Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 28
Filtrar
Mais filtros

Base de dados
Tipo de documento
Intervalo de ano de publicação
1.
Dev Cell ; 58(22): 2428-2446.e9, 2023 11 20.
Artigo em Inglês | MEDLINE | ID: mdl-37652013

RESUMO

Thymus is necessary for lifelong immunological tolerance and immunity. It displays a distinctive epithelial complexity and undergoes age-dependent atrophy. Nonetheless, it also retains regenerative capacity, which, if harnessed appropriately, might permit rejuvenation of adaptive immunity. By characterizing cortical and medullary compartments in the human thymus at single-cell resolution, in this study we have defined specific epithelial populations, including those that share properties with bona fide stem cells (SCs) of lifelong regenerating epidermis. Thymic epithelial SCs display a distinctive transcriptional profile and phenotypic traits, including pleiotropic multilineage potency, to give rise to several cell types that were not previously considered to have shared origin. Using here identified SC markers, we have defined their cortical and medullary niches and shown that, in vitro, the cells display long-term clonal expansion and self-organizing capacity. These data substantively broaden our knowledge of SC biology and set a stage for tackling thymic atrophy and related disorders.


Assuntos
Células-Tronco , Timo , Humanos , Diferenciação Celular , Células-Tronco/metabolismo , Timo/metabolismo , Células Cultivadas , Células Epiteliais/metabolismo , Atrofia/metabolismo
2.
Biomaterials ; 301: 122203, 2023 10.
Artigo em Inglês | MEDLINE | ID: mdl-37515903

RESUMO

Lung infections are one of the leading causes of death worldwide, and this situation has been exacerbated by the emergence of COVID-19. Pre-clinical modelling of viral infections has relied on cell cultures that lack 3D structure and the context of lung extracellular matrices. Here, we propose a bioreactor-based, whole-organ lung model of viral infection. The bioreactor takes advantage of an automated system to achieve efficient decellularization of a whole rat lung, and recellularization of the scaffold using primary human bronchial cells. Automatization allowed for the dynamic culture of airway epithelial cells in a breathing-mimicking setup that led to an even distribution of lung epithelial cells throughout the distal regions. In the sealed bioreactor system, we demonstrate proof-of-concept for viral infection within the epithelialized lung by infecting primary human airway epithelial cells and subsequently injecting neutrophils. Moreover, to assess the possibility of drug screening in this model, we demonstrate the efficacy of the broad-spectrum antiviral remdesivir. This whole-organ scale lung infection model represents a step towards modelling viral infection of human cells in a 3D context, providing a powerful tool to investigate the mechanisms of the early stages of pathogenic infections and the development of effective treatment strategies for respiratory diseases.


Assuntos
COVID-19 , Pneumonia , Viroses , Ratos , Humanos , Animais , Pulmão , Células Epiteliais , Alicerces Teciduais/química
3.
Nat Cell Biol ; 5(4): 330-5, 2003 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-12640462

RESUMO

Homeostasis of blood glucose is maintained by hormone secretion from the pancreatic islets of Langerhans. Glucose stimulates insulin secretion from beta-cells but suppresses the release of glucagon, a hormone that raises blood glucose, from alpha-cells. The mechanism by which nutrients stimulate insulin secretion has been studied extensively: ATP has been identified as the main messenger and the ATP-sensitive potassium channel as an essential transducer in this process. By contrast, much less is known about the mechanisms by which nutrients modulate glucagon secretion. Here we use conventional pancreas perfusion and a transcriptional targeting strategy to analyse cell-type-specific signal transduction and the relationship between islet alpha- and beta-cells. We find that pyruvate, a glycolytic intermediate and principal substrate of mitochondria, stimulates glucagon secretion. Our analyses indicate that, although alpha-cells, like beta-cells, possess the inherent capacity to respond to nutrients, secretion from alpha-cells is normally suppressed by the simultaneous activation of beta-cells. Zinc released from beta-cells may be implicated in this suppression. Our results define the fundamental mechanisms of differential responses to identical stimuli between cells in a microorgan.


