RESUMO
Human pancreatic islets engrafted into immunodeficient mice serve as an important model for in vivo human diabetes studies. Following engraftment, islet function can be monitored in vivo by measuring circulating glucose and human insulin; however, it will be important to recover viable cells for more complex graft analyses. Moreover, RNA analyses of dissected grafts have not distinguished which hormone-specific cell types contribute to gene expression. We developed a method for recovering live cells suitable for fluorescence-activated cell sorting from human islets engrafted in mice. Although yields of recovered islet cells were relatively low, the ratios of bulk-sorted ß, α, and δ cells and their respective hormone-specific RNA-Seq transcriptomes are comparable pretransplant and posttransplant, suggesting that the cellular characteristics of islet grafts posttransplant closely mirror the original donor islets. Single-cell RNA-Seq transcriptome analysis confirms the presence of appropriate ß, α, and δ cell subsets. In addition, ex vivo perifusion of recovered human islet grafts demonstrated glucose-stimulated insulin secretion. Viable cells suitable for patch-clamp analysis were recovered from transplanted human embryonic stem cell-derived ß cells. Together, our functional and hormone-specific transcriptome analyses document the broad applicability of this system for longitudinal examination of human islet cells undergoing developmental/metabolic/pharmacogenetic manipulation in vivo and may facilitate the discovery of treatments for diabetes.
Assuntos
Células Endócrinas/fisiologia , Ilhotas Pancreáticas/fisiologia , Transcriptoma/fisiologia , Adulto , Animais , Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Experimental/patologia , Células Endócrinas/metabolismo , Feminino , Perfilação da Expressão Gênica/métodos , Sobrevivência de Enxerto/fisiologia , Humanos , Insulina/metabolismo , Células Secretoras de Insulina/metabolismo , Células Secretoras de Insulina/fisiologia , Ilhotas Pancreáticas/metabolismo , Transplante das Ilhotas Pancreáticas/métodos , Masculino , Camundongos , Transplante Heterólogo/métodos , Adulto JovemRESUMO
Previous studies revealed a paradox whereby mitogen-activated protein kinase kinase kinase kinase 4 (Map4k4) acted as a negative regulator of insulin sensitivity in chronically obese mice, yet systemic deletion of Map4k4 did not improve glucose tolerance. Here, we report markedly reduced glucose-responsive plasma insulin and C-peptide levels in whole body Map4k4-depleted mice (M4K4 iKO) as well as an impaired first phase of insulin secretion from islets derived from M4K4 iKO mice ex vivo After long-term high fat diet (HFD), M4K4 iKO mice pancreata also displayed reduced ß cell mass, fewer proliferating ß cells and reduced islet-specific gene mRNA expression compared with controls, although insulin content was normal. Interestingly, the reduced plasma insulin in M4K4 iKO mice exposed to chronic (16 weeks) HFD was not observed in response to acute HFD challenge or short term treatment with the insulin receptor antagonist S961. Furthermore, the improved insulin sensitivity in obese M4K4 iKO mice was abrogated by high exogenous insulin over the course of a euglycemic clamp study, indicating that hypoinsulinemia promotes insulin sensitivity in chronically obese M4K4 iKO mice. These results demonstrate that protein kinase Map4k4 drives obesity-induced hyperinsulinemia and insulin resistance in part by promoting insulin secretion from ß cells in mice.
Assuntos
Resistência à Insulina , Células Secretoras de Insulina/metabolismo , Insulina/metabolismo , Obesidade/enzimologia , Proteínas Serina-Treonina Quinases/metabolismo , Animais , Gorduras na Dieta/efeitos adversos , Gorduras na Dieta/farmacologia , Insulina/genética , Secreção de Insulina , Células Secretoras de Insulina/patologia , Camundongos , Camundongos Knockout , Obesidade/induzido quimicamente , Obesidade/genética , Obesidade/patologia , Peptídeos/farmacologia , Proteínas Serina-Treonina Quinases/genética , Quinase Induzida por NF-kappaBRESUMO
Individuals with schizophrenia and their first-degree relatives have higher rates of type 2 diabetes (T2D) than the general population (18-30 vs. 1.2-6.3%), independent of body mass index and antipsychotic medication, suggesting shared genetic components may contribute to both diseases. The cause of this association remains unknown. Mutations in disrupted in schizophrenia 1 (DISC1) increase the risk of developing psychiatric disorders [logarithm (base 10) of odds = 7.1]. Here, we identified DISC1 as a major player controlling pancreatic ß-cell proliferation and insulin secretion via regulation of glycogen synthase kinase-3ß (GSK3ß). DISC1 expression was enriched in developing mouse and human pancreas and adult ß- and ductal cells. Loss of DISC1 function, through siRNA-mediated depletion or expression of a dominant-negative truncation that models the chromosomal translocation of human DISC1 in schizophrenia, resulted in decreased ß-cell proliferation (3 vs. 1%; P < 0.01), increased apoptosis (0.1 vs. 0.6%; P < 0.01), and glucose intolerance in transgenic mice. Insulin secretion was reduced (0.5 vs. 0.1 ng/ml; P < 0.05), and critical ß-cell transcription factors Pdx1 and Nkx6.1 were significantly decreased. Impaired DISC1 allowed inappropriate activation of GSK3ß in ß cells, and antagonizing GSK3ß (SB216763; IC50 = 34.3 nM) rescued the ß-cell defects. These results uncover an unexpected role for DISC1 in normal ß-cell physiology and suggest that DISC1 dysregulation contributes to T2D independently of its importance for cognition.
Assuntos
Proliferação de Células , Quinase 3 da Glicogênio Sintase/metabolismo , Células Secretoras de Insulina/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Animais , Diabetes Mellitus Tipo 2/genética , Diabetes Mellitus Tipo 2/metabolismo , Diabetes Mellitus Tipo 2/patologia , Regulação da Expressão Gênica , Quinase 3 da Glicogênio Sintase/genética , Glicogênio Sintase Quinase 3 beta , Humanos , Células Secretoras de Insulina/patologia , Camundongos , Camundongos Transgênicos , Proteínas do Tecido Nervoso/genética , Esquizofrenia/genética , Esquizofrenia/metabolismo , Esquizofrenia/patologia , Translocação GenéticaRESUMO
Knockout technology has proven useful for delineating functional roles of specific genes. Here we describe and provide an explanation for striking pathology that occurs in a subset of genetically engineered mice expressing a rat CaVß2a transgene under control of the cardiac α-myosin heavy chain promoter. Lesions were limited to mice homozygous for transgene and independent of native Cacnb2 genomic copy number. Gross findings included an atrophied pancreas; decreased adipose tissue; thickened, orange intestines; and enlarged liver, spleen, and abdominal lymph nodes. Immune cell infiltration and cell engulfment by macrophages were associated with loss of pancreatic acinar cells. Foamy macrophages diffusely infiltrated the small intestine's lamina propria, while similar macrophage aggregates packed liver and splenic red pulp sinusoids. Periodic acid-Schiff-positive, diastase-resistant, iron-negative, Oil Red O-positive, and autofluorescent cytoplasm was indicative of a lipid storage disorder. Electron microscopic analysis revealed liver sinusoids distended by clusters of macrophages containing intracellular myelin "swirls" and hepatocytes with enlarged lysosomes. Additionally, build up of cholesterol, cholesterol esters, and triglycerides, along with changes in liver metabolic enzyme levels, were consistent with a lipid processing defect. Because of this complex pathology, we examined the transgene insertion site. Multiple transgene copies inserted into chromosome 19; at this same site, an approximate 180,000 base pair deletion occurred, ablating cholesterol 25-hydroxylase and partially deleting lysosomal acid lipase and CD95 Loss of gene function can account for the altered lipid processing, along with hypertrophy of the immune system, which define this phenotype, and serendipitously provides a novel mouse model of lysosomal storage disorder.
Assuntos
Canais de Cálcio Tipo L/genética , Colesterol/metabolismo , Doenças por Armazenamento dos Lisossomos/genética , Triglicerídeos/metabolismo , Animais , Modelos Animais de Doenças , Hepatócitos/metabolismo , Hepatócitos/patologia , Fígado/metabolismo , Fígado/patologia , Doenças por Armazenamento dos Lisossomos/metabolismo , Doenças por Armazenamento dos Lisossomos/patologia , Lisossomos/metabolismo , Lisossomos/patologia , Macrófagos/metabolismo , Macrófagos/patologia , Camundongos , Camundongos Transgênicos , Regiões Promotoras Genéticas , Baço/metabolismo , Baço/patologiaRESUMO
Primary cilia play an essential role in modulating signaling cascades that shape cellular responses to environmental cues to maintain proper tissue development. Mutations in primary cilium proteins have been linked to several rare developmental disorders, collectively known as ciliopathies. Together with other disorders associated with dysfunctional cilia/centrosomes, affected individuals have increased risk of developing metabolic syndrome, neurologic disorders, and diabetes. In pancreatic tissues, cilia are found exclusively in islet and ductal cells where they play an essential role in pancreatic tissue organization. Their absence or disorganization leads to pancreatic duct abnormalities, acinar cell loss, polarity defects, and dysregulated insulin secretion. Cilia in pancreatic tissues are hubs for cellular signaling. Many signaling components, such as Hh, Notch, and Wnt, localize to pancreatic primary cilia and are necessary for proper development of pancreatic epithelium and ß-cell morphogenesis. Receptors for neuroendocrine hormones, such as Somatostatin Receptor 3, also localize to the cilium and may play a more direct role in controlling insulin secretion due to somatostatin's inhibitory function. Finally, unique calcium signaling, which is at the heart of ß-cell function, also occurs in primary cilia. Whereas voltage-gated calcium channels trigger insulin secretion and serve a variety of homeostatic functions in ß-cells, transient receptor potential channels regulate calcium levels within the cilium that may serve as a feedback mechanism, regulating insulin secretion. This review article summarizes our current understanding of the role of primary cilia in normal pancreas function and in the diseased state.
Assuntos
Cílios/patologia , Cílios/fisiologia , Pâncreas/fisiologia , Pâncreas/fisiopatologia , Transdução de Sinais , Animais , Canais de Cálcio/metabolismo , Modelos Animais de Doenças , Humanos , Células Secretoras de Insulina/metabolismoRESUMO
AIMS/HYPOTHESIS: We hypothesised that pathological endoplasmic reticulum (ER) stress contributes to beta cell death during development of type 1 diabetes. In this study, we investigated the occurrence of beta cell ER stress and the signalling pathways involved during discrete stages of autoimmune diabetes progression. The virus-inducible BBDR rat model was used to systematically interrogate the three main ER stress signalling pathways (IRE1 [inositol-requiring protein-1], PERK [double-stranded RNA-dependent protein kinase (PKR)-like ER kinase] and ATF6 [activating transcription factor 6]) in pancreatic beta cells during type 1 diabetes development. METHODS: ER stress and apoptotic markers were assessed by immunoblot analyses of isolated pancreatic islets and immunofluorescence staining of pancreas sections from control and virus-induced rats. Various time points were analysed: (1) early stages preceding the development of insulitis and (2) a late stage during onset and progression of insulitis, which precedes overt hyperglycaemia. RESULTS: The IRE1 pathway, including its downstream component X-box-binding protein 1, was specifically activated in pancreatic beta cells of virus-induced rats at early stages preceding the development of insulitis. Furthermore, ER stress-specific pro-apoptotic caspase 12 and effector caspase 3 were also activated at this stage. Activation of PERK and its downstream effector pro-apoptotic CHOP (CCAAT/-enhancer-binding-protein homologous protein), only occurred during late stages of diabetes induction concurrent with insulitis, whereas ATF6 activation in pancreatic beta cells was similar in control and virus-induced rats. CONCLUSIONS/INTERPRETATION: Activation of the IRE1 pathway and ER stress-specific pro-apoptotic caspase 12, before the development of insulitis, are indicative of ER stress-mediated beta cell damage. The early occurrence of pathological ER stress and death in pancreatic beta cells may contribute to the initiation and/or progression of virus-induced autoimmune diabetes.
Assuntos
Diabetes Mellitus Experimental/patologia , Diabetes Mellitus Tipo 1/patologia , Estresse do Retículo Endoplasmático , Células Secretoras de Insulina/metabolismo , Proteínas de Membrana/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Fator 6 Ativador da Transcrição/metabolismo , Animais , Apoptose , Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Tipo 1/metabolismo , Feminino , Immunoblotting , Células Secretoras de Insulina/patologia , Masculino , Ratos , Transdução de Sinais , Fator de Transcrição CHOP/metabolismo , eIF-2 Quinase/metabolismoRESUMO
Zebrafish embryos are emerging as models of glucose metabolism. However, patterns of endogenous glucose levels, and the role of the islet in glucoregulation, are unknown. We measured absolute glucose levels in zebrafish and mouse embryos, and demonstrate similar, dynamic glucose fluctuations in both species. Further, we show that chemical and genetic perturbations elicit mammalian-like glycemic responses in zebrafish embryos. We show that glucose is undetectable in early zebrafish and mouse embryos, but increases in parallel with pancreatic islet formation in both species. In zebrafish, increasing glucose is associated with activation of gluconeogenic phosphoenolpyruvate carboxykinase1 (pck1) transcription. Non-hepatic Pck1 protein is expressed in mouse embryos. We show using RNA in situ hybridization, that zebrafish pck1 mRNA is similarly expressed in multiple cell types prior to hepatogenesis. Further, we demonstrate that the Pck1 inhibitor 3-mercaptopicolinic acid suppresses normal glucose accumulation in early zebrafish embryos. This shows that pre- and extra-hepatic pck1 is functional, and provides glucose locally to rapidly developing tissues. To determine if the primary islet is glucoregulatory in early fish embryos, we injected pdx1-specific morpholinos into transgenic embryos expressing GFP in beta cells. Most morphant islets were hypomorphic, not a genetic, but embryos still exhibited persistent hyperglycemia. We conclude from these data that the early zebrafish islet is functional, and regulates endogenous glucose. In summary, we identify mechanisms of glucoregulation in zebrafish embryos that are conserved with embryonic and adult mammals. These observations justify use of this model in mechanistic studies of human metabolic disease.
Assuntos
Embrião não Mamífero/metabolismo , Glucose/metabolismo , Peixe-Zebra/metabolismo , Animais , Animais Geneticamente Modificados , Desenvolvimento Embrionário/efeitos dos fármacos , Proteínas de Fluorescência Verde/análise , Hibridização In Situ , Células Secretoras de Insulina/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Fosfoenolpiruvato Carboxiquinase (GTP)/genética , Fosfoenolpiruvato Carboxiquinase (GTP)/metabolismo , Fosfoenolpiruvato Carboxiquinase (GTP)/fisiologia , Filogenia , Ácidos Picolínicos/farmacologia , RNA Mensageiro/metabolismo , Peixe-Zebra/genética , Peixe-Zebra/crescimento & desenvolvimentoRESUMO
Disrupted in Schizophrenia 1 (DISC1) participates in a wide variety of developmental processes of central neurons. It also serves critical roles that underlie cognitive functioning in adult central neurons. Here we summarize DISC1's general properties and discuss its use as a model system for understanding major mental illnesses (MMIs). We then discuss the cellular actions of DISC1 that involve or regulate Ca2+ signaling in adult central neurons. In particular, we focus on the tethering role DISC1 plays in transporting RNA particles containing Ca2+ channel subunit RNAs, including IP3R1, CACNA1C and CACNA2D1, and in transporting mitochondria into dendritic and axonal processes. We also review DISC1's role in modulating IP3R1 activity within mitochondria-associated ER membrane (MAM). Finally, we discuss DISC1-glycogen synthase kinase 3ß (GSK3ß) signaling that regulates functional expression of voltage-gated Ca2+ channels (VGCCs) at central synapses. In each case, DISC1 regulates the movement of molecules that impact Ca2+ signaling in neurons.
RESUMO
Enteroviral infections are implicated in islet autoimmunity and type 1 diabetes (T1D) pathogenesis. Significant ß-cell stress and damage occur with viral infection, leading to cells that are dysfunctional and vulnerable to destruction. Human stem cell-derived ß (SC-ß) cells are insulin-producing cell clusters that closely resemble native ß cells. To better understand the events precipitated by enteroviral infection of ß cells, we investigated transcriptional and proteomic changes in SC-ß cells challenged with coxsackie B virus (CVB). We confirmed infection by demonstrating that viral protein colocalized with insulin-positive SC-ß cells by immunostaining. Transcriptome analysis showed a decrease in insulin gene expression following infection, and combined transcriptional and proteomic analysis revealed activation of innate immune pathways, including type I interferon (IFN), IFN-stimulated genes, nuclear factor-kappa B (NF-κB) and downstream inflammatory cytokines, and major histocompatibility complex (MHC) class I. Finally, insulin release by CVB4-infected SC-ß cells was impaired. These transcriptional, proteomic, and functional findings are in agreement with responses in primary human islets infected with CVB ex vivo. Human SC-ß cells may serve as a surrogate for primary human islets in virus-induced diabetes models. Because human SC-ß cells are more genetically tractable and accessible than primary islets, they may provide a preferred platform for investigating T1D pathogenesis and developing new treatments.
RESUMO
Primary cilia are nonmotile microtubule structures that assemble from basal bodies by a process called intraflagellar transport (IFT) and are associated with several human diseases. Here, we show that the centrosome protein pericentrin (Pcnt) colocalizes with IFT proteins to the base of primary and motile cilia. Immunogold electron microscopy demonstrates that Pcnt is on or near basal bodies at the base of cilia. Pcnt depletion by RNA interference disrupts basal body localization of IFT proteins and the cation channel polycystin-2 (PC2), and inhibits primary cilia assembly in human epithelial cells. Conversely, silencing of IFT20 mislocalizes Pcnt from basal bodies and inhibits primary cilia assembly. Pcnt is found in spermatocyte IFT fractions, and IFT proteins are found in isolated centrosome fractions. Pcnt antibodies coimmunoprecipitate IFT proteins and PC2 from several cell lines and tissues. We conclude that Pcnt, IFTs, and PC2 form a complex in vertebrate cells that is required for assembly of primary cilia and possibly motile cilia and flagella.
Assuntos
Antígenos/metabolismo , Proteínas de Transporte/metabolismo , Cílios/metabolismo , Células Epiteliais/metabolismo , Proteínas de Membrana/metabolismo , Animais , Antígenos/ultraestrutura , Proteínas de Transporte/genética , Movimento Celular/fisiologia , Células Cultivadas , Cílios/ultraestrutura , Células Epiteliais/ultraestrutura , Imuno-Histoquímica , Substâncias Macromoleculares , Masculino , Proteínas de Membrana/ultraestrutura , Camundongos , Microscopia Eletrônica , Transporte Proteico/fisiologia , Interferência de RNA , Espermatócitos/metabolismo , Espermatócitos/ultraestrutura , Canais de Cátion TRPPRESUMO
Centrosomes nucleate microtubules and contribute to mitotic spindle organization and function. They also participate in cytokinesis and cell cycle progression in ways that are poorly understood. Here we describe a novel human protein called centriolin that localizes to the maternal centriole and functions in both cytokinesis and cell cycle progression. Centriolin silencing induces cytokinesis failure by a novel mechanism whereby cells remain interconnected by long intercellular bridges. Most cells continue to cycle, reenter mitosis, and form multicellular syncytia. Some ultimately divide or undergo apoptosis specifically during the protracted period of cytokinesis. At later times, viable cells arrest in G1/G0. The cytokinesis activity is localized to a centriolin domain that shares homology with Nud1p and Cdc11p, budding and fission yeast proteins that anchor regulatory pathways involved in progression through the late stages of mitosis. The Nud1p-like domain of centriolin binds Bub2p, another component of the budding yeast pathway. We conclude that centriolin is required for a late stage of vertebrate cytokinesis, perhaps the final cell cleavage event, and plays a role in progression into S phase.
Assuntos
Proteínas de Ciclo Celular/isolamento & purificação , Divisão Celular/genética , Centríolos/metabolismo , Células Eucarióticas/metabolismo , Fase S/genética , Sequência de Aminoácidos/genética , Animais , Anticorpos/farmacologia , Sequência de Bases/genética , Células COS , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Centríolos/ultraestrutura , Proteínas do Citoesqueleto/genética , Proteínas do Citoesqueleto/metabolismo , DNA Complementar/análise , Desoxirribonucleases/genética , Desoxirribonucleases/metabolismo , Embrião não Mamífero/efeitos dos fármacos , Embrião não Mamífero/metabolismo , Células Eucarióticas/ultraestrutura , Células HeLa , Humanos , Imuno-Histoquímica , Microscopia Eletrônica , Microtúbulos/genética , Microtúbulos/metabolismo , Dados de Sequência Molecular , Ligação Proteica/genética , Estrutura Terciária de Proteína/genética , RNA Interferente Pequeno , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , tRNA MetiltransferasesRESUMO
T cell receptor (TCR) ligation (signal one) in the presence of co-stimulation (signal two) results in downstream signals that increase protein production enabling naïve T cells to fully activate and gain effector function. Enhanced production of proteins by a cell requires an increase in endoplasmic reticulum (ER) chaperone expression, which is accomplished through activation of a cellular mechanism known as the ER stress response. The ER stress response is initiated during the cascade of events that occur for the activation of many cells; however, this process has not been comprehensively studied for T cell function. In this study, we used primary T cells and mice circulating TCR transgenic CD8(+) T cells to investigate ER chaperone expression in which TCR signaling was initiated in the presence or absence of co-stimulation. In the presence of both signals, in vitro and in vivo analyses demonstrated induction of the ER stress response, as evidenced by elevated expression of GRP78 and other ER chaperones. Unexpectedly, ER chaperones were also increased in T cells exposed only to signal one, a treatment known to cause T cells to enter the 'nonresponsive' states of anergy and tolerance. Treatment of T cells with an inhibitor to protein kinase C (PKC), a serine/threonine protein kinase found downstream of TCR signaling, indicated PKC is involved in the induction of the ER stress response during the T cell activation process, thus revealing a previously unknown role for this signaling protein in T cells. Collectively, these data suggest that induction of the ER stress response through PKC signaling is an important component for the preparation of a T cell response to antigen.
Assuntos
Retículo Endoplasmático/enzimologia , Ativação Linfocitária/imunologia , Proteína Quinase C/metabolismo , Transdução de Sinais , Estresse Fisiológico , Linfócitos T/enzimologia , Linfócitos T/imunologia , Animais , Retículo Endoplasmático/efeitos dos fármacos , Chaperona BiP do Retículo Endoplasmático , Ativação Enzimática/efeitos dos fármacos , Proteínas de Choque Térmico/metabolismo , Tolerância Imunológica/efeitos dos fármacos , Interleucina-2/biossíntese , Ionomicina/farmacologia , Ativação Linfocitária/efeitos dos fármacos , Masculino , Camundongos , Modelos Biológicos , Chaperonas Moleculares/metabolismo , Dibutirato de 12,13-Forbol/farmacologia , Proteína Quinase C/antagonistas & inibidores , Inibidores de Proteínas Quinases/farmacologia , Receptores de Antígenos de Linfócitos T/metabolismo , Transdução de Sinais/efeitos dos fármacos , Estresse Fisiológico/efeitos dos fármacos , Linfócitos T/citologia , Linfócitos T/efeitos dos fármacos , Tapsigargina/farmacologia , Regulação para Cima/efeitos dos fármacosRESUMO
PURPOSE OF REVIEW: Understanding the relationship between viral infections and the development of type 1 diabetes (T1D) is essential for T1D prevention. Virus-induced innate immune responses, specifically type I interferon (IFN-I) and the IFN gene signature, orchestrate early events of ß-cell dysfunction preceding islet autoimmunity. We summarize recent advances in how IFN-I and the IFN gene signature can drive T1D development. RECENT FINDINGS: IFN-I, particularly IFN-α, and the IFN gene signature have been detected in islets and peripheral blood of T1D patients. T1D risk genes in the IFN-I signaling pathway regulate antiviral responses in ß cells driven by IFN-I and proinflammatory cytokines. Polymorphisms in these genes may cause chronic dysregulated IFN signaling in islets, characterized by hyperexpression of IFN-I, the IFN gene signature, and major histocompatibility complex class I during viral infection. Islet-cell inflammation mediated by aberrant IFN signaling drives ß-cell apoptosis by initiating autoreactivity against ß-cell antigens. The profound elevation in IFN-I and the IFN gene signature observed in some forms of T1D are also seen in a novel group of human autoimmune and autoinflammatory diseases called interferonopathies. SUMMARY: Despite significant advances, further studies are required to functionally dissect the mechanisms by which excessive IFN-I contributes to the evolution of autoimmunity that destroys ß cells.
Assuntos
Diabetes Mellitus Tipo 1/etiologia , Interferon Tipo I/fisiologia , Animais , Apoptose/genética , Apoptose/imunologia , Autoimunidade/genética , Diabetes Mellitus Tipo 1/imunologia , Diabetes Mellitus Tipo 1/patologia , Humanos , Imunidade Inata/fisiologia , Inflamação/metabolismo , Células Secretoras de Insulina/imunologia , Células Secretoras de Insulina/metabolismo , Células Secretoras de Insulina/patologia , Ilhotas Pancreáticas/imunologia , Ilhotas Pancreáticas/metabolismo , Transdução de Sinais/fisiologiaRESUMO
Recombinant adeno-associated viruses (rAAVs) serve as vectors for in vivo gene delivery in both mice and humans, and have broad applicability for the treatment of genetic diseases. Clinical trials with AAV vectors have demonstrated promise and safety in several human diseases. However, the in vivo validation of novel AAV constructs expressing products that act specifically on human cells and tissues is limited by a paucity of effective translatable models. Humanized mice that are engrafted with human cells, tissues, and immune systems offer strong potential to test the biological effectiveness of AAV vectors on human cells and tissues. Using the BLT (bone marrow, liver, thymus) humanized NOD-scid Il2rgnull (NSG) mouse model, which enables efficient development of HLA-restricted effector and regulatory T cells (Tregs), we have evaluated the delivery and function of human interleukin (IL)-2 by an AAV vector. Humanized mice treated with an AAV vector expressing human IL-2 showed a significant and sustained increase in the number of functional human FOXP3+CD4+ Tregs. The expression of human IL-2 did not significantly change the levels or activation status of conventional T-cell subsets. Numbers of activated human natural killer cells were also increased significantly in humanized mice treated with the IL-2 vector. These data recapitulate observations in clinical trials of IL-2 therapy and collectively show that humanized mouse models offer a translational platform for testing the efficacy of AAV vectors targeting human immune cells.
Assuntos
Dependovirus , Expressão Gênica , Terapia Genética , Homeostase , Interleucina-2 , Linfócitos T Reguladores/imunologia , Animais , Dependovirus/genética , Dependovirus/imunologia , Vetores Genéticos/genética , Vetores Genéticos/imunologia , Homeostase/genética , Homeostase/imunologia , Humanos , Interleucina-2/biossíntese , Interleucina-2/genética , Interleucina-2/imunologia , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Linfócitos T Reguladores/patologiaRESUMO
Enteroviral infections have been associated with the development of type 1 diabetes (T1D), a chronic inflammatory disease characterized by autoimmune destruction of insulin-producing pancreatic beta cells. Cultured human islets, including the insulin-producing beta cells, can be infected with coxsackievirus B4 (CVB4) and thus are useful for understanding cellular responses to infection. We performed quantitative mass spectrometry analysis on cultured primary human islets infected with CVB4 to identify molecules and pathways altered upon infection. Corresponding uninfected controls were included in the study for comparative protein expression analyses. Proteins were significantly and differentially regulated in human islets challenged with virus compared with their uninfected counterparts. Complementary analyses of gene transcripts in CVB4-infected primary islets over a time course validated the induction of RNA transcripts for many of the proteins that were increased in the proteomics studies. Notably, infection with CVB4 results in a considerable decrease in insulin. Genes/proteins modulated during CVB4 infection also include those involved in activation of immune responses, including type I interferon pathways linked to T1D pathogenesis and with antiviral, cell repair, and inflammatory properties. Our study applies proteomics analyses to cultured human islets challenged with virus and identifies target proteins that could be useful in T1D interventions.
RESUMO
BACKGROUND/AIMS: Studies of human cadaveric pancreas specimens indicate that pancreas inflammation plays an important role in type 1 diabetes pathogenesis. Due to the inaccessibility of pancreas in living patients, imaging technology to visualize pancreas inflammation is much in need. In this study, we investigated the feasibility of utilizing ultrasound imaging to assess pancreas inflammation longitudinally in living rats during the progression leading to type 1 diabetes onset. METHODS: The virus-inducible BBDR type 1 diabetes rat model was used to systematically investigate pancreas changes that occur prior to and during development of autoimmunity. The nearly 100% diabetes incidence upon virus induction and the highly consistent time course of this rat model make longitudinal imaging examination possible. A combination of histology, immunoblotting, flow cytometry, and ultrasound imaging technology was used to identify stage-specific pancreas changes. RESULTS: Our histology data indicated that exocrine pancreas tissue of the diabetes-induced rats underwent dramatic changes, including blood vessel dilation and increased CD8+ cell infiltration, at a very early stage of disease initiation. Ultrasound imaging data revealed significant acute and persistent pancreas inflammation in the diabetes-induced rats. The pancreas micro-vasculature was significantly dilated one day after diabetes induction, and large blood vessel (superior mesenteric artery in this study) dilation and inflammation occurred several days later, but still prior to any observable autoimmune cell infiltration of the pancreatic islets. CONCLUSIONS: Our data demonstrate that ultrasound imaging technology can detect pancreas inflammation in living rats during the development of type 1 diabetes. Due to ultrasound's established use as a non-invasive diagnostic tool, it may prove useful in a clinical setting for type 1 diabetes risk prediction prior to autoimmunity and to assess the effectiveness of potential therapeutics.
Assuntos
Diabetes Mellitus Tipo 1/diagnóstico por imagem , Diabetes Mellitus Tipo 1/patologia , Pancreatite/diagnóstico por imagem , Pancreatite/patologia , Ultrassonografia , Animais , Apoptose , Resistência Capilar , Caspase 3/metabolismo , Diabetes Mellitus Experimental , Diabetes Mellitus Tipo 1/etiologia , Modelos Animais de Doenças , Humanos , Ilhotas Pancreáticas/imunologia , Ilhotas Pancreáticas/metabolismo , Ilhotas Pancreáticas/patologia , Microvasos , Pâncreas/irrigação sanguínea , Pâncreas/metabolismo , Pâncreas/patologia , Pancreatite/complicações , Pancreatite/metabolismo , Prognóstico , Ratos , Subpopulações de Linfócitos T/imunologia , Subpopulações de Linfócitos T/metabolismo , Subpopulações de Linfócitos T/patologia , Ultrassonografia/métodosRESUMO
OBJECTIVE: Glucagon-like peptide-1 induces glucose-dependent insulin secretion and, in rodents, increases proliferation and survival of pancreatic beta cells. To investigate the effects on human beta cells, we used immunodeficient mice transplanted with human islets. The goal was to determine whether lixisenatide, a glucagon-like peptide-1 receptor agonist, improves human islet function and survival in vivo. METHODS: Five independent transplant studies were conducted with human islets from five individual donors. Diabetic human islet-engrafted immunodeficient mice were treated with lixisenatide (50, 150, and 500 µg/kg) or vehicle. Islet function was determined by blood glucose, plasma human insulin/C-peptide, and glucose tolerance tests. Grafts were analyzed for total beta- and alpha-cell number, percent proliferation, and levels of apoptosis. RESULTS: Diabetic mice transplanted with marginal human islet mass and treated with lixisenatide were restored to euglycemia more rapidly than vehicle-treated mice. Glucose tolerance tests, human plasma insulin, and glucose-stimulation indices of lixisenatide-treated mice were significantly improved compared to vehicle-treated mice. The percentages of proliferating or apoptotic beta cells at graft recovery were not different between lixisenatide-treated and vehicle-treated mice. Nevertheless, in one experiment we found a significant twofold to threefold increase in human beta-cell numbers in lixisenatide-treated compared to vehicle-treated mice. CONCLUSION: Diabetic human islet-engrafted immunodeficient mice treated with lixisenatide show improved restoration of normoglycemia, human plasma insulin, and glucose tolerance compared to vehicle-treated mice engrafted with the same donor islets. Because the proliferative capacity of human beta cells is limited, improved beta-cell survival coupled with enhanced beta-cell function following lixisenatide treatment may provide the greatest benefit for diabetic patients with reduced functional islet mass.
RESUMO
PURPOSE OF REVIEW: Therapies that increase functional ß-cell mass may be the best long-term treatment for diabetes. Significant resources are devoted toward this goal, and progress is occurring at a rapid pace. Here, we summarize recent advances relevant to human ß-cell regeneration. RECENT FINDINGS: New ß-cells arise from proliferation of pre-existing ß-cells or transdifferentiation from other cell types. In addition, dedifferentiated ß-cells may populate islets in diabetes, possibly representing a pool of cells that could redifferentiate into functional ß-cells. Advances in finding strategies to drive ß-cell proliferation include new insight into proproliferative factors, both circulating and local, and elements intrinsic to the ß-cell, such as cell cycle machinery and regulation of gene expression through epigenetic modification and noncoding RNAs. Controversy continues in the arena of generation of ß-cells by transdifferentiation from exocrine, ductal, and alpha cells, with studies producing both supporting and opposing data. Progress has been made in redifferentiation of ß-cells that have lost expression of ß-cell markers. SUMMARY: Although significant progress has been made, and promising avenues exist, more work is needed to achieve the goal of ß-cell regeneration as a treatment for diabetes.
Assuntos
Diabetes Mellitus Tipo 1/fisiopatologia , Diabetes Mellitus Tipo 2/fisiopatologia , Células Secretoras de Insulina , Engenharia Celular , Proliferação de Células , Transdiferenciação Celular , Diabetes Mellitus Tipo 1/genética , Diabetes Mellitus Tipo 2/genética , Feminino , Regulação da Expressão Gênica , Humanos , Células Secretoras de Insulina/fisiologia , Células Secretoras de Insulina/transplante , Masculino , Fatores de Crescimento Neural/metabolismo , RegeneraçãoRESUMO
Majewski osteodysplastic primordial dwarfism type II (MOPDII) is caused by mutations in the centrosome gene pericentrin (PCNT) that lead to severe pre- and postnatal growth retardation. As in MOPDII patients, disruption of pericentrin (Pcnt) in mice caused a number of abnormalities including microcephaly, aberrant hemodynamics analyzed by in utero echocardiography, and cardiovascular anomalies; the latter being associated with mortality, as in the human condition. To identify the mechanisms underlying these defects, we tested for changes in cell and molecular function. All Pcnt(-/-) mouse tissues and cells examined showed spindle misorientation. This mouse phenotype was associated with misdirected ventricular septal growth in the heart, decreased proliferative symmetric divisions in brain neural progenitors, and increased misoriented divisions in fibroblasts; the same phenotype was seen in fibroblasts from three MOPDII individuals. Misoriented spindles were associated with disrupted astral microtubules and near complete loss of a unique set of centrosome proteins from spindle poles (ninein, Cep215, centriolin). All these proteins appear to be crucial for microtubule anchoring and all interacted with Pcnt, suggesting that Pcnt serves as a molecular scaffold for this functionally linked set of spindle pole proteins. Importantly, Pcnt disruption had no detectable effect on localization of proteins involved in the cortical polarity pathway (NuMA, p150(glued), aPKC). Not only do these data reveal a spindle-pole-localized complex for spindle orientation, but they identify key spindle symmetry proteins involved in the pathogenesis of MOPDII.
Assuntos
Antígenos/genética , Nanismo/genética , Retardo do Crescimento Fetal/genética , Microcefalia/genética , Microtúbulos/genética , Mitose , Osteocondrodisplasias/genética , Fuso Acromático/genética , Animais , Antígenos/metabolismo , Centrossomo/metabolismo , Modelos Animais de Doenças , Nanismo/fisiopatologia , Embrião de Mamíferos/embriologia , Retardo do Crescimento Fetal/fisiopatologia , Humanos , Camundongos , Microcefalia/fisiopatologia , Osteocondrodisplasias/fisiopatologia , Fuso Acromático/metabolismo , Polos do Fuso/genética , Polos do Fuso/metabolismoRESUMO
PURPOSE: Dipeptidyl-peptidase-4 (DPP-4) inhibitors are known to increase insulin secretion and beta cell proliferation in rodents. To investigate the effects on human beta cells in vivo, we utilize immunodeficient mice transplanted with human islets. The study goal was to determine the efficacy of alogliptin, a DPP-4 inhibitor, to enhance human beta cell function and proliferation in an in vivo context using diabetic immunodeficient mice engrafted with human pancreatic islets. METHODS: Streptozotocin-induced diabetic NOD-scid IL2rγ(null) (NSG) mice were transplanted with adult human islets in three separate trials. Transplanted mice were treated daily by gavage with alogliptin (30 mg/kg/day) or vehicle control. Islet graft function was compared using glucose tolerance tests and non-fasting plasma levels of human insulin and C-peptide; beta cell proliferation was determined by bromodeoxyuridine (BrdU) incorporation. RESULTS: Glucose tolerance tests were significantly improved by alogliptin treatment for mice transplanted with islets from two of the three human islet donors. Islet-engrafted mice treated with alogliptin also had significantly higher plasma levels of human insulin and C-peptide compared to vehicle controls. The percentage of insulin+BrdU+ cells in human islet grafts from alogliptin-treated mice was approximately 10-fold more than from vehicle control mice, consistent with a significant increase in human beta cell proliferation. CONCLUSION: Human islet-engrafted immunodeficient mice treated with alogliptin show improved human insulin secretion and beta cell proliferation compared to control mice engrafted with the same donor islets. Immunodeficient mice transplanted with human islets provide a useful model to interrogate potential therapies to improve human islet function and survival in vivo.