological examination of testicular cross sections from Irs2(-/-) mice revealed normal cellular associations without obvious abnormalities in the seminiferous epithelium. Reduced testicular weight was associated with fewer Sertoli cells, spermatogonia, spermatocytes, elongated spermatids, and epididymal spermatozoa. However, Leydig cell number and the concentration of serum testosterone were equivalent between Irs2-deficient and control males. Testicular weight was reduced similarly in non-diabetic and diabetic Irs2(-/-) mice, indicating that hyperglycemia does not compound the effects of Irs2 deletion on impaired testis development. Expression of Irs1, Irs3, and Irs4 was comparable between experimental groups. Collectively, our results demonstrate that IRS2 plays a critical role in testicular development, potentially by mediating IGF1 signalling during embryonic and early postnatal development.
Assuntos
Regulação da Expressão Gênica no Desenvolvimento , Proteínas Substratos do Receptor de Insulina/genética , Fator de Crescimento Insulin-Like I/genética , Organogênese/genética , Transdução de Sinais , Animais , Animais Recém-Nascidos , Peso Corporal , Diabetes Mellitus Experimental/genética , Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Experimental/patologia , Embrião de Mamíferos , Feminino , Proteínas Substratos do Receptor de Insulina/deficiência , Fator de Crescimento Insulin-Like I/metabolismo , Células Intersticiais do Testículo/citologia , Células Intersticiais do Testículo/metabolismo , Masculino , Camundongos , Camundongos Knockout , Tamanho do Órgão , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Células de Sertoli/metabolismo , Células de Sertoli/patologia , Espermátides/metabolismo , Espermátides/patologia , Espermatócitos/metabolismo , Espermatócitos/patologia , Espermatogônias/metabolismo , Espermatogônias/patologiaRESUMO
PURPOSE: Mice with deletion of insulin receptor substrate (IRS) 2 develop type 2 diabetes and photoreceptor degeneration. Loss of protein tyrosine phosphatase 1B (PTP1B) in diabetic IRS2(-/-) mice restores insulin sensitivity and normalizes glucose homeostasis. Since insulin-like growth factor (IGF)-IR promotes survival of photoreceptors and is a substrate of PTP1B, we investigated IGF-IR-mediated survival signaling and visual function in PTP1B(-/-) and double mutant IRS2(-/-)/PTP1B(-/-) mice. METHODS: IGF-IR-mediated Akt signaling was evaluated in IGF-I-stimulated retinal explants. Histologic and electroretinogram analysis was performed in wild-type (WT), IRS2(-/-), PTP1B(-/-), and the double mutant IRS2(-/-)/PTP1B(-/-) mice. RESULTS: IGF-I stimulated the tyrosine phosphorylation of its receptor and Akt activation in retinal explants of WT mice. In PTP1B(-/-) retinal explants, these responses were enhanced. Conversely, in retinas from IRS2(-/-) mice, expression of PTP1B was increased, coincident with decreased IGF-I-mediated Akt serine 473 phosphorylation. PTP1B deletion in IRS2(-/-) mice also enhanced IGF-IR tyrosine phosphorylation but, unexpectedly, did not rescue Akt activation in response to IGF-I. One potential explanation is that PTEN was increased in retinas of IRS2(-/-) and IRS2(-/-)/PTP1B(-/-) mice. Histologic evaluation revealed alterations in various structures of the retina in IRS2(-/-) and IRS2(-/-)/PTP1B(-/-) mice, specifically in the outer nuclear layer (ONL) and retinal outer segments (ROS). Electroretinogram (ERG) analysis confirmed that PTP1B deficiency did not restore visual function in IRS2(-/-) mice. CONCLUSIONS: Although loss of PTP1B enhances tyrosine phosphorylation of the IGF-IR in retinal explants of IRS2(-/-) mice, Akt activation remains defective owing to elevated PTEN levels and, thus, structural and functional visual defects persist in this model.
Assuntos
Proteínas Substratos do Receptor de Insulina/deficiência , Fator de Crescimento Insulin-Like I/metabolismo , Proteína Tirosina Fosfatase não Receptora Tipo 1/fisiologia , Receptores Proteína Tirosina Quinases/metabolismo , Receptor IGF Tipo 1/metabolismo , Doenças Retinianas/fisiopatologia , Animais , Modelos Animais de Doenças , Feminino , Resistência à Insulina/fisiologia , Masculino , Camundongos , Camundongos Knockout , Fosforilação/efeitos dos fármacos , Proteína Tirosina Fosfatase não Receptora Tipo 1/deficiência , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais/efeitos dos fármacosRESUMO
Recent genome-wide association studies have linked type-2 diabetes mellitus to a genomic region in chromosome 9p21 near the Ink4/Arf locus, which encodes tumor suppressors that are up-regulated in a variety of mammalian organs during aging. However, it is unclear whether the susceptibility to type-2 diabetes is associated with altered expression of the Ink4/Arf locus. In the present study, we investigated the role of Ink4/Arf in age-dependent alterations of insulin and glucose homeostasis using Super-Ink4/Arf mice which bear an extra copy of the entire Ink4/Arf locus. We find that, in contrast to age-matched wild-type controls, Super-Ink4/Arf mice do not develop glucose intolerance with aging. Insulin tolerance tests demonstrated increased insulin sensitivity in Super-Ink4/Arf compared with wild-type mice, which was accompanied by higher activation of the insulin receptor substrate (IRS)-PI3K-AKT pathway in liver, skeletal muscle and heart. Glucose uptake studies in Super-Ink4/Arf mice showed a tendency toward increased (18)F-fluorodeoxyglucose uptake in skeletal muscle compared with wild-type mice (P = 0.079). Furthermore, a positive correlation between glucose uptake and baseline glucose levels was observed in Super-Ink4/Arf mice (P < 0.008) but not in wild-type mice. Our studies reveal a protective role of the Ink4/Arf locus against the development of age-dependent insulin resistance and glucose intolerance.
Assuntos
Fatores de Ribosilação do ADP/genética , Inibidor p16 de Quinase Dependente de Ciclina/genética , Intolerância à Glucose , Resistência à Insulina , Fatores de Ribosilação do ADP/metabolismo , Envelhecimento/genética , Envelhecimento/metabolismo , Animais , Inibidor p16 de Quinase Dependente de Ciclina/metabolismo , Estudo de Associação Genômica Ampla , Glucose/metabolismo , Insulina/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos TransgênicosRESUMO
Insulin and insulin-like growth factor-I play important roles in the development and maintenance of neurons and glial cells of the nervous system. Both factors activate tyrosine kinase receptors, which signal through adapter proteins of the insulin receptor substrate (IRS) family. Although insulin and insulin-like growth factor-I receptors are expressed in dorsal root ganglia (DRG), the function of IRS-mediated signalling in these structures has not been studied. Here we address the role of IRS2-mediated signalling in murine DRG. Studies in cultured DRG neurons from different embryonic stages indicated that a subset of nerve growth factor-responsive neurons is also dependent on insulin for survival at very early time points. Consistent with this, increased apoptosis during gangliogenesis resulted in a partial loss of trkA-positive neurons in DRG of Irs2 mutant embryos. Analyses in adult Irs2(-/-) mice revealed that unmyelinated fibre afferents, which express calcitonin gene-related peptide/substance P and isolectin B4, as well as some myelinated afferents to the skin were affected by the mutation. The diminished innervation of glabrous skin in adult Irs2(-/-) mice correlated with longer paw withdrawal latencies in the hot-plate assay. Collectively, these findings indicate that IRS2 signalling is required for the proper development of spinal sensory neurons involved in the perception of pain.
