RESUMO
Implantable medical devices have revolutionized modern medicine. However, immune-mediated foreign body response (FBR) to the materials of these devices can limit their function or even induce failure. Here we describe long-term controlled-release formulations for local anti-inflammatory release through the development of compact, solvent-free crystals. The compact lattice structure of these crystals allows for very slow, surface dissolution and high drug density. These formulations suppress FBR in both rodents and non-human primates for at least 1.3 years and 6 months, respectively. Formulations inhibited fibrosis across multiple implant sites-subcutaneous, intraperitoneal and intramuscular. In particular, incorporation of GW2580, a colony stimulating factor 1 receptor inhibitor, into a range of devices, including human islet microencapsulation systems, electrode-based continuous glucose-sensing monitors and muscle-stimulating devices, inhibits fibrosis, thereby allowing for extended function. We believe that local, long-term controlled release with the crystal formulations described here enhances and extends function in a range of medical devices and provides a generalized solution to the local immune response to implanted biomaterials.
Assuntos
Fibrose/etiologia , Fibrose/prevenção & controle , Próteses e Implantes/efeitos adversos , Animais , Preparações de Ação Retardada , Composição de Medicamentos , Macrófagos/efeitos dos fármacos , RoedoresRESUMO
The efficacy of implanted biomedical devices is often compromised by host recognition and subsequent foreign body responses. Here, we demonstrate the role of the geometry of implanted materials on their biocompatibility in vivo. In rodent and non-human primate animal models, implanted spheres 1.5 mm and above in diameter across a broad spectrum of materials, including hydrogels, ceramics, metals and plastics, significantly abrogated foreign body reactions and fibrosis when compared with smaller spheres. We also show that for encapsulated rat pancreatic islet cells transplanted into streptozotocin-treated diabetic C57BL/6 mice, islets prepared in 1.5-mm alginate capsules were able to restore blood-glucose control for up to 180 days, a period more than five times longer than for transplanted grafts encapsulated within conventionally sized 0.5-mm alginate capsules. Our findings suggest that the in vivo biocompatibility of biomedical devices can be significantly improved simply by tuning their spherical dimensions.
Assuntos
Reação a Corpo Estranho/imunologia , Animais , Camundongos , Camundongos Endogâmicos C57BL , PrimatasRESUMO
Diabetes is a pathological condition characterized by relative insulin deficiency, persistent hyperglycemia, and, consequently, diffuse micro- and macrovascular disease. One therapeutic strategy is to amplify insulin-secretion capacity by increasing the number of the insulin-producing ß cells without triggering a generalized proliferative response. Here, we present the development of a small-molecule screening platform for the identification of molecules that increase ß-cell replication. Using this platform, we identify a class of compounds [adenosine kinase inhibitors (ADK-Is)] that promote replication of primary ß cells in three species (mouse, rat, and pig). Furthermore, the replication effect of ADK-Is is cell type-selective: treatment of islet cell cultures with ADK-Is increases replication of ß cells but not that of α cells, PP cells, or fibroblasts. Short-term in vivo treatment with an ADK-I also increases ß-cell replication but not exocrine cell or hepatocyte replication. Therefore, we propose ADK inhibition as a strategy for the treatment of diabetes.
Assuntos
Adenosina Quinase/farmacologia , Regulação da Expressão Gênica , Células Secretoras de Insulina/citologia , Animais , Feminino , Fibroblastos/metabolismo , Receptor do Peptídeo Semelhante ao Glucagon 1 , Glucose/metabolismo , Hepatócitos/citologia , Insulina/metabolismo , Ilhotas Pancreáticas/citologia , Camundongos , Camundongos Endogâmicos C57BL , Ratos , Ratos Sprague-Dawley , Receptores de Glucagon/metabolismo , Suínos , Serina-Treonina Quinases TOR/metabolismoRESUMO
Biomedical devices such as islet-encapsulating systems are used for treatment of type 1 diabetes (T1D). Despite recent strides in preventing biomaterial fibrosis, challenges remain for biomaterial scaffolds due to limitations on cells contained within. The study demonstrates that proliferation and function of insulinoma (INS-1) cells as well as pancreatic rat islets may be improved in alginate hydrogels with optimized gel%, crosslinking, and stiffness. Quantitative polymerase chain reaction (qPCR)-based graft phenotyping of encapsulated INS-1 cells and pancreatic islets identified a hydrogel stiffness range between 600 and 1000 Pa that improved insulin Ins and Pdx1 gene expression as well as glucose-sensitive insulin-secretion. Barium chloride (BaCl2) crosslinking time is also optimized due to toxicity of extended exposure. Despite possible benefits to cell viability, calcium chloride (CaCl2)-crosslinked hydrogels exhibited a sharp storage modulus loss in vitro. Despite improved stability, BaCl2-crosslinked hydrogels also exhibited stiffness losses over the same timeframe. It is believed that this is due to ion exchange with other species in culture media, as hydrogels incubated in dIH2O exhibited significantly improved stability. To maintain cell viability and function while increasing 3D matrix stability, a range of useful media:dIH2O dilution ratios for use are identified. Such findings have importance to carry out characterization and optimization of cell microphysiological systems with high fidelity in vitro.
Assuntos
Alginatos , Sobrevivência Celular , Diabetes Mellitus Tipo 1 , Hidrogéis , Alginatos/química , Alginatos/farmacologia , Animais , Diabetes Mellitus Tipo 1/metabolismo , Diabetes Mellitus Tipo 1/terapia , Ratos , Sobrevivência Celular/efeitos dos fármacos , Hidrogéis/química , Hidrogéis/farmacologia , Ilhotas Pancreáticas/metabolismo , Ilhotas Pancreáticas/efeitos dos fármacos , Cloretos/metabolismo , Cloretos/farmacologia , Transplante das Ilhotas Pancreáticas/métodos , Insulina/metabolismo , Alicerces Teciduais/química , Linhagem Celular Tumoral , Compostos de Bário/farmacologia , Compostos de Bário/químicaRESUMO
Replenishment of pancreatic beta cells is a key to the cure for diabetes. Beta cells regeneration is achieved predominantly by self-replication especially in rodents, but it was also shown that pancreatic duct cells can transdifferentiate into beta cells. How pancreatic duct cells undergo transdifferentiated and whether we could manipulate the transdifferentiation to replenish beta cell mass is not well understood. Using a genome-wide CRISPR screen, we discovered that loss-of-function of ALDH3B2 is sufficient to transdifferentiate human pancreatic duct cells into functional beta-like cells. The transdifferentiated cells have significant increase in beta cell marker genes expression, secrete insulin in response to glucose, and reduce blood glucose when transplanted into diabetic mice. Our study identifies a novel gene that could potentially be targeted in human pancreatic duct cells to replenish beta cell mass for diabetes therapy.
RESUMO
Pancreatic ß-cells are specialized for coupling glucose metabolism to insulin peptide production and secretion. Acute glucose exposure robustly and coordinately increases translation of proinsulin and proteins required for secretion of mature insulin peptide. By contrast, chronically elevated glucose levels that occur during diabetes impair ß-cell insulin secretion and have been shown experimentally to suppress insulin translation. Whether translation of other genes critical for insulin secretion are similarly downregulated by chronic high glucose is unknown. Here, we used high-throughput ribosome profiling and nascent proteomics in MIN6 insulinoma cells to elucidate the genome-wide impact of sustained high glucose on ß-cell mRNA translation. Prior to induction of ER stress or suppression of global translation, sustained high glucose suppressed glucose-stimulated insulin secretion and downregulated translation of not only insulin, but also of mRNAs related to insulin secretory granule formation, exocytosis, and metabolism-coupled insulin secretion. Translation of these mRNAs was also downregulated in primary rat and human islets following ex-vivo incubation with sustained high glucose and in an in vivo model of chronic mild hyperglycemia. Furthermore, translational downregulation decreased cellular abundance of these proteins. Our findings uncover a translational regulatory circuit during ß-cell glucose toxicity that impairs expression of proteins with critical roles in ß-cell function.
RESUMO
Pancreatic ß cells are specialized for coupling glucose metabolism to insulin peptide production and secretion. Acute glucose exposure robustly and coordinately increases translation of proinsulin and proteins required for secretion of mature insulin peptide. By contrast, chronically elevated glucose levels that occur during diabetes impair ß cell insulin secretion and have been shown experimentally to suppress insulin translation. Whether translation of other genes critical for insulin secretion is similarly downregulated by chronic high glucose is unknown. Here, we used high-throughput ribosome profiling and nascent proteomics in MIN6 insulinoma cells to elucidate the genome-wide impact of sustained high glucose on ß cell mRNA translation. Before induction of ER stress or suppression of global translation, sustained high glucose suppressed glucose-stimulated insulin secretion and downregulated translation of not only insulin, but also mRNAs related to insulin secretory granule formation, exocytosis, and metabolism-coupled insulin secretion. Translation of these mRNAs was also downregulated in primary rat and human islets following ex vivo incubation with sustained high glucose and in an in vivo model of chronic mild hyperglycemia. Furthermore, translational downregulation decreased cellular abundance of these proteins. Our study uncovered a translational regulatory circuit during ß cell glucose toxicity that impairs expression of proteins with critical roles in ß cell function.
Assuntos
Hiperglicemia , Células Secretoras de Insulina , Ilhotas Pancreáticas , Neoplasias Pancreáticas , Ratos , Humanos , Animais , Secreção de Insulina , RNA Mensageiro/metabolismo , Insulina/metabolismo , Hiperglicemia/genética , Hiperglicemia/metabolismo , Glucose/metabolismo , Células Secretoras de Insulina/metabolismo , Peptídeos/metabolismo , Neoplasias Pancreáticas/metabolismo , Ilhotas Pancreáticas/metabolismoRESUMO
Biomedical devices comprise a major component of modern medicine, however immune-mediated fibrosis and rejection can limit their function over time. Here, we describe a humanized mouse model that recapitulates fibrosis following biomaterial implantation. Cellular and cytokine responses to multiple biomaterials were evaluated across different implant sites. Human innate immune macrophages were verified as essential to biomaterial rejection in this model and were capable of cross-talk with mouse fibroblasts for collagen matrix deposition. Cytokine and cytokine receptor array analysis confirmed core signaling in the fibrotic cascade. Foreign body giant cell formation, often unobserved in mice, was also prominent. Last, high-resolution microscopy coupled with multiplexed antibody capture digital profiling analysis supplied spatial resolution of rejection responses. This model enables the study of human immune cell-mediated fibrosis and interactions with implanted biomaterials and devices.
Assuntos
Materiais Biocompatíveis , Corpos Estranhos , Humanos , Animais , Camundongos , Reação a Corpo Estranho/etiologia , Modelos Animais de Doenças , Citocinas , FibroseRESUMO
OBJECTIVE: As diabetes develops, marked reductions of insulin secretion are associated with very modest elevations of glucose. We wondered if these glucose changes disrupt beta cell differentiation enough to account for the altered function. METHODS: Rats were subjected to 90% partial pancreatectomies and those with only mild glucose elevations 4 weeks or 10 weeks after surgery had major alterations of gene expression in their islets as determined by RNAseq. RESULTS: Changes associated with glucose toxicity demonstrated that many of the critical genes responsible for insulin secretion were downregulated while the expression of normally suppressed genes increased. Also, there were marked changes in genes associated with replication, aging, senescence, stress, inflammation, and increased expression of genes controlling both class I and II MHC antigens. CONCLUSIONS: These findings suggest that mild glucose elevations in the early stages of diabetes lead to phenotypic changes that adversely affect beta cell function, growth, and vulnerability.
Assuntos
Glicemia/metabolismo , Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Tipo 2/metabolismo , Hiperglicemia/metabolismo , Células Secretoras de Insulina/metabolismo , Animais , Diferenciação Celular , Diabetes Mellitus Experimental/etiologia , Diabetes Mellitus Tipo 2/etiologia , Modelos Animais de Doenças , Regulação para Baixo , Expressão Gênica , Hiperglicemia/etiologia , Insulina/metabolismo , Secreção de Insulina/genética , Transplante das Ilhotas Pancreáticas/efeitos adversos , Transplante das Ilhotas Pancreáticas/métodos , Masculino , Pancreatectomia/efeitos adversos , Pancreatectomia/métodos , RNA Mensageiro/genética , Ratos , Ratos Endogâmicos LewRESUMO
Type 1 diabetes (T1D) is caused by the autoimmune destruction of pancreatic beta cells. Pluripotent stem cells can now be differentiated into beta cells, thus raising the prospect of a cell replacement therapy for T1D. However, autoimmunity would rapidly destroy newly transplanted beta cells. Using a genome-scale CRISPR screen in a mouse model for T1D, we show that deleting RNLS, a genome-wide association study candidate gene for T1D, made beta cells resistant to autoimmune killing. Structure-based modelling identified the U.S. Food and Drug Administration-approved drug pargyline as a potential RNLS inhibitor. Oral pargyline treatment protected transplanted beta cells in diabetic mice, thus leading to disease reversal. Furthermore, pargyline prevented or delayed diabetes onset in several mouse models for T1D. Our results identify RNLS as a modifier of beta cell vulnerability and as a potential therapeutic target to avert beta cell loss in T1D.
Assuntos
Sistemas CRISPR-Cas , Diabetes Mellitus Tipo 1/tratamento farmacológico , Estudo de Associação Genômica Ampla , Células Secretoras de Insulina/efeitos dos fármacos , Monoaminoxidase/efeitos dos fármacos , Animais , Autoimunidade/efeitos dos fármacos , Diabetes Mellitus Tipo 1/imunologia , Diabetes Mellitus Tipo 1/patologia , Estresse do Retículo Endoplasmático , Inibidores Enzimáticos/farmacologia , Feminino , Células-Tronco Pluripotentes Induzidas/imunologia , Células Secretoras de Insulina/imunologia , Células Secretoras de Insulina/patologia , Transplante das Ilhotas Pancreáticas , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos NOD , Camundongos Knockout , Mutação , Pargilina/farmacologiaRESUMO
The long-term function of transplanted therapeutic cells typically requires systemic immune suppression. Here, we show that a retrievable implant comprising a silicone reservoir and a porous polymeric membrane protects human cells encapsulated in it after implant transplantation in the intraperitoneal space of immunocompetent mice. Membranes with pores 1 µm in diameter allowed host macrophages to migrate into the device without the loss of transplanted cells, whereas membranes with pore sizes <0.8 µm prevented their infiltration by immune cells. A synthetic polymer coating prevented fibrosis and was necessary for the long-term function of the device. For >130 days, the device supported human cells engineered to secrete erythropoietin in immunocompetent mice, as well as transgenic human cells carrying an inducible gene circuit for the on-demand secretion of erythropoietin. Pancreatic islets from rats encapsulated in the device and implanted in diabetic mice restored normoglycaemia in the mice for over 75 days. The biocompatible device provides a retrievable solution for the transplantation of engineered cells in the absence of immunosuppression.
Assuntos
Transplante de Células/métodos , Sobrevivência de Enxerto , Próteses e Implantes , Animais , Cápsulas , Transplante de Células/instrumentação , Materiais Revestidos Biocompatíveis , Diabetes Mellitus Experimental/terapia , Desenho de Equipamento , Eritropoetina/genética , Eritropoetina/metabolismo , Reação a Corpo Estranho/prevenção & controle , Células HEK293 , Humanos , Ilhotas Pancreáticas , Transplante das Ilhotas Pancreáticas/instrumentação , Transplante das Ilhotas Pancreáticas/métodos , Camundongos , Permeabilidade , Ratos , Transplante HeterólogoRESUMO
Traditionally, studies of cells and tissues have been performed on isolated primary cells or immortalized cell lines by culturing them in 2D culture dishes or flasks. However, a caveat regarding 2D culture is that the cells poorly recapitulate their in vivo counterparts, mainly due to a lack of 3D cell-cell and cell-extracellular matrix interactions. In recent years, the development of in vitro organoids as 3D culture has gained substantial attention as a model to study different tissues. In adults, pancreatic ductal cells are considered as a source of stem or progenitor cells, so developing new methods to study ductal cells would be useful. Here, we provide a protocol to isolate mouse pancreatic ductal cells and a cost-effective protocol to generate 3D organoid structures from such ductal cells. Additionally, we have devised a protocol for immunostaining of intact whole organoids without sectioning. © 2018 by John Wiley & Sons, Inc.
Assuntos
Organoides/citologia , Ductos Pancreáticos/citologia , Técnicas de Cultura de Tecidos , Animais , Camundongos , Camundongos Endogâmicos C57BLRESUMO
Type 2 diabetes (T2D) is an age-related disease. Although changes in function and proliferation of aged ß cells resemble those preceding the development of diabetes, the contribution of ß cell aging and senescence remains unclear. We generated a ß cell senescence signature and found that insulin resistance accelerates ß cell senescence leading to loss of function and cellular identity and worsening metabolic profile. Senolysis (removal of senescent cells), using either a transgenic INK-ATTAC model or oral ABT263, improved glucose metabolism and ß cell function while decreasing expression of markers of aging, senescence, and senescence-associated secretory profile (SASP). Beneficial effects of senolysis were observed in an aging model as well as with insulin resistance induced both pharmacologically (S961) and physiologically (high-fat diet). Human senescent ß cells also responded to senolysis, establishing the foundation for translation. These novel findings lay the framework to pursue senolysis of ß cells as a preventive and alleviating strategy for T2D.
Assuntos
Diabetes Mellitus Tipo 2/tratamento farmacológico , Diabetes Mellitus Tipo 2/metabolismo , Glucose/metabolismo , Células Secretoras de Insulina/metabolismo , Compostos de Anilina/uso terapêutico , Animais , Peso Corporal/fisiologia , Células Cultivadas , Senescência Celular/fisiologia , Citometria de Fluxo , Humanos , Técnicas In Vitro , Resistência à Insulina/fisiologia , Células Secretoras de Insulina/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos C57BL , Sulfonamidas/uso terapêuticoRESUMO
Pancreatic duct epithelial cells have been suggested as a source of progenitors for pancreatic growth and regeneration. However, genetic lineage-tracing experiments with pancreatic duct-specific Cre expression have given conflicting results. Using immunofluorescence and flow cytometry, we show heterogeneous expression of both HNF1ß and SOX9 in adult human and murine ductal epithelium. Their expression was dynamic and diminished significantly after induced replication. Purified pancreatic duct cells formed organoid structures in 3D culture, and heterogeneity of expression of Hnf1ß and Sox9 was maintained even after passaging. Using antibodies against a second cell surface molecule CD51 (human) or CD24 (mouse), we could isolate living subpopulations of duct cells enriched for high or low expression of HNF1ß and SOX9. Only the CD24high (Hnfßhigh/Sox9high) subpopulation was able to form organoids.
Assuntos
Fator 1-beta Nuclear de Hepatócito/metabolismo , Ductos Pancreáticos/metabolismo , Fatores de Transcrição SOX9/metabolismo , Adulto , Idoso , Animais , Antígeno CD24/metabolismo , Células Epiteliais/metabolismo , Humanos , Integrina alfaV/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Pessoa de Meia-Idade , Organoides/metabolismoRESUMO
The in vivo microenvironment of tissues provides myriad unique signals to cells. Thus, following isolation, many cell types change in culture, often preserving some but not all of their in vivo characteristics in culture. At least some of the in vivo microenvironment may be mimicked by providing specific cues to cultured cells. Here, we show that after isolation and during maintenance in culture, adherent rat islets reduce expression of key ß-cell transcription factors necessary for ß-cell function and that soluble pancreatic decellularized matrix (DCM) can enhance ß-cell gene expression. Following chromatographic fractionation of pancreatic DCM, we performed proteomics to identify soluble factors that can maintain ß-cell stability and function. We identified Apolipoprotein E (ApoE) as an extracellular protein that significantly increased the expression of key ß-cell genes. The ApoE effect on beta cells was mediated at least in part through the JAK/STAT signaling pathway. Together, these results reveal a role for ApoE as an extracellular factor that can maintain the mature ß-cell gene expression profile.
Assuntos
Apolipoproteínas E/metabolismo , Espaço Extracelular/metabolismo , Regulação da Expressão Gênica/fisiologia , Células Secretoras de Insulina/metabolismo , Animais , Células Cultivadas , Proteoglicanas de Heparan Sulfato/metabolismo , Ilhotas Pancreáticas/metabolismo , Janus Quinases/metabolismo , Proteoma , Proteômica , Ratos Sprague-Dawley , Receptores de LDL/metabolismo , Fatores de Transcrição STAT/metabolismo , Técnicas de Cultura de TecidosRESUMO
The transplantation of pancreatic islet cells could restore glycaemic control in patients with type-I diabetes. Microspheres for islet encapsulation have enabled long-term glycaemic control in diabetic rodent models; yet human patients transplanted with equivalent microsphere formulations have experienced only transient islet-graft function, owing to a vigorous foreign-body reaction (FBR), to pericapsular fibrotic overgrowth (PFO) and, in upright bipedal species, to the sedimentation of the microspheres within the peritoneal cavity. Here, we report the results of the testing, in non-human primate (NHP) models, of seven alginate formulations that were efficacious in rodents, including three that led to transient islet-graft function in clinical trials. Although one month post-implantation all formulations elicited significant FBR and PFO, three chemically modified, immune-modulating alginate formulations elicited reduced FBR. In conjunction with a minimally invasive transplantation technique into the bursa omentalis of NHPs, the most promising chemically modified alginate derivative (Z1-Y15) protected viable and glucose-responsive allogeneic islets for 4 months without the need for immunosuppression. Chemically modified alginate formulations may enable the long-term transplantation of islets for the correction of insulin deficiency.
RESUMO
We hypothesized that the known heterogeneity of pancreatic ß cells was due to subpopulations of ß cells at different stages of their life cycle with different functional capacities and that further changes occur with metabolic stress and aging. We identified new markers of aging in ß cells, including IGF1R. In ß cells IGF1R expression correlated with age, dysfunction, and expression of known age markers p16ink4a, p53BP1, and senescence-associated ß-galactosidase. The new markers showed striking heterogeneity both within and between islets in both mouse and human pancreas. Acute induction of insulin resistance with an insulin receptor antagonist or chronic ER stress resulted in increased expression of aging markers, providing insight into how metabolic stress might accelerate dysfunction and decline of ß cells. These novel findings about ß cell and islet heterogeneity, and how they change with age, open up an entirely new set of questions about the pathogenesis of type 2 diabetes.
Assuntos
Biomarcadores/metabolismo , Senescência Celular , Resistência à Insulina , Células Secretoras de Insulina/metabolismo , Células Secretoras de Insulina/patologia , Adolescente , Adulto , Idoso , Envelhecimento/metabolismo , Animais , Diabetes Mellitus Tipo 2/metabolismo , Diabetes Mellitus Tipo 2/patologia , Feminino , Citometria de Fluxo , Regulação da Expressão Gênica no Desenvolvimento , Glucose/metabolismo , Proteínas de Fluorescência Verde/metabolismo , Técnica de Placa Hemolítica , Humanos , Insulina/metabolismo , Masculino , Camundongos Endogâmicos C57BL , Pessoa de Meia-Idade , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptor IGF Tipo 1/metabolismo , Estresse Fisiológico , Proteína 1 de Ligação à Proteína Supressora de Tumor p53/metabolismo , Adulto JovemRESUMO
Congenital hyperinsulinism of infancy (CHI) can be caused by inactivating mutations in the gene encoding short-chain 3-hydroxyacyl-CoA dehydrogenase (SCHAD), a ubiquitously expressed enzyme involved in fatty acid oxidation. The hypersecretion of insulin may be explained by a loss of interaction between SCHAD and glutamate dehydrogenase in the pancreatic ß-cells. However, there is also a general accumulation of metabolites specific for the enzymatic defect in affected individuals. It remains to be explored whether hypoglycemia in SCHAD CHI can be uncoupled from the systemic effect on fatty acid oxidation. We therefore transplanted islets from global SCHAD knockout (SCHADKO) mice into mice with streptozotocin-induced diabetes. After transplantation, SCHADKO islet recipients exhibited significantly lower random and fasting blood glucose compared with mice transplanted with normal islets or nondiabetic, nontransplanted controls. Furthermore, intraperitoneal glucose tolerance was improved in animals receiving SCHADKO islets compared with those receiving normal islets. Graft ß-cell proliferation and apoptosis rates were similar in the two transplantation groups. We conclude that hypoglycemia in SCHAD-CHI is islet cell-autonomous.
Assuntos
3-Hidroxiacil-CoA Desidrogenase/deficiência , Hiperinsulinismo Congênito/enzimologia , Hipoglicemia/enzimologia , Células Secretoras de Insulina/metabolismo , Fenótipo , Animais , Hiperinsulinismo Congênito/genética , Glutamato Desidrogenase/metabolismo , Hipoglicemia/genética , Insulina/metabolismo , Secreção de Insulina , Masculino , Camundongos , Camundongos KnockoutRESUMO
The capacity of pancreatic ß cells to maintain glucose homeostasis during chronic physiologic and immunologic stress is important for cellular and metabolic homeostasis. Insulin receptor substrate 2 (IRS2) is a regulated adapter protein that links the insulin and IGF1 receptors to downstream signaling cascades. Since strategies to maintain or increase IRS2 expression can promote ß cell growth, function, and survival, we conducted a screen to find small molecules that can increase IRS2 mRNA in isolated human pancreatic islets. We identified 77 compounds, including 15 that contained a tricyclic core. To establish the efficacy of our approach, one of the tricyclic compounds, trimeprazine tartrate, was investigated in isolated human islets and in mouse models. Trimeprazine is a first-generation antihistamine that acts as a partial agonist against the histamine H1 receptor (H1R) and other GPCRs, some of which are expressed on human islets. Trimeprazine promoted CREB phosphorylation and increased the concentration of IRS2 in islets. IRS2 was required for trimeprazine to increase nuclear Pdx1, islet mass, ß cell replication and function, and glucose tolerance in mice. Moreover, trimeprazine synergized with anti-CD3 Abs to reduce the progression of diabetes in NOD mice. Finally, it increased the function of human islet transplants in streptozotocin-induced (STZ-induced) diabetic mice. Thus, trimeprazine, its analogs, or possibly other compounds that increase IRS2 in islets and ß cells without adverse systemic effects might provide mechanism-based strategies to prevent the progression of diabetes.
RESUMO
The transplantation of glucose-responsive, insulin-producing cells offers the potential for restoring glycemic control in individuals with diabetes. Pancreas transplantation and the infusion of cadaveric islets are currently implemented clinically, but these approaches are limