RESUMO
Novel strategies in diabetes therapy would obviously benefit from the use of beta (beta) cell stem/progenitor cells. However, whether or not adult beta cell progenitors exist is one of the most controversial issues in today's diabetes research. Guided by the expression of Neurogenin 3 (Ngn3), the earliest islet cell-specific transcription factor in embryonic development, we show that beta cell progenitors can be activated in injured adult mouse pancreas and are located in the ductal lining. Differentiation of the adult progenitors is Ngn3 dependent and gives rise to all islet cell types, including glucose responsive beta cells that subsequently proliferate, both in situ and when cultured in embryonic pancreas explants. Multipotent progenitor cells thus exist in the pancreas of adult mice and can be activated cell autonomously to increase the functional beta cell mass by differentiation and proliferation rather than by self-duplication of pre-existing beta cells only.
Assuntos
Células Secretoras de Insulina/citologia , Pâncreas/citologia , Pâncreas/lesões , Células-Tronco/citologia , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/isolamento & purificação , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Diferenciação Celular , Núcleo Celular/metabolismo , Proliferação de Células , Expressão Gênica , Genes Reporter , Vetores Genéticos , Proteínas de Fluorescência Verde/metabolismo , Imuno-Histoquímica , Insulina/análise , Insulina/metabolismo , Células Secretoras de Insulina/metabolismo , Queratinas/metabolismo , Lentivirus/genética , Ligadura , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Transgênicos , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/isolamento & purificação , Proteínas do Tecido Nervoso/metabolismo , Técnicas de Cultura de Órgãos , Ductos Pancreáticos/cirurgia , Células-Tronco/metabolismo , Fatores de Tempo , beta-Galactosidase/metabolismoRESUMO
Clinical evidence suggests that patients with chronic pancreatitis (CP) are prone to development of diabetes (chronic pancreatitis-related diabetes; CPRD), whereas the underlying mechanisms are not fully determined. Recently, we showed that the gradual loss of functional beta-cells in a mouse model for CPRD, partial pancreatic duct ligation (PDL), results from a transforming growth factor ß1 (TGFß1)-triggered beta-cell epithelial-mesenchymal transition (EMT), rather than from apoptotic beta-cell death. Here, the role of angiogenesis in CPRD-associated beta-cell EMT was addressed. We detected enhanced angiogenesis in the inflamed pancreas from CP patients by bioinformatic analysis and from PDL-mice. Inhibition of angiogenesis by specific antisera for vascular endothelial growth factor receptor 2 (VEGFR2), DC101, did not alter the loss of beta-cells and the fibrotic process in PDL-pancreas. However, DC101-mediated inhibition of angiogenesis abolished pancreatitis-induced beta-cell EMT and rendered it to apoptotic beta-cell death. Thus, our data suggest that angiogenesis promotes beta-cell survival in the inflamed pancreas, while suppression of angiogenesis turns beta-cell EMT into apoptotic beta-cell death. This finding could be informative during development of intervention therapies for CPRD.
Assuntos
Diabetes Mellitus/genética , Transição Epitelial-Mesenquimal/genética , Células Secretoras de Insulina/metabolismo , Neovascularização Patológica/genética , Pancreatite Crônica/genética , Animais , Anticorpos Monoclonais/farmacologia , Apoptose/efeitos dos fármacos , Apoptose/genética , Diabetes Mellitus/etiologia , Diabetes Mellitus/metabolismo , Modelos Animais de Doenças , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Feminino , Perfilação da Expressão Gênica/métodos , Humanos , Insulina/metabolismo , Células Secretoras de Insulina/efeitos dos fármacos , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Neovascularização Patológica/metabolismo , Neovascularização Patológica/prevenção & controle , Pancreatite Crônica/complicações , Pancreatite Crônica/metabolismo , Molécula-1 de Adesão Celular Endotelial a Plaquetas/metabolismoRESUMO
The Cre/loxP system has been used extensively in mouse models with a limitation of one lineage at a time. Differences in function and other properties among populations of adult ß-cells is termed ß-cell heterogeneity, which was recently associated with diabetic phenotypes. Nevertheless, the presence of a developmentally derived ß-cell heterogeneity is unclear. Here, we have developed a novel dual lineage-tracing technology, using a combination of two recombinase systems, Dre/RoxP and Cre/LoxP, to independently trace green fluorescent Pdx1-lineage cells and red fluorescent Ptf1a-lineage cells in the developing and adult mouse pancreas. We detected a few Pdx1+/Ptf1a- lineage cells in addition to the vast majority of Pdx1+/Ptf1a+ lineage cells in the pancreas. Moreover, Pdx1+/Ptf1a+ lineage ß-cells had fewer Ki-67+ proliferating ß-cells, and expressed higher mRNA levels of insulin, Glut2, Pdx1, MafA and Nkx6.1, but lower CCND1 and CDK4 levels, compared with Pdx1+/Ptf1a- lineage ß-cells. Furthermore, more TSQ-high, SSC-high cells were detected in the Pdx1+Ptf1a+ lineage population than in the Pdx1+Ptf1a- lineage population. Together, these data suggest that differential activation of Ptf1a in the developing pancreas may correlate with this ß-cell heterogeneity.
Assuntos
Linhagem da Célula , Rastreamento de Células/métodos , Células Secretoras de Insulina/citologia , Pâncreas/citologia , Células-Tronco/citologia , Animais , Diferenciação Celular/genética , Linhagem da Célula/genética , Separação Celular/métodos , Células Cultivadas , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Células Secretoras de Insulina/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Imagem Molecular/métodos , Organogênese/genética , Pâncreas/embriologia , Pâncreas/crescimento & desenvolvimento , Pâncreas/metabolismo , Células-Tronco/metabolismo , Transativadores/genética , Transativadores/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
AIM: To show that depletion of pancreatic macrophages impairs gestational beta cell proliferation and leads to glucose intolerance. MATERIALS AND METHODS: Genetic animal models were applied to study the effects of depletion of pancreatic macrophges on gestational beta-cell proliferaiton and glucose response. The crosstalk between macrophages and beta-cells was studied in vivo using beta-cell-specific extracellular-signal-regulated kinase 5 (ERK5) knockout and epidermal growth receptor (EGFR) knockout mice, and in vitro using a co-culture system. RESULTS: Beta cell-derived placental growth factor (PlGF) recruited naïve macrophages and polarized them towards an M2-like phenotype. These macrophages then secreted epidermal growth factor (EGF), which activated extracellular signal-regulated kinase 5 (ERK5) signalling in beta cells to promote gestational beta cell proliferation. On the other hand, activation of ERK5 signalling in beta cells likely, in turn, enhanced the production and secretion of PlGF by beta cells. CONCLUSIONS: Our study shows a regulatory loop between macrophages and beta cells through PlGF/EGF/ERK5 signalling cascades to regulate gestational beta cell growth.
Assuntos
Fator de Crescimento Epidérmico , Proteína Quinase 7 Ativada por Mitógeno , Animais , Proliferação de Células , Fator de Crescimento Epidérmico/metabolismo , Fator de Crescimento Epidérmico/farmacologia , Feminino , Macrófagos/metabolismo , Camundongos , Proteína Quinase 7 Ativada por Mitógeno/metabolismo , Fator de Crescimento Placentário/metabolismoRESUMO
BACKGROUND & AIMS: Calcineurin is a ubiquitously expressed central Ca2+-responsive signaling molecule that mediates acute pancreatitis, but little is known about its effects. We compared the effects of calcineurin expression by hematopoietic cells vs pancreas in mouse models of pancreatitis and pancreatitis-associated lung inflammation. METHODS: We performed studies with mice with hematopoietic-specific or pancreas-specific deletion of protein phosphatase 3, regulatory subunit B, alpha isoform (PPP3R1, also called CNB1), in mice with deletion of CNB1 (Cnb1UBCâ³/â³) and in the corresponding controls for each deletion of CNB1. Acute pancreatitis was induced in mice by administration of caerulein or high-pressure infusion of radiocontrast into biliopancreatic ducts; some mice were also given intraductal infusions of an adeno-associated virus vector that expressed nuclear factor of activated T -cells (NFAT)-luciferase into pancreas. Pancreas, bone marrow, liver, kidney, heart, and lung were collected and analyzed by histopathology, immunohistochemistry, and immunoblots; levels of cytokines were measured in serum. Mouse and human primary pancreatic acinar cells were transfected with a vector that expressed NFAT-luciferase and incubated with an agent that blocks interaction of NFAT with calcineurin; cells were analyzed by immunofluorescence. Calcineurin-mediated neutrophil chemotaxis and reactive oxygen species production were measured in neutrophils from mice. RESULTS: Mice with hematopoietic-specific deletion of CNB1 developed the same level of local pancreatic inflammation as control mice after administration of caerulein or infusion of radiocontrast into biliopancreatic ducts. Cnb1UBCâ³/â³ mice or mice with pancreas-specific deletion of CNB1 developed less severe pancreatitis and reduced pancreatic inflammation after administration of caerulein or infusion of radiocontrast into biliopancreatic ducts compared with control mice. NFAT was activated in pancreas of Swiss Webster mice given caerulein or infusions of radiocontrast into biliopancreatic ducts. Blocking the interaction between calcineurin and NFAT did not reduce pancreatic acinar cell necrosis in response to caerulein or infusions of radiocontrast. Mice with hematopoietic-specific deletion of CNB1 (but not mice with pancreas-specific deletion of CNB1) had reduced infiltration of lung tissues by neutrophils. Neutrophil chemotaxis and production of reactive oxygen species were decreased after incubation with a calcineurin inhibitor. CONCLUSIONS: Hematopoietic and neutrophil expression of calcineurin promotes pancreatitis-associated lung inflammation, whereas pancreatic calcineurin promotes local pancreatic inflammation. The findings indicate that the protective effects of blocking or deleting calcineurin on pancreatitis are mediated by the source of its expression. This information should be used in the development of strategies to inhibit calcineurin for the prevention of pancreatitis and pancreatitis-associated lung inflammation.
Assuntos
Lesão Pulmonar Aguda/imunologia , Inibidores de Calcineurina/uso terapêutico , Calcineurina/metabolismo , Proteínas de Ligação ao Cálcio/metabolismo , Proteínas Musculares/metabolismo , Pancreatite/imunologia , Células Acinares/metabolismo , Lesão Pulmonar Aguda/sangue , Lesão Pulmonar Aguda/patologia , Lesão Pulmonar Aguda/prevenção & controle , Animais , Células da Medula Óssea/imunologia , Células da Medula Óssea/metabolismo , Calcineurina/genética , Calcineurina/imunologia , Proteínas de Ligação ao Cálcio/genética , Células Cultivadas , Ceruletídeo/administração & dosagem , Ceruletídeo/toxicidade , Citocinas/sangue , Citocinas/metabolismo , Modelos Animais de Doenças , Feminino , Humanos , Masculino , Camundongos , Camundongos Transgênicos , Proteínas Musculares/genética , Fatores de Transcrição NFATC/antagonistas & inibidores , Fatores de Transcrição NFATC/metabolismo , Neutrófilos/imunologia , Neutrófilos/metabolismo , Pâncreas/citologia , Pâncreas/imunologia , Pâncreas/metabolismo , Pancreatite/induzido quimicamente , Pancreatite/complicações , Pancreatite/tratamento farmacológico , Cultura Primária de CélulasRESUMO
BACKGROUND & AIMS: Pancreatitis after endoscopic retrograde cholangiopancreatography (PEP) is thought to be provoked by pancreatic ductal hypertension, via unknown mechanisms. We investigated the effects of hydrostatic pressures on the development of pancreatitis in mice. METHODS: We performed studies with Swiss Webster mice, B6129 mice (controls), and B6129 mice with disruption of the protein phosphatase 3, catalytic subunit, ßisoform gene (Cnab-/- mice). Acute pancreatitis was induced in mice by retrograde biliopancreatic ductal or intraductal infusion of saline with a constant hydrostatic pressure while the proximal common bile duct was clamped -these mice were used as a model of PEP. Some mice were given pancreatic infusions of adeno-associated virus 6-nuclear factor of activated T-cells-luciferase to monitor calcineurin activity or the calcineurin inhibitor FK506. Blood samples and pancreas were collected at 6 and 24 hours and analyzed by enzyme-linked immunosorbent assay, histology, immunohistochemistry, or fluorescence microscopy. Ca2+ signaling and mitochondrial permeability were measured in pancreatic acinar cells isolated 15 minutes after PEP induction. Ca2+-activated phosphatase calcineurin within the pancreas was tracked in vivo over 24 hours. RESULTS: Intraductal pressures of up to 130 mm Hg were observed in the previously reported model of PEP; we found that application of hydrostatic pressures of 100 and 150 mm Hg for 10 minutes consistently induced pancreatitis. Pancreatic tissues had markers of inflammation (increased levels of interleukin [IL] 6, IL1B, and tumor necrosis factor), activation of signal transducer and activator of transcription 3, increased serum amylase and IL6, and loss of tight junction integrity. Transiently high pressures dysregulated Ca2+ processing (reduced Ca2+ oscillations and an increased peak plateau Ca2+ signal) and reduced the mitochondrial membrane potential. We observed activation of pancreatic calcineurin in the pancreas in mice. Cnab-/- mice, which lack the catalytic subunit of calcineurin, and mice given FK506 did not develop pressure-induced pancreatic inflammation, edema, or loss of tight junction integrity. CONCLUSIONS: Transient high ductal pressure produces pancreatic inflammation and loss of tight junction integrity in a mouse model of PEP. These processes require calcineurin signaling. Calcineurin inhibitors might be used to prevent acute pancreatitis that results from obstruction.
Assuntos
Ampola Hepatopancreática/enzimologia , Calcineurina/metabolismo , Sinalização do Cálcio , Mecanotransdução Celular , Pancreatite/enzimologia , Junções Íntimas/enzimologia , Ampola Hepatopancreática/efeitos dos fármacos , Ampola Hepatopancreática/patologia , Amilases/sangue , Animais , Calcineurina/deficiência , Calcineurina/genética , Inibidores de Calcineurina/farmacologia , Sinalização do Cálcio/efeitos dos fármacos , Colangiopancreatografia Retrógrada Endoscópica , Modelos Animais de Doenças , Feminino , Pressão Hidrostática , Interleucina-1beta/metabolismo , Interleucina-6/sangue , Masculino , Mecanotransdução Celular/efeitos dos fármacos , Potencial da Membrana Mitocondrial , Camundongos Knockout , Mitocôndrias/metabolismo , Pancreatite/etiologia , Pancreatite/patologia , Pancreatite/prevenção & controle , Fator de Transcrição STAT3/metabolismo , Tacrolimo/farmacologia , Junções Íntimas/efeitos dos fármacos , Junções Íntimas/patologia , Fatores de Tempo , Fator de Necrose Tumoral alfa/metabolismoRESUMO
The mechanisms underlying the effects of exocrine dysfunction on the development of diabetes remain largely unknown. Here we show that pancreatic depletion of SMAD7 resulted in age-dependent increases in ß cell dysfunction with accelerated glucose intolerance, followed by overt diabetes. The accelerated ß cell dysfunction and loss of proliferation capacity, two features of ß cell aging, appeared to be non-cell-autonomous, secondary to the adjacent exocrine failure as a "bystander effect." Increased Forkhead box protein 1 (FoxO1) acetylation and nuclear retention was followed by progressive FoxO1 loss in ß cells that marked the onset of diabetes. Moreover, forced FoxO1 expression in ß cells prevented ß cell dysfunction and loss in this model. Thus, we present a model of accelerated ß cell aging that may be useful for studying the mechanisms underlying ß cell failure in diabetes. Moreover, we provide evidence highlighting a critical role of FoxO1 in maintaining ß cell identity in the context of SMAD7 failure.
Assuntos
Diabetes Mellitus/metabolismo , Proteína Forkhead Box O1/metabolismo , Células Secretoras de Insulina/patologia , Proteína Smad7/metabolismo , Animais , Proliferação de Células , Senescência Celular , Diabetes Mellitus/genética , Diabetes Mellitus/patologia , Proteína Forkhead Box O1/genética , Células Secretoras de Insulina/metabolismo , Masculino , Camundongos , Camundongos Knockout , Camundongos SCID , Mutação , Pâncreas/metabolismo , Pâncreas/patologia , Transporte Proteico , Proteína Smad7/genéticaRESUMO
Diabetic retinopathy (DR), a major complication of diabetes caused by vascular damage and pathological proliferation of retinal vessels, often progresses to vision loss. Vascular endothelial growth factor (VEGF) signaling plays a pivotal role in the development of DR, but the exact underlying molecular mechanisms remain ill-defined. Cellular prion protein (PrPc) is a surface protein expressed by vascular endothelial cells, and the increased expression of PrPc is associated with physiological and pathological vascularization. Nevertheless, a role for PrPc in the development of DR has not been appreciated. Here, we addressed this question. We found that the development of streptozocin (STZ)-induced DR, but not the STZ-induced hyperglycemia/diabetes itself, was significantly attenuated in PrPc-KO mice, compared to control wildtype (WT) mice, evident by measurement of retinal vascular leakage, retinal neovascularization, a retinopathy score and visual acuity assessment. Moreover, the attenuation of DR severity seemingly resulted from attenuation of retinal neovascularization via VEGF/ras/rac signaling. Together, our study suggests a previously unappreciated role for PrPc in the development of DR.
Assuntos
Diabetes Mellitus Experimental/metabolismo , Retinopatia Diabética/metabolismo , Proteínas PrPC/metabolismo , Neovascularização Retiniana/metabolismo , Animais , Diabetes Mellitus Experimental/genética , Diabetes Mellitus Experimental/patologia , Retinopatia Diabética/genética , Retinopatia Diabética/patologia , Camundongos , Camundongos Knockout , Proteínas PrPC/genética , Neovascularização Retiniana/genética , Neovascularização Retiniana/patologiaRESUMO
A series of chalcones containing naphthalene moiety 4a-4p have been synthesized, characterized by 1H NMR and 13C NMR and evaluated for their in vitro anticancer activity. The majority of the screened compounds displayed potent anticancer activity against both HCT116 and HepG2 human cancer cell lines. Among the series, compound 4h with a diethylamino group at the para position of the phenyl ring exhibited the most potent anticancer activity against HCT116 and HepG2 cell lines with IC50 values of 1.20⯱â¯0.07 and 1.02⯱â¯0.04⯵M, respectively. The preliminary structure-activity relationship has been summarized. Tubulin polymerization experiments indicated that 4h effectively inhibited tubulin polymerization and flow cytometric assay revealed that 4h arrests HepG2 cells at the G2/M phase in a dose-dependent manner. Furthermore, molecular docking studies suggested that 4h binds to the colchicine binding site of tubulin.
Assuntos
Antineoplásicos/farmacologia , Chalconas/farmacologia , Naftalenos/farmacologia , Antineoplásicos/síntese química , Antineoplásicos/química , Antineoplásicos/metabolismo , Sítios de Ligação , Linhagem Celular Tumoral , Chalconas/síntese química , Chalconas/química , Chalconas/metabolismo , Ensaios de Seleção de Medicamentos Antitumorais , Pontos de Checagem da Fase G2 do Ciclo Celular/efeitos dos fármacos , Humanos , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Estrutura Molecular , Naftalenos/síntese química , Naftalenos/química , Naftalenos/metabolismo , Ligação Proteica , Relação Estrutura-Atividade , Tubulina (Proteína)/química , Tubulina (Proteína)/metabolismo , Moduladores de Tubulina/síntese química , Moduladores de Tubulina/química , Moduladores de Tubulina/metabolismo , Moduladores de Tubulina/farmacologiaRESUMO
Diabetes is a global epidemic and affects millions of individuals in the United States. Devising novel treatments for diabetes continues to be a great medical challenge. Postnatal beta cell growth or compensation is largely attributed to beta cell proliferation, which declines continuously with age. To boost beta cell proliferation to regenerate an adequate functional mass, there is a need to understand the signaling pathways that regulate beta cell proliferation for creating practical strategies to promote the process. Transforming growth factor ß (TGFß) belongs to a signaling superfamily that governs pancreatic development and the regeneration of beta cells after pancreatic diseases. TGFß exerts its functions by activation of downstream Smad proteins and through its crosstalk with other pathways. Accumulating data demonstrate that the TGFß receptor signaling pathway also participates in the control of beta cell proliferation. This review details the role of the TGFß receptor signaling pathway in beta cell proliferation physiologically and in the pathogenesis of diabetes.
Assuntos
Células Secretoras de Insulina/citologia , Receptores de Fatores de Crescimento Transformadores beta/metabolismo , Proteínas Smad/metabolismo , Fator de Crescimento Transformador beta/metabolismo , Adulto , Animais , Ciclo Celular , Proliferação de Células , Humanos , Células Secretoras de Insulina/metabolismo , Transdução de SinaisRESUMO
AIMS/HYPOTHESIS: The Cre/loxP system, which enables tissue-specific manipulation of genes, is widely used in mice for diabetes research. Our aim was to develop a new Cre-driver mouse line for the specific and efficient manipulation of genes in pancreatic alpha cells. METHODS: A Gcg CreERT2 knockin mouse, which expresses a tamoxifen-inducible form of Cre from the endogenous preproglucagon (Gcg) gene locus, was generated by homologous recombination. The new Gcg CreERT2 mouse line was crossed to the Rosa26 tdTomato (R26 tdTomato ) Cre reporter mouse line in order to evaluate the tissue specificity, efficiency and tamoxifen dependency of Gcg CreERT2 -mediated recombination. Cell types of pancreatic islets were identified using immunohistochemistry. Biochemical and physiological data, including blood glucose levels, plasma glucagon and glucagon-like peptide (GLP)-1 levels, and pancreatic glucagon content, were collected and used to assess the overall effect of Gcg gene targeting on Gcg CreERT2/w heterozygous mice. RESULTS: Tamoxifen-treated Gcg CreERT2/w ;R26 tdTomato/w mice displayed Cre reporter activity, i.e. expression of tdTomato red fluorescent protein (RFP) in all known cells that produce proglucagon-derived peptides. In the adult pancreas, RFP was detected in 94-97% of alpha cells, whereas it was detected in a negligible (~ 0.2%) proportion of beta cells. While more than 98% of cells labelled with tamoxifen-induced RFP were glucagon-positive cells, 14-25% of pancreatic polypeptide (PP)-positive cells were also positive for RFP, indicating the presence of glucagon/PP bihormonal cell population. Tamoxifen-independent expression of RFP occurred in approximately 6% of alpha cells. In contrast to alpha cells and GLP-1-producing neurons, in which RFP expression persisted for at least 5 months after tamoxifen administration (presumably due to rare neogenesis in these cell types in adulthood), nearly half of RFP-positive intestinal L cells were replaced with RFP-negative L cells over the first 2 weeks after tamoxifen administration. Heterozygous Gcg CreERT2/w mice showed reduced Gcg mRNA levels in islets, but maintained normal levels of pancreatic and plasma glucagon. The mice did not exhibit any detectable baseline physiological abnormalities, at least in young adulthood. CONCLUSIONS/INTERPRETATION: The newly developed Gcg CreERT2 knockin mouse shows faithful expression of CreERT2 in pancreatic alpha cells, intestinal L cells and GLP-1-producing neurons. This mouse line will be particularly useful for manipulating genes in alpha cells, due to highly specific and efficient CreERT2-mediated recombination in this cell type in the pancreas.
Assuntos
Células Secretoras de Glucagon/metabolismo , Proglucagon/metabolismo , Animais , Feminino , Glucagon/sangue , Peptídeo 1 Semelhante ao Glucagon/sangue , Imuno-Histoquímica , Masculino , Camundongos , Camundongos Transgênicos , Proglucagon/genética , Tamoxifeno/farmacologiaRESUMO
A thorough understanding of the signaling pathways involved in the regulation of ß cell proliferation is an important initial step in restoring ß cell mass in the diabetic patient. Here, we show that epidermal growth factor receptor 1 (EGFR) was significantly up-regulated in the islets of C57BL/6 mice after 50% partial pancreatectomy (PPx), a model for workload-induced ß cell proliferation. Specific deletion of EGFR in the ß cells of adult mice impaired ß cell proliferation at baseline and after 50% PPx, suggesting that the EGFR signaling pathway plays an essential role in adult ß cell proliferation. Further analyses showed that ß cell-specific depletion of EGFR resulted in impaired expression of cyclin D1 and impaired suppression of p27 after PPx, both of which enhance ß cell proliferation. These data highlight the importance of EGFR signaling and its downstream signaling cascade in postnatal ß cell growth.
Assuntos
Proliferação de Células/fisiologia , Receptores ErbB/metabolismo , Células Secretoras de Insulina/metabolismo , Transdução de Sinais/fisiologia , Animais , Ciclina D1/genética , Ciclina D1/metabolismo , Receptores ErbB/genética , Camundongos , Camundongos TransgênicosRESUMO
Hypertriglyceridemia results from increased production and decreased clearance of triglyceride-rich very low-density lipoproteins, a pathological condition that accounts for heightened risk of ischemic vascular diseases in obesity and type 2 diabetes. Despite its intimate association with insulin resistance, whether hypertriglyceridemia constitutes an independent risk for beta cell dysfunction in diabetes is unknown. Answering this fundamental question is stymied by the fact that hypertriglyceridemia is intertwined with hyperglycemia and insulin resistance in obese and diabetic subjects. To circumvent this limitation, we took advantage of apolipoprotein C3 (ApoC3)-transgenic mice, a model with genetic predisposition to hypertriglyceridemia. We showed that ApoC3-transgenic mice, as opposed to age/sex-matched wild-type littermates, develop hypertriglyceridemia with concomitant elevations in plasma cholesterol and non-esterified fatty acid levels. Anti-insulin and anti-glucagon dual immunohistochemistry in combination with morphometric analysis revealed that ApoC3-transgenic and wild-type littermates had similar beta cell and alpha cell masses as well as islet size and architecture. These effects correlated with similar amplitudes of glucose-stimulated insulin secretion and similar degrees of postprandial glucose excursion in ApoC3-transgenic versus wild-type littermates. Oil Red O histology did not visualize lipid infiltration into islets, correlating with the lack of ectopic triglyceride and cholesterol depositions in the pancreata of ApoC3-transgenic versus wild-type littermates. ApoC3-transgenic mice, despite persistent hypertriglyceridemia, maintained euglycemia under both fed and fasting conditions without manifestation of insulin resistance and fasting hyperinsulinemia. Thus, hypertriglyceridemia per se is not an independent risk factor for beta cell dysfunction in ApoC3 transgenic mice.
Assuntos
Apolipoproteína C-III/metabolismo , Diabetes Mellitus Tipo 2/complicações , Hipertrigliceridemia/complicações , Células Secretoras de Insulina/patologia , Animais , Apolipoproteína C-III/genética , Glicemia/análise , Diabetes Mellitus Tipo 2/sangue , Diabetes Mellitus Tipo 2/metabolismo , Diabetes Mellitus Tipo 2/patologia , Feminino , Glucose/metabolismo , Humanos , Hipertrigliceridemia/sangue , Hipertrigliceridemia/metabolismo , Hipertrigliceridemia/patologia , Resistência à Insulina , Células Secretoras de Insulina/metabolismo , Metabolismo dos Lipídeos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , ObesidadeRESUMO
Determination of signaling pathways that regulate beta-cell replication is critical for beta-cell therapy. Here, we show that blocking pancreatic macrophage infiltration after pancreatic duct ligation (PDL) completely inhibits beta-cell proliferation. The TGFß superfamily signaling inhibitor SMAD7 was significantly up-regulated in beta cells after PDL. Beta cells failed to proliferate in response to PDL in beta-cell-specific SMAD7 mutant mice. Forced expression of SMAD7 in beta cells by itself was sufficient to promote beta-cell proliferation in vivo. M2, rather than M1 macrophages, seem to be the inducers of SMAD7-mediated beta-cell proliferation. M2 macrophages not only release TGFß1 to directly induce up-regulation of SMAD7 in beta cells but also release EGF to activate EGF receptor signaling that inhibits TGFß1-activated SMAD2 nuclear translocation, resulting in TGFß signaling inhibition. SMAD7 promotes beta-cell proliferation by increasing CyclinD1 and CyclinD2, and by inducing nuclear exclusion of p27. Our study thus reveals a molecular pathway to potentially increase beta-cell mass through enhanced SMAD7 activity induced by extracellular stimuli.
Assuntos
Células Secretoras de Insulina/metabolismo , Células Secretoras de Insulina/patologia , Macrófagos/metabolismo , Proteína Smad7/metabolismo , Regulação para Cima , Animais , Movimento Celular , Núcleo Celular/metabolismo , Proliferação de Células , Inibidor de Quinase Dependente de Ciclina p27/metabolismo , Modelos Animais de Doenças , Receptores ErbB/metabolismo , Inflamação/metabolismo , Inflamação/patologia , Ligadura , Camundongos , Camundongos Endogâmicos C57BL , Modelos Biológicos , Transdução de Sinais , Fator de Crescimento Transformador beta/metabolismoRESUMO
Nuclear factor κB (NF-κB) is an important signaling molecule that plays a critical role in the development of acute pancreatitis. Current methods for examining NF-κB activation involve infection of an adenoviral NF-κB-luciferase reporter into cell lines or electrophoretic mobility shift assay of lysate. The use of adeno-associated viruses (AAVs) has proven to be an effective method of transfecting whole organs in live animals. We examined whether intrapancreatic duct infusion of AAV containing an NF-κB-luciferase reporter (AAV-NF-κB-luciferase) can reliably measure pancreatic NF-κB activation. We confirmed the infectivity of the AAV-NF-κB-luciferase reporter in HEK293 cells using a traditional luciferase readout. Mice were infused with AAV-NF-κB-luciferase 5 weeks before induction of pancreatitis (caerulein, 50 µg/kg). Unlike transgenic mice that globally express NF-κB-luciferase, AAV-infused mice showed a 15-fold increase in pancreas-specific NF-κB bioluminescence following 12 h of caerulein compared with baseline luminescence (p < 0.05). The specificity of the NF-κB-luciferase signal to the pancreas was confirmed by isolating the pancreas and adjacent organs and observing a predominant bioluminescent signal in the pancreas compared with liver, spleen, and stomach. A complementary mouse model of post-ERCP-pancreatitis also induced pancreatic NF-κB signals. Taken together these data provide the first demonstration that NF-κB activation can be examined in a live, dynamic fashion during pancreatic inflammation. We believe this technique offers a valuable tool to study real-time activation of NF-κB in vivo.
Assuntos
Dependovirus/genética , Medições Luminescentes , Imagem Molecular , NF-kappa B/metabolismo , Pâncreas/metabolismo , Pâncreas/virologia , Animais , Ceruletídeo/metabolismo , Dependovirus/fisiologia , Células HEK293 , Humanos , Luciferases/genética , Camundongos , Camundongos Transgênicos , NF-kappa B/genética , Especificidade de Órgãos , Transdução de SinaisRESUMO
AIMS/HYPOTHESIS: Vascular endothelial growth factor (VEGF) is essential for proper pancreatic development, islet vascularisation and insulin secretion. In the adult pancreas, VEGF is thought to be predominantly secreted by beta cells. Although human duct cells have previously been shown to secrete VEGF at angiogenic levels in culture, an analysis of the kinetics of VEGF synthesis and secretion, as well as elucidation of an in vivo role for this ductal VEGF in affecting islet function and physiology, has been lacking. METHODS: We analysed purified duct cells independently prepared by flow cytometry, surgical isolation or laser-capture microdissection. We infected duct cells in vivo with Vegf (also known as Vegfa) short hairpin RNA (shRNA) in an intrapancreatic ductal infusion system and examined the effect of VEGF knockdown in duct cells in vitro and in vivo. RESULTS: Pancreatic duct cells express high levels of Vegf mRNA. Compared with beta cells, duct cells had a much higher ratio of secreted to intracellular VEGF. As a bioassay, formation of tubular structures by human umbilical vein endothelial cells was essentially undetectable when cultured alone and was substantially increased when co-cultured with pancreatic duct cells but significantly reduced when co-cultured with duct cells pretreated with Vegf shRNA. Compared with islets transplanted alone, improved vascularisation and function was detected in the islets co-transplanted with duct cells but not in islets co-transplanted with duct cells pretreated with Vegf shRNA. CONCLUSIONS/INTERPRETATION: Human islet preparations for transplantation typically contain some contaminating duct cells and our findings suggest that the presence of duct cells in the islet preparation may improve transplantation outcomes.
Assuntos
Células Secretoras de Insulina/metabolismo , Ductos Pancreáticos/metabolismo , Fator A de Crescimento do Endotélio Vascular/metabolismo , Animais , Células Epiteliais/citologia , Técnicas de Silenciamento de Genes , Células Endoteliais da Veia Umbilical Humana , Humanos , Ilhotas Pancreáticas/citologia , Transplante das Ilhotas Pancreáticas , Camundongos , Neovascularização Fisiológica , RNA Interferente Pequeno/metabolismo , Fatores de Transcrição SOX9/genética , Fator A de Crescimento do Endotélio Vascular/genéticaRESUMO
Expansion of the pancreatic endocrine cell population occurs during both embryonic development and during post-natal pancreatic growth and regeneration. Mechanisms of the expansion of endocrine cells during embryonic development are not completely understood, and no clear mechanistic link has been established between growth of the embryonic endocrine pancreas and the islet cell replication that occurs in an adult animal. We found that transforming growth factor-beta (TGF-ß) superfamily signaling, which has been implicated in many developmental processes, plays a key role in regulating pancreatic endocrine maturation and development. Specifically, the intracellular mediators of TGF-ß signaling, smad2 and smad3, along with their inhibitor smad7, appear to mediate this process. Smad2, smad3 and smad7 were all broadly expressed throughout the early embryonic pancreatic epithelium. However, during later stages of development, smad2 and smad3 became strongly localized to the nuclei of the endocrine positive cells, whereas the inhibitory smad7 became absent in the endocrine component. Genetic inactivation of smad2 and smad3 led to a significant expansion of the embryonic endocrine compartment, whereas genetic inactivation of smad7 led to a significant decrease in the endocrine compartment. In vitro antisense studies further corroborated these results and supported the possibility that interplay between the inhibitory smad7 and the intracellular mediators smad2/3 is a control point for pancreatic endocrine development. These results should provide a better understanding of the key control mechanisms for ß-cell development.
Assuntos
Ilhotas Pancreáticas/metabolismo , Proteína Smad2/metabolismo , Proteína Smad3/metabolismo , Proteína Smad7/metabolismo , Animais , Western Blotting , Proliferação de Células , Epitélio/metabolismo , Perfilação da Expressão Gênica , Regulação da Expressão Gênica no Desenvolvimento , Técnicas de Silenciamento de Genes , Imuno-Histoquímica , Ilhotas Pancreáticas/citologia , Ilhotas Pancreáticas/embriologia , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Microscopia de Fluorescência , Fosforilação , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais/genética , Proteína Smad2/genética , Proteína Smad3/genética , Proteína Smad7/genética , Fatores de Tempo , Fator de Crescimento Transformador beta/metabolismoRESUMO
VEGF-A expression in beta cells is critical for pancreatic development, formation of islet-specific vasculature, and Insulin secretion. However, two key questions remain. First, is VEGF-A release from beta cells coupled to VEGF-A production in beta cells? Second, how is the VEGF-A response by beta cells affected by metabolic signals? Here, we show that VEGF-A secretion, but not gene transcription, in either cultured islets or purified pancreatic beta cells, was significantly reduced early on during low glucose conditions. In vivo, a sustained hypoglycemia in mice was induced with Insulin pellets, resulting in a significant reduction in beta cell mass. This loss of beta cell mass could be significantly rescued with continuous delivery of exogenous VEGF-A, which had no effect on beta cell mass in normoglycemic mice. In addition, an increase in apoptotic endothelial cells during hypoglycemia preceded an increase in apoptotic beta cells. Both endothelial and beta cell apoptosis were prevented by exogenous VEGF-A, suggesting a possible causative relationship between reduced VEGF-A and the loss of islet vasculature and beta cells. Furthermore, in none of these experimental groups did beta cell proliferation and islet vessel density change, suggesting a tightly regulated balance between these two cellular compartments. The average islet size decreased in hypoglycemia, which was also prevented by exogenous VEGF-A. Taken together, our data suggest that VEGF-A release in beta cells is independent of VEGF-A synthesis. Beta cell mass can be regulated through modulated release of VEGF-A from beta cells based on physiological need.
Assuntos
Hipoglicemia/metabolismo , Células Secretoras de Insulina/metabolismo , Fator A de Crescimento do Endotélio Vascular/biossíntese , Animais , Apoptose , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Proliferação de Células , Células Cultivadas , Expressão Gênica , Células Secretoras de Insulina/fisiologia , Transferases Intramoleculares/genética , Transferases Intramoleculares/metabolismo , Camundongos , Pâncreas/irrigação sanguínea , Pâncreas/patologia , Fator A de Crescimento do Endotélio Vascular/genética , Fator A de Crescimento do Endotélio Vascular/metabolismo , Fator A de Crescimento do Endotélio Vascular/fisiologiaRESUMO
It remains controversial whether adult pancreatic ducts harbor facultative beta cell progenitors. Because neurogenin3 (Ngn3) is a key determinant of pancreatic endocrine cell neogenesis during embryogenesis, many studies have also relied upon Ngn3 expression as evidence of beta cell neogenesis in adults. Recently, however, Ngn3 as a marker of adult beta cell neogenesis has been called into question by reports of Ngn3 expression in fully-developed beta cells. Nevertheless, direct evidence as to whether Ngn3 activation in adult pancreatic duct cells may lead to duct-to-beta cell transdifferentiation is lacking. Here we studied two models of Ngn3 activation in adult pancreatic duct cells (low-dose alloxan treatment and pancreatic duct ligation) and lineage-traced Ngn3-activated duct cells by labeling them through intraductal infusion with a cell-tagging dye, CFDA-SE No dye-labeled beta cells were found during the follow-up in either model, suggesting that activation of Ngn3 in duct cells is not sufficient to direct their transdifferentiation into beta cells. Therefore, Ngn3 activation in duct cells is not a signature for adult beta cell neogenesis.
Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Transdiferenciação Celular/fisiologia , Células Secretoras de Insulina/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Ductos Pancreáticos/metabolismo , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Fluoresceínas/farmacologia , Corantes Fluorescentes/farmacologia , Células Secretoras de Insulina/citologia , Camundongos , Camundongos Transgênicos , Proteínas do Tecido Nervoso/genética , Ductos Pancreáticos/citologia , Succinimidas/farmacologiaRESUMO
The antioxidant activities of lycopene (LY), lutein (LU), chlorogenic acid (CA), and delphinidin (DP) were tested in vitro on H9c2 cell-based models. Some indicators, such as the generation of reactive oxygen (ROS), the quantification of cell antioxidant activity (CAA), and the expressions of SOD, GSH-Px, and CAT, were calculated to examine their antioxidant interactions. From our results, the phytochemical mixtures (M1: CA-LU: F3/10, M2: DP-CA: F7/10, M3: DP-LY: F5/10) displayed strong synergistic effects based on the generation of ROS and the quantification of CAA. However, great antagonistic bioactivities were seen in the combinations of LY-LU: F5/10 (M4), CA-LU: F9/10 (M5), and DP-LY: F7/10 (M6). Western blotting analysis indicated that the possible mechanism underlying the synergistic antioxidant interactions among phytochemical combinations was to enhance the accumulation of Nrf2 in the nucleus and the expression of its downstream antioxidant enzymes, HO-1 and GCLC. The combinations (M1-M3 groups) showed significant protection against the loss of mitochondrial membrane potential than individual groups to avoid excessive ROS production. The M4-M6 groups exerted antagonistic protective effects compared with the individual groups. In addition, lutein and lycopene absorption was improved more because of the presence of chlorogenic acid and delphinidin in the M1 and M3 groups, respectively. However, delphinidin significantly reduced the cellular uptake of lycopene in the M6 group. It appeared that antioxidant interactions of phytochemical combinations may contribute to the restoration of cellular redox homeostasis and lead to an improvement in diet quality and collocation.