Assuntos
Gânglios Espinais/citologia , Proteínas Substratos do Receptor de Insulina/metabolismo , Nociceptores/fisiologia , Células Receptoras Sensoriais/fisiologia , Transdução de Sinais/fisiologia , Animais , Comportamento Animal/fisiologia , Peptídeo Relacionado com Gene de Calcitonina/metabolismo , Embrião de Mamíferos/citologia , Feminino , Proteínas Substratos do Receptor de Insulina/genética , Lectinas/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Nociceptores/citologia , Medição da Dor , Gravidez , Receptor trkA/metabolismo , Células Receptoras Sensoriais/citologia , Pele/citologia , Pele/inervação , Pele/metabolismoAssuntos
Técnicas de Cultura de Células , Células-Tronco Embrionárias/fisiologia , Matriz Extracelular/metabolismo , Fibroblastos/metabolismo , Engenharia Tecidual/métodos , Adesão Celular , Movimento Celular , Células Cultivadas , Técnicas de Cocultura , Células-Tronco Embrionárias/metabolismo , Matriz Extracelular/ultraestrutura , Proteínas da Matriz Extracelular/isolamento & purificação , Proteínas da Matriz Extracelular/metabolismo , Prepúcio do Pênis/citologia , Humanos , Cariótipo , MasculinoRESUMO
The beneficial effects of insulin and insulin-like growth factor I on cognition have been documented in humans and animal models. Conversely, obesity, hyperinsulinemia, and diabetes increase the risk for neurodegenerative disorders including Alzheimer's disease (AD). However, the mechanisms by which insulin regulates synaptic plasticity are not well understood. Here, we report that complete disruption of insulin receptor substrate 2 (Irs2) in mice impairs long-term potentiation (LTP) of synaptic transmission in the hippocampus. Basal synaptic transmission and paired-pulse facilitation were similar between the 2 groups of mice. Induction of LTP by high-frequency conditioning tetanus did not activate postsynaptic N-methyl-D-aspartate (NMDA) receptors in hippocampus slices from Irs2(-/-) mice, although the expression of NR2A, NR2B, and PSD95 was equivalent to wild-type controls. Activation of Fyn, AKT, and MAPK in response to tetanus stimulation was defective in Irs2(-/-) mice. Interestingly, IRS2 was phosphorylated during induction of LTP in control mice, revealing a potential new component of the signaling machinery which modulates synaptic plasticity. Given that IRS2 expression is diminished in Type 2 diabetics as well as in AD patients, these data may reveal an explanation for the prevalence of cognitive decline in humans with metabolic disorders by providing a mechanistic link between insulin resistance and impaired synaptic transmission.
Assuntos
Proteínas Substratos do Receptor de Insulina/metabolismo , Potenciação de Longa Duração/fisiologia , Receptores de N-Metil-D-Aspartato/metabolismo , Transmissão Sináptica/fisiologia , Animais , Western Blotting , Feminino , Hipocampo/metabolismo , Imunoprecipitação , Proteínas Substratos do Receptor de Insulina/deficiência , Camundongos , Camundongos Knockout , Técnicas de Patch-ClampRESUMO
The insulin receptor substrate (IRS) proteins are key mediators of insulin and insulinlike growth factor 1 (IGF-1) signaling. Protein tyrosine phosphatase (PTP)-1B dephosphorylates and inactivates both insulin and IGF-1 receptors. IRS2-deficient mice present altered hepatic insulin signaling and ß-cell failure and develop type 2-like diabetes. In addition, IRS2 deficiency leads to developmental defects in the nervous system. IGF1 gene mutations cause syndromic sensorineural hearing loss in humans and mice. However, the involvement of IRS2 and PTP1B, two IGF-1 downstream signaling mediators, in hearing onset and loss has not been studied. Our objective was to study the hearing function and cochlear morphology of Irs2-null mice and the impact of PTP1B deficiency. We have studied the auditory brainstem responses and the cochlear morphology of systemic Irs2â»/â»Ptpn1âº/âº, Irs2âº/âºPtpn1â»/â» and Irs2â»/â»Ptpn1â»/â» mice at different postnatal ages. The results indicated that Irs2â»/â»Ptpn1âº/⺠mice present a profound congenital sensorineural deafness before the onset of diabetes and altered cochlear morphology with hypoinnervation of the cochlear ganglion and aberrant stria vascularis, compared with wild-type mice. Simultaneous PTP1B deficiency in Irs2â»/â»Ptpn1â»/â» mice delays the onset of deafness. We show for the first time that IRS2 is essential for hearing and that PTP1B inhibition may be useful for treating deafness associated with hyperglycemia and type 2 diabetes.
Assuntos
Cóclea/metabolismo , Perda Auditiva/metabolismo , Proteínas Substratos do Receptor de Insulina/deficiência , Proteína Tirosina Fosfatase não Receptora Tipo 1/deficiência , Animais , Cóclea/patologia , Cóclea/fisiopatologia , Perda Auditiva/fisiopatologia , Proteínas Substratos do Receptor de Insulina/genética , Masculino , Camundongos , Camundongos Transgênicos , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Proteína Tirosina Fosfatase não Receptora Tipo 1/genéticaRESUMO
The diabetic phenotype caused by the deletion of insulin receptor substrate-2 (Irs-2) in mice displays a sexual dimorphism. Whereas the majority of male Irs-2(-/-) mice are overtly diabetic by 12 weeks of age, female Irs-2(-/-) animals develop mild obesity and progress less rapidly to diabetes. Here we investigated ß-cell function and lipolysis as potential explanations for the gender-related differences in this model. Glucose-stimulated insulin secretion was enhanced in islets from male null mice as compared to male WT whereas this response in female Irs-2(-/-) islets was identical to that of female controls. The ability of α(2)-adrenoceptor (α(2)-AR) agonists to inhibit insulin secretion was attenuated in male Irs2 null mice. Consistent with this, the expression of the α(2A)-AR was reduced in male Irs-2(-/-) islets. The response of male Irs-2(-/-) islets to forskolin was enhanced, owing to increased production of cAMP. Basal lipolysis was increased in male Irs-2(-/-) but decreased in female Irs-2(-/-) mice, concordant with the observation that adipose tissue is sparse in males whereas female Irs2 null mice are mildly obese. Adipocytes from both male and female Irs-2(-/-) were resistant to the anti-lipolytic effects of insulin but female Irs-2(-/-) fat cells were additionally resistant to the catabolic effects of beta-adrenergic agonists. This catecholamine resistance was associated with impaired generation of cAMP. Consequently, targets of cAMP-dependent protein kinase (PKA) which mediate lipolysis were not phosphorylated in adipose tissue of female Irs-2(-/-) mice. Our findings suggest that IRS-2 deficiency in mice alters the expression and/or sensitivity of components of adrenergic signaling.
Assuntos
Agonistas de Receptores Adrenérgicos alfa 2/farmacologia , Diabetes Mellitus/genética , Diabetes Mellitus/metabolismo , Proteínas Substratos do Receptor de Insulina/genética , Proteínas Substratos do Receptor de Insulina/metabolismo , Caracteres Sexuais , Adenilil Ciclases , Adipócitos/metabolismo , Animais , Células Cultivadas , Colforsina/farmacologia , AMP Cíclico/metabolismo , Feminino , Regulação da Expressão Gênica/fisiologia , Insulina/metabolismo , Insulina/farmacologia , Ilhotas Pancreáticas/fisiologia , Lipólise/fisiologia , Masculino , Camundongos , Camundongos Knockout , Receptores Adrenérgicos alfa 2/metabolismo , Transdução de Sinais , Esterol Esterase/metabolismoRESUMO
Early development of mammalian embryos occurs in an environment of relative hypoxia. Nevertheless, human embryonic stem cells (hESC), which are derived from the inner cell mass of blastocyst, are routinely cultured under the same atmospheric conditions (21% O(2)) as somatic cells. We hypothesized that O(2) levels modulate gene expression and differentiation potential of hESC, and thus, we performed gene profiling of hESC maintained under normoxic or hypoxic (1% or 5% O(2)) conditions. Our analysis revealed that hypoxia downregulates expression of pluripotency markers in hESC but increases significantly the expression of genes associated with angio- and vasculogenesis including vascular endothelial growth factor and angiopoitein-like proteins. Consequently, we were able to efficiently differentiate hESC to functional endothelial cells (EC) by varying O(2) levels; after 24 hours at 5% O(2), more than 50% of cells were CD34+. Transplantation of resulting endothelial-like cells improved both systolic function and fractional shortening in a rodent model of myocardial infarction. Moreover, analysis of the infarcted zone revealed that transplanted EC reduced the area of fibrous scar tissue by 50%. Thus, use of hypoxic conditions to specify the endothelial lineage suggests a novel strategy for cellular therapies aimed at repair of damaged vasculature in pathologies such as cerebral ischemia and myocardial infarction.
Assuntos
Diferenciação Celular/fisiologia , Células-Tronco Embrionárias/metabolismo , Células Endoteliais/metabolismo , Regulação da Expressão Gênica/fisiologia , Oxigênio/metabolismo , Angiopoietina-1/genética , Animais , Biomarcadores/análise , Biomarcadores/metabolismo , Técnicas de Cultura de Células , Hipóxia Celular/genética , Transplante de Células/métodos , Células Cultivadas , Regulação para Baixo/genética , Células-Tronco Embrionárias/citologia , Células Endoteliais/citologia , Perfilação da Expressão Gênica/métodos , Humanos , Masculino , Infarto do Miocárdio/cirurgia , Neovascularização Fisiológica/genética , Células-Tronco Pluripotentes/citologia , Células-Tronco Pluripotentes/metabolismo , Ratos , Ratos Nus , Fator A de Crescimento do Endotélio Vascular/genéticaRESUMO
OBJECTIVE: Mice with complete deletion of insulin receptor substrate 2 (IRS2) develop hyperglycemia, impaired hepatic insulin signaling, and elevated gluconeogenesis, whereas mice deficient for protein tyrosine phosphatase (PTP)1B display an opposing hepatic phenotype characterized by increased sensitivity to insulin. To define the relationship between these two signaling pathways in the regulation of liver metabolism, we used genetic and pharmacological approaches to study the effects of inhibiting PTP1B on hepatic insulin signaling and expression of gluconeogenic enzymes in IRS2(-/-) mice. RESEARCH DESIGN AND METHODS: We analyzed glucose homeostasis and insulin signaling in liver and isolated hepatocytes from IRS2(-/-) and IRS2(-/-)/PTP1B(-/-) mice. Additionally, hepatic insulin signaling was assessed in control and IRS2(-/-) mice treated with resveratrol, an antioxidant present in red wine. RESULTS: In livers of hyperglycemic IRS2(-/-) mice, the expression levels of PTP1B and its association with the insulin receptor (IR) were increased. The absence of PTP1B in the double-mutant mice restored hepatic IRS1-mediated phosphatidylinositol (PI) 3-kinase/Akt/Foxo1 signaling. Moreover, resveratrol treatment of hyperglycemic IRS2(-/-) mice decreased hepatic PTP1B mRNA and inhibited PTP1B activity, thereby restoring IRS1-mediated PI 3-kinase/Akt/Foxo1 signaling and peripheral insulin sensitivity. CONCLUSIONS: By regulating the phosphorylation state of IR, PTB1B determines sensitivity to insulin in liver and exerts a unique role in the interplay between IRS1 and IRS2 in the modulation of hepatic insulin action.
Assuntos
Proteínas Substratos do Receptor de Insulina/genética , Proteínas Substratos do Receptor de Insulina/metabolismo , Fígado/enzimologia , Proteína Tirosina Fosfatase não Receptora Tipo 1 , Transdução de Sinais/fisiologia , Animais , Antioxidantes/farmacologia , Células Cultivadas , Feminino , Proteína Forkhead Box O1 , Fatores de Transcrição Forkhead/metabolismo , Glucosefosfato Desidrogenase/metabolismo , Hepatócitos/citologia , Hepatócitos/enzimologia , Insulina/metabolismo , Células Secretoras de Insulina/citologia , Células Secretoras de Insulina/enzimologia , Fígado/citologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Mutantes , Fenótipo , Fosfatidilinositol 3-Quinases/genética , Fosfatidilinositol 3-Quinases/metabolismo , Fosfoenolpiruvato Carboxiquinase (GTP)/metabolismo , Fosforilação/fisiologia , Proteína Tirosina Fosfatase não Receptora Tipo 1/antagonistas & inibidores , Proteína Tirosina Fosfatase não Receptora Tipo 1/genética , Proteína Tirosina Fosfatase não Receptora Tipo 1/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Resveratrol , Estilbenos/farmacologiaRESUMO
Atherosclerosis and related forms of cardiovascular disease (CVD) are associated with several genetic and environmental risk factors, including hypercholesterolemia, diabetes mellitus (DM), hypertension, obesity and smoking. Human DM is a multi-system disorder that results from progressive failure of insulin production and insulin resistance. Most diabetic patients die from complications of atherosclerosis and CVD, and DM is also associated with increased risk of restenosis post-angioplasty. Furthermore, the incidence of DM, particularly type 2-DM, is expected to increase significantly during the next decades owing to the unhealthy effects of modern life-style habits (e.g., obesity and lack of physical exercise). Thus, it is of utmost importance to develop novel preventive and therapeutic strategies to reduce the social and health-care burden of CVD and DM. Although a number of physiological alterations thought to promote atherosclerosis have been identified in diabetic patients, the precise molecular mechanisms that link DM and atherosclerosis are largely unknown. Thus, the aim of this review is to discuss current murine models of combined DM and atherosclerosis and to explore how these experimental systems are being utilized to gain mechanistic insights into diabetes-induced neointimal lesion development, as well as their potential use in evaluating the efficacy of new therapies. Our discussion includes models generated by streptozotocin treatment and those resulting from naturally occurring or targeted mutations in the mouse.