Assuntos
Comunicação Celular/fisiologia , Glucagon/metabolismo , Insulina/metabolismo , Ilhotas Pancreáticas/metabolismo , Trifosfato de Adenosina/metabolismo , Animais , Glicemia/fisiologia , Sinalização do Cálcio/fisiologia , Células Cultivadas , Glucose/metabolismo , Glucose/farmacologia , Glicólise/fisiologia , Homeostase/fisiologia , Secreção de Insulina , Ilhotas Pancreáticas/efeitos dos fármacos , Masculino , Potenciais da Membrana/fisiologia , Ácido Pirúvico/metabolismo , Ácido Pirúvico/farmacologia , Ratos , Ratos Wistar , Transdução de Sinais/fisiologia , Zinco/metabolismo , Zinco/farmacologia
4.
iScience ; 23(12): 101808, 2020 Dec 18.
Artigo em Inglês | MEDLINE | ID: mdl-33305175

RESUMO

Explosion of gene therapy approaches for treating rare monogenic and common liver disorders created an urgent need for disease models able to replicate human liver cellular environment. Available models lack 3D liver structure or are unable to survive in long-term culture. We aimed to generate and test a 3D culture system that allows long-term maintenance of human liver cell characteristics. The in vitro whole-organ "Bioreactor grown Artificial Liver Model" (BALM) employs a custom-designed bioreactor for long-term 3D culture of human induced pluripotent stem cells-derived hepatocyte-like cells (hiHEPs) in a mouse decellularized liver scaffold. Adeno-associated viral (AAV) and lentiviral (LV) vectors were introduced by intravascular injection. Substantial AAV and LV transgene expression in the BALM-grown hiHEPs was detected. Measurement of secreted proteins in the media allowed non-invasive monitoring of the system. We demonstrated that humanized whole-organ BALM is a valuable tool to generate pre-clinical data for investigational medicinal products.

5.
Nat Commun ; 11(1): 6372, 2020 12 11.
Artigo em Inglês | MEDLINE | ID: mdl-33311516

RESUMO

The thymus is a primary lymphoid organ, essential for T cell maturation and selection. There has been long-standing interest in processes underpinning thymus generation and the potential to manipulate it clinically, because alterations of thymus development or function can result in severe immunodeficiency and autoimmunity. Here, we identify epithelial-mesenchymal hybrid cells, capable of long-term expansion in vitro, and able to reconstitute an anatomic phenocopy of the native thymus, when combined with thymic interstitial cells and a natural decellularised extracellular matrix (ECM) obtained by whole thymus perfusion. This anatomical human thymus reconstruction is functional, as judged by its capacity to support mature T cell development in vivo after transplantation into humanised immunodeficient mice. These findings establish a basis for dissecting the cellular and molecular crosstalk between stroma, ECM and thymocytes, and offer practical prospects for treating congenital and acquired immunological diseases.


Assuntos
Células Estromais , Timo/imunologia , Animais , Autoimunidade , Diferenciação Celular , Células Epiteliais/imunologia , Matriz Extracelular , Feminino , Humanos , Masculino , Camundongos , Camundongos Nus , Ratos , Regeneração , Timócitos , Timo/patologia , Timo/transplante , Alicerces Teciduais
6.
Antimicrob Agents Chemother ; 53(7): 3150-2, 2009 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-19364855

RESUMO

Twice-daily 7-day regimens of tigecycline (7 mg/kg) and vancomycin (50 mg/kg) were compared in a rat tissue cage model of chronic foreign-body infection due to methicillin (meticillin)-resistant Staphylococcus aureus strain MRGR3. Subcutaneously administered tigecycline reached levels in tissue cage fluid that were nearly equivalent or slightly superior to the antibiotic MIC (0.5 microg/ml) for strain MRGR3. After 7 days, equivalent, significant reductions in bacterial counts were recorded for tigecycline-treated and vancomycin-treated rats, compared with those for untreated animals.


Assuntos
Antibacterianos/uso terapêutico , Corpos Estranhos/tratamento farmacológico , Corpos Estranhos/microbiologia , Staphylococcus aureus Resistente à Meticilina/fisiologia , Minociclina/análogos & derivados , Infecções Estafilocócicas/tratamento farmacológico , Infecções Estafilocócicas/microbiologia , Vancomicina/uso terapêutico , Animais , Minociclina/uso terapêutico , Ratos , Tigeciclina
7.
Diabetes ; 56(4): 1078-86, 2007 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-17395748

RESUMO

We studied the effect of gap junctional coupling on the excitability of beta-cells in slices of pancreas, which provide a normal environment for islet cells. The electrophysiological properties of beta-cells from mice (C57Bl/6 background) lacking the gap junction protein connexin36 (Cx36(-/-)) were compared with heterozygous (Cx36(+/-)) and wild-type littermates (Cx36(+/+)) and with frequently used wild-type NMRI mice. Most electrophysiological characteristics of beta-cells were found to be unchanged after the knockout of Cx36, except the density of Ca(2+) channels, which was increased in uncoupled cells. With closed ATP-sensitive K(+) (K(ATP)) channels, the electrically coupled beta-cells of Cx36(+/+) and Cx36(+/-) mice were hyperpolarized by the membrane potential of adjacent, inactive cells. Additionally, the hyperpolarization of one beta-cell could attenuate or even stop the electrical activity of nearby coupled cells. In contrast, beta-cells of Cx36(-/-) littermates with blocked K(ATP) channels rapidly depolarized and exhibited a continuous electrical activity. Absence of electrical coupling modified the electrophysiological properties of beta-cells consistent with the reported increase in basal insulin release and altered the switch on/off response of beta-cells during an acute drop of the glucose concentration. Our data indicate an important role for Cx36-gap junctions in modulating stimulation threshold and kinetics of insulin release.


Assuntos
Conexinas/fisiologia , Glucose/fisiologia , Células Secretoras de Insulina/fisiologia , Insulina/metabolismo , Animais , Conexinas/deficiência , Conexinas/genética , Eletrofisiologia , Secreção de Insulina , Células Secretoras de Insulina/metabolismo , Cinética , Potenciais da Membrana , Camundongos , Camundongos Endogâmicos , Camundongos Knockout , Técnicas de Patch-Clamp , Proteína delta-2 de Junções Comunicantes
8.
Sci Rep ; 8(1): 8398, 2018 05 30.
Artigo em Inglês | MEDLINE | ID: mdl-29849047

RESUMO

Pathological conditions affecting skeletal muscle function may lead to irreversible volumetric muscle loss (VML). Therapeutic approaches involving acellular matrices represent an emerging and promising strategy to promote regeneration of skeletal muscle following injury. Here we investigated the ability of three different decellularised skeletal muscle scaffolds to support muscle regeneration in a xenogeneic immune-competent model of VML, in which the EDL muscle was surgically resected. All implanted acellular matrices, used to replace the resected muscles, were able to generate functional artificial muscles by promoting host myogenic cell migration and differentiation, as well as nervous fibres, vascular networks, and satellite cell (SC) homing. However, acellular tissue mainly composed of extracellular matrix (ECM) allowed better myofibre three-dimensional (3D) organization and the restoration of SC pool, when compared to scaffolds which also preserved muscular cytoskeletal structures. Finally, we showed that fibroblasts are indispensable to promote efficient migration and myogenesis by muscle stem cells across the scaffolds in vitro. This data strongly support the use of xenogeneic acellular muscles as device to treat VML conditions in absence of donor cell implementation, as well as in vitro model for studying cell interplay during myogenesis.


Assuntos
Movimento Celular , Músculo Esquelético/citologia , Músculo Esquelético/fisiologia , Regeneração , Engenharia Tecidual , Animais , Diferenciação Celular , Camundongos , Camundongos Endogâmicos C57BL , Ratos , Células-Tronco/citologia
9.
Nat Commun ; 9(1): 4286, 2018 10 16.
Artigo em Inglês | MEDLINE | ID: mdl-30327457

RESUMO

A tissue engineered oesophagus could overcome limitations associated with oesophageal substitution. Combining decellularized scaffolds with patient-derived cells shows promise for regeneration of tissue defects. In this proof-of-principle study, a two-stage approach for generation of a bio-artificial oesophageal graft addresses some major challenges in organ engineering, namely: (i) development of multi-strata tubular structures, (ii) appropriate re-population/maturation of constructs before transplantation, (iii) cryopreservation of bio-engineered organs and (iv) in vivo pre-vascularization. The graft comprises decellularized rat oesophagus homogeneously re-populated with mesoangioblasts and fibroblasts for the muscle layer. The oesophageal muscle reaches organised maturation after dynamic culture in a bioreactor and functional integration with neural crest stem cells. Grafts are pre-vascularised in vivo in the omentum prior to mucosa reconstitution with expanded epithelial progenitors. Overall, our optimised two-stage approach produces a fully re-populated, structurally organized and pre-vascularized oesophageal substitute, which could become an alternative to current oesophageal substitutes.


Assuntos
Esôfago/citologia , Esôfago/fisiologia , Músculo Esquelético/citologia , Engenharia Tecidual/métodos , Alicerces Teciduais , Animais , Técnicas de Cultura de Células , Diferenciação Celular , Criança , Pré-Escolar , Criopreservação/métodos , Células Epiteliais , Matriz Extracelular/fisiologia , Humanos , Lactente , Recém-Nascido , Masculino , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Crista Neural/transplante , Ratos Sprague-Dawley
10.
J Clin Invest ; 113(4): 635-45, 2004 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-14966573

RESUMO

The role of the gluco-incretin hormones GIP and GLP-1 in the control of beta cell function was studied by analyzing mice with inactivation of each of these hormone receptor genes, or both. Our results demonstrate that glucose intolerance was additively increased during oral glucose absorption when both receptors were inactivated. After intraperitoneal injections, glucose intolerance was more severe in double- as compared to single-receptor KO mice, and euglycemic clamps revealed normal insulin sensitivity, suggesting a defect in insulin secretion. When assessed in vivo or in perfused pancreas, insulin secretion showed a lack of first phase in Glp-1R(-/-) but not in Gipr(-/-) mice. In perifusion experiments, however, first-phase insulin secretion was present in both types of islets. In double-KO islets, kinetics of insulin secretion was normal, but its amplitude was reduced by about 50% because of a defect distal to plasma membrane depolarization. Thus, gluco-incretin hormones control insulin secretion (a) by an acute insulinotropic effect on beta cells after oral glucose absorption (b) through the regulation, by GLP-1, of in vivo first-phase insulin secretion, probably by an action on extra-islet glucose sensors, and (c) by preserving the function of the secretory pathway, as evidenced by a beta cell autonomous secretion defect when both receptors are inactivated.


Assuntos
Polipeptídeo Inibidor Gástrico/metabolismo , Glucagon/metabolismo , Insulina/metabolismo , Fragmentos de Peptídeos/metabolismo , Precursores de Proteínas/metabolismo , Receptores dos Hormônios Gastrointestinais/metabolismo , Receptores de Glucagon/metabolismo , Animais , Glicemia/metabolismo , Carbacol/metabolismo , AMP Cíclico/metabolismo , Feminino , Peptídeo 1 Semelhante ao Glucagon , Teste de Tolerância a Glucose , Secreção de Insulina , Masculino , Camundongos , Camundongos Knockout , Pâncreas/metabolismo , Receptores dos Hormônios Gastrointestinais/genética , Receptores de Glucagon/genética
11.
PLoS One ; 12(12): e0189586, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-29261712

RESUMO

Liver transplantation is the definitive treatment of liver failure but donor organ shortage limits its availability. Stem cells are highly expandable and have the potential to differentiate into any specialist cell. Use of patient-derived induced Pluripotent Stem Cells (hiPSCs) has the additional advantage for organ regeneration therapies by removing the need for immunosuppression. We compared hepatocyte differentiation of human embryonic stem cells (hESCs) and hiPSCs in a mouse decellularised liver scaffold (3D) with standard in vitro protocol (2D). Mouse livers were decellularised preserving micro-architecture, blood vessel network and extracellular matrix. hESCs and hiPSCs were primed towards the definitive endoderm. Cells were then seeded either in 3D or 2D cultures and the hepatocyte differentiation was continued. Both hESCs and hiPSCs differentiated more efficiently in 3D than in 2D, with higher and earlier expression of mature hepatocyte marker albumin, lipid and glycogen synthesis associated with a decrease in expression of fetal hepatocyte marker alpha-fetoprotein. Thus we conclude that stem cell hepatocyte differentiation in 3D culture promotes faster cell maturation. This finding suggests that optimised 3D protocols could allow generation of mature liver cells not achieved so far in standard 2D conditions and lead to improvement in cell models of liver disease and regenerative medicine applications.


Assuntos
Diferenciação Celular , Células-Tronco Embrionárias/citologia , Hepatócitos/citologia , Células-Tronco Pluripotentes Induzidas/citologia , Alicerces Teciduais , Animais , Matriz Extracelular , Humanos , Camundongos
12.
Diabetes ; 54(6): 1808-15, 2005 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-15919803

RESUMO

Glucagon, secreted from islet alpha-cells, mobilizes liver glucose. During hyperglycemia, glucagon secretion is inhibited by paracrine factors from other islet cells, but in type 1 and type 2 diabetic patients, this suppression is lost. We investigated the effects of beta-cell secretory products zinc and insulin on isolated rat alpha-cells, intact islets, and perfused pancreata. Islet glucagon secretion was markedly zinc sensitive (IC(50) = 2.7 micromol/l) more than insulin release (IC(50) = 10.7 micromol/l). Glucose, the mitochondrial substrate pyruvate, and the ATP-sensitive K(+) channel (K(ATP) channel) inhibitor tolbutamide stimulated isolated alpha-cell electrical activity and glucagon secretion. Zinc opened K(ATP) channels and inhibited both electrical activity and pyruvate (but not arginine)-stimulated glucagon secretion in alpha-cells. Insulin transiently increased K(ATP) channel activity, inhibited electrical activity and glucagon secretion in alpha-cells, and inhibited pancreatic glucagon output. Insulin receptor and K(ATP) channel subunit transcripts were more abundant in alpha- than beta-cells. Transcript for the glucagon-like peptide 1 (GLP-1) receptor was not detected in alpha-cells nor did GLP-1 stimulate alpha-cell glucagon release. beta-Cell secretory products zinc and insulin therefore inhibit glucagon secretion most probably by direct activation of K(ATP) channels, thereby masking an alpha-cell metabolism secretion coupling pathway similar to beta-cells.


Assuntos
Glucagon/metabolismo , Ilhotas Pancreáticas/fisiologia , Canais de Potássio/fisiologia , Trifosfato de Adenosina/fisiologia , Animais , Técnicas In Vitro , Insulina/fisiologia , Ilhotas Pancreáticas/metabolismo , Masculino , Ácido Pirúvico/metabolismo , Ratos , Ratos Wistar , Zinco/fisiologia
13.
Diabetes ; 54(6): 1798-807, 2005 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-15919802

RESUMO

Normal insulin secretion requires the coordinated functioning of beta-cells within pancreatic islets. This coordination depends on a communications network that involves the interaction of beta-cells with extracellular signals and neighboring cells. In particular, adjacent beta-cells are coupled via channels made of connexin36 (Cx36). To assess the function of this protein, we investigated islets of transgenic mice in which the Cx36 gene was disrupted by homologous recombination. We observed that compared with wild-type and heterozygous littermates that expressed Cx36 and behaved as nontransgenic controls, mice homozygous for the Cx36 deletion (Cx36(-/-)) featured beta-cells devoid of gap junctions and failing to exchange microinjected Lucifer yellow. During glucose stimulation, islets of Cx36(-/-) mice did not display the regular oscillations of intracellular calcium concentrations ([Ca(2+)](i)) seen in controls due to the loss of cell-to-cell synchronization of [Ca(2+)](i) changes. The same islets did not release insulin in a pulsatile fashion, even though the overall output of the hormone in response to glucose stimulation was normal. However, under nonstimulatory conditions, islets lacking Cx36 showed increased basal release of insulin. These data show that Cx36-dependent signaling is essential for the proper functioning of beta-cells, particularly for the pulsatility of [Ca(2+)](i) and insulin secretion during glucose stimulation.


Assuntos
Cálcio/metabolismo , Conexinas/fisiologia , Glucose/fisiologia , Insulina/metabolismo , Ilhotas Pancreáticas/metabolismo , Animais , Conexinas/genética , Feminino , Heterozigoto , Homozigoto , Masculino , Camundongos , Camundongos Knockout , Proteína delta-2 de Junções Comunicantes
14.
Nat Commun ; 7: 12111, 2016 07 20.
Artigo em Inglês | MEDLINE | ID: mdl-27435297

RESUMO

Post-translational modifications are necessary for collagen precursor molecules (procollagens) to acquire final shape and function. However, the mechanism and contribution of collagen modifications that occur outside the endoplasmic reticulum and Golgi are not understood. We discovered that VIPAR, with its partner proteins, regulate sorting of lysyl hydroxylase 3 (LH3, also known as PLOD3) into newly identified post-Golgi collagen IV carriers and that VIPAR-dependent sorting is essential for modification of lysines in multiple collagen types. Identification of structural and functional collagen abnormalities in cells and tissues from patients and murine models of the autosomal recessive multisystem disorder Arthrogryposis, Renal dysfunction and Cholestasis syndrome caused by VIPAR and VPS33B deficiencies confirmed our findings. Thus, regulation of post-Golgi LH3 trafficking is essential for collagen homeostasis and for the development and function of multiple organs and tissues.


Assuntos
Colágeno/metabolismo , Complexo de Golgi/metabolismo , Homeostase , Pró-Colágeno-Lisina 2-Oxoglutarato 5-Dioxigenase/metabolismo , Animais , Artrogripose/metabolismo , Artrogripose/patologia , Modelos Animais de Doenças , Regulação da Expressão Gênica , Técnicas de Silenciamento de Genes , Complexo de Golgi/ultraestrutura , Células HEK293 , Humanos , Camundongos , Fenótipo , Ligação Proteica , Transporte Proteico , Proteínas de Transporte Vesicular/química , Proteínas de Transporte Vesicular/metabolismo , Proteínas rab de Ligação ao GTP/metabolismo , Rede trans-Golgi/metabolismo
15.
Diabetes ; 51 Suppl 1: S99-102, 2002 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-11815466

RESUMO

It is intriguing that the kinetics of glucose-stimulated insulin secretion from the in situ perfused pancreas differ between the rat and the mouse. Here we confirm that insulin release in the rat is clearly biphasic, whereas in the mouse glucose essentially elicits a transient monophasic insulin release. Glucose-derived glutamate has been suggested to participate in the full development of the secretory response. The present report shows that the expression of glutamate dehydrogenase is lower in mouse than in rat or human islets, paralleling the insulin secretion profile. Addition of glutamic acid dimethyl ester mainly enhances insulin release at an intermediate glucose concentration in the rat pancreas. In the mouse preparation, glutamic acid dimethyl ester induces a sustained secretory response, both at 7.0 and 16.7 mmol/l glucose. These results are compatible with a role for glucose-derived glutamate principally in the sustained phase of nutrient-stimulated insulin secretion.


Assuntos
Glutamatos/farmacologia , Ácido Glutâmico/metabolismo , Insulina/metabolismo , Ilhotas Pancreáticas/metabolismo , Animais , Glucose/farmacologia , Glutamato Desidrogenase/metabolismo , Humanos , Secreção de Insulina , Cinética , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Perfusão , Ratos , Ratos Wistar
16.
Endocrinology ; 143(10): 3766-72, 2002 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-12239086

RESUMO

Insulin and GH can activate common signaling elements in many tissues and cell lines. We investigated the possibility of overlap in signaling pathways activated by insulin and GH in a key target cell, the hepatocyte. In primary cultures of rat hepatocytes, GH caused a dose- and time-dependent increase in tyrosine phosphorylation of signal transducer and activator of transcription 5. This was accompanied by the induction of the mRNA encoding suppressor of cytokine signaling 2. Neither of these effects took place in companion hepatocytes challenged with insulin. By contrast, insulin caused a rapid and sustained phosphorylation of protein kinase B, accompanied by a massive induction of the mRNA encoding glucokinase. GH had no detectable effect on phosphorylation of protein kinase B or level of glucokinase mRNA. Insulin also elicited brief hyperphosphorylation of ERK1 and 2, an effect not seen in GH-stimulated hepatocytes. Thus, there was a clear demarcation of signaling events triggered in hepatocytes by insulin and GH, and this was accompanied by hormone-specific responses with respect to the induction of gene expression. Additionally, the current results show that signal transducer and activator of transcription 5 activation is neither necessary nor sufficient for the insulin-dependent induction of hepatic glucokinase.


Assuntos
Regulação da Expressão Gênica/fisiologia , Hormônio do Crescimento/fisiologia , Hepatócitos/fisiologia , Insulina/fisiologia , Proteínas do Leite , Proteínas Serina-Treonina Quinases , Proteínas Repressoras , Transdução de Sinais/fisiologia , Animais , Células Cultivadas , Proteínas de Ligação a DNA/metabolismo , Relação Dose-Resposta a Droga , Expressão Gênica/efeitos dos fármacos , Glucoquinase/genética , Hormônio do Crescimento/administração & dosagem , Hormônio do Crescimento/farmacologia , Hepatócitos/efeitos dos fármacos , Insulina/farmacologia , Masculino , Fosforilação/efeitos dos fármacos , Proteínas/genética , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas c-akt , RNA Mensageiro/metabolismo , Ratos , Ratos Wistar , Fator de Transcrição STAT5 , Proteínas Supressoras da Sinalização de Citocina , Transativadores/metabolismo
17.
Diabetes ; 62(10): 3488-99, 2013 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-23863811

RESUMO

Prohibitins are highly conserved proteins mainly implicated in the maintenance of mitochondrial function and architecture. Their dysfunctions are associated with aging, cancer, obesity, and inflammation. However, their possible role in pancreatic ß-cells remains unknown. The current study documents the expression of prohibitins in human and rodent islets and their key role for ß-cell function and survival. Ablation of Phb2 in mouse ß-cells sequentially resulted in impairment of mitochondrial function and insulin secretion, loss of ß-cells, progressive alteration of glucose homeostasis, and, ultimately, severe diabetes. Remarkably, these events progressed over a 3-week period of time after weaning. Defective insulin supply in ß-Phb2(-/-) mice was contributed by both ß-cell dysfunction and apoptosis, temporarily compensated by increased ß-cell proliferation. At the molecular level, we observed that deletion of Phb2 caused mitochondrial abnormalities, including reduction of mitochondrial DNA copy number and respiratory chain complex IV levels, altered mitochondrial activity, cleavage of L-optic atrophy 1, and mitochondrial fragmentation. Overall, our data demonstrate that Phb2 is essential for metabolic activation of mitochondria and, as a consequence, for function and survival of ß-cells.


Assuntos
DNA Mitocondrial/metabolismo , Diabetes Mellitus Tipo 2/metabolismo , Células Secretoras de Insulina/metabolismo , Mitocôndrias/metabolismo , Proteínas Repressoras/metabolismo , Animais , Apoptose , Glicemia/metabolismo , Proliferação de Células , Sobrevivência Celular , DNA Mitocondrial/genética , Diabetes Mellitus Tipo 2/genética , Progressão da Doença , Feminino , GTP Fosfo-Hidrolases/metabolismo , Deleção de Genes , Humanos , Insulina/metabolismo , Secreção de Insulina , Masculino , Camundongos , Camundongos Knockout , Mitocôndrias/genética , Proibitinas , Proteínas Repressoras/genética
18.
PLoS One ; 7(9): e45844, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-23029270

RESUMO

The absence of the transcriptional repressor RE-1 Silencing Transcription Factor (REST) in insulin-secreting beta cells is a major cue for the specific expression of a large number of genes. These REST target genes were largely ascribed to a function of neurotransmission in a neuronal context, whereas their role in pancreatic beta cells has been poorly explored. To identify their functional significance, we have generated transgenic mice expressing REST in beta cells (RIP-REST mice), and previously discovered that REST target genes are essential to insulin exocytosis. Herein we characterized a novel line of RIP-REST mice featuring diabetes. In diabetic RIP-REST mice, high levels of REST were associated with postnatal beta cell apoptosis, which resulted in gradual beta cell loss and sustained hyperglycemia in adults. Moreover, adenoviral REST transduction in INS-1E cells led to increased cell death under control conditions, and sensitized cells to death induced by cytokines. Screening for REST target genes identified several anti-apoptotic genes bearing the binding motif RE-1 that were downregulated upon REST expression in INS-1E cells, including Gjd2, Mapk8ip1, Irs2, Ptprn, and Cdk5r2. Decreased levels of Cdk5r2 in beta cells of RIP-REST mice further confirmed that it is controlled by REST, in vivo. Using siRNA-mediated knock-down in INS-1E cells, we showed that Cdk5r2 protects beta cells against cytokines and palmitate-induced apoptosis. Together, these data document that a set of REST target genes, including Cdk5r2, is important for beta cell survival.


Assuntos
Proteínas de Transporte/genética , Sobrevivência Celular , Regulação da Expressão Gênica , Células Secretoras de Insulina/fisiologia , Proteínas Repressoras/fisiologia , Animais , Apoptose , Sequência de Bases , Proteínas de Transporte/metabolismo , Linhagem Celular Tumoral , Sequência Consenso , Proteínas do Citoesqueleto , Diabetes Mellitus/metabolismo , Diabetes Mellitus/patologia , Expressão Gênica , Técnicas de Silenciamento de Genes , Glucose/metabolismo , Homeostase , Hiperglicemia/metabolismo , Hiperglicemia/patologia , Insulina/metabolismo , Secreção de Insulina , Células Secretoras de Insulina/metabolismo , Proteínas Ligadas a Lipídeos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Pâncreas/metabolismo , Pâncreas/patologia , Interferência de RNA , Ratos , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Elementos de Resposta
19.
Nutr Metab (Lond) ; 8(1): 2, 2011 Jan 18.
Artigo em Inglês | MEDLINE | ID: mdl-21244699

RESUMO

Current notions about mechanisms by which catch-up growth predisposes to later type 2 diabetes center upon those that link hyperinsulinemia with an accelerated rate of fat deposition (catch-up fat). Using a rat model of semistarvation-refeeding in which catch-up fat is driven solely by elevated metabolic efficiency associated with hyperinsulinemia, we previously reported that insulin-stimulated glucose utilization is diminished in skeletal muscle but increased in white adipose tissue. Here, we investigated the possibility that hyperinsulinemia during catch-up fat can be contributed by changes in the secretory response of pancreatic beta-cells to glucose. Using the rat model of semistarvation-refeeding showing catch-up fat and hyperinsulinemia, we compared isocalorically refed and control groups for potential differences in pancreatic morphology and in glucose-stimulated insulin secretion during in situ pancreas perfusions as well as ex vivo isolated islet perifusions. Between refed and control animals, no differences were found in islet morphology, insulin content, and the secretory responses of perifused isolated islets upon glucose stimulation. By contrast, the rates of insulin secretion from in situ perfused pancreas showed that raising glucose from 2.8 to 16.7 mmol/l produced a much more pronounced increase in insulin release in refed than in control groups (p < 0.01). These results indicate a role for islet secretory hyperresponsiveness to glucose in the thrifty mechanisms that drive catch-up fat through glucose redistribution between skeletal muscle and adipose tissue. Such beta-cell hyperresponsiveness to glucose may be a key event in the link between catch-up growth, hyperinsulinemia and risks for later type 2 diabetes.

20.
Mol Cell Endocrinol ; 338(1-2): 46-57, 2011 May 16.
Artigo em Inglês | MEDLINE | ID: mdl-21371522

RESUMO

Glutamate is generated during nutrient stimulation of pancreatic islets and has been proposed to act both as an intra- and extra-cellular messenger molecule. We demonstrate that glutamate is not co-secreted with the hormones from intact islets or purified α- and ß-cells. Fractional glutamate release was 5-50 times higher than hormone secretion. Furthermore, various hormone secretagogues did not elicit glutamate efflux. Interestingly, epinephrine even decreased glutamate release while increasing glucagon secretion. Rather than being co-secreted with hormones, we show that glutamate is mainly released via plasma membrane excitatory amino acid transporters (EAAT) by uptake reversal. Transcripts for EAAT1, 2 and 3 were present in both rat α- and ß-cells. Inhibition of EAATs by L-trans-pyrrolidine-2,4-dicarboxylate augmented intra-cellular glutamate and α-ketoglutarate contents and potentiated glucose-stimulated insulin secretion from islets and purified ß-cells without affecting glucagon secretion from α-cells. In conclusion, intra-cellular glutamate-derived metabolite pools are linked to glucose-stimulated insulin but not glucagon secretion.


Assuntos
Células Secretoras de Glucagon/metabolismo , Glucagon/metabolismo , Proteínas de Transporte de Glutamato da Membrana Plasmática/antagonistas & inibidores , Ácido Glutâmico/metabolismo , Células Secretoras de Insulina/metabolismo , Insulina/metabolismo , Alanina/metabolismo , Animais , Ácido Aspártico/metabolismo , Membrana Celular/metabolismo , Células Cultivadas , Ácidos Dicarboxílicos/farmacologia , Epinefrina/farmacologia , Glucose/metabolismo , Glucose/farmacologia , Proteínas de Transporte de Glutamato da Membrana Plasmática/genética , Proteínas de Transporte de Glutamato da Membrana Plasmática/metabolismo , Glutamina/metabolismo , Secreção de Insulina , Ácidos Cetoglutáricos/metabolismo , Malatos/metabolismo , Masculino , Pirrolidinas/farmacologia , Ratos , Ratos Wistar , Transcrição Gênica , Ácido gama-Aminobutírico/metabolismo
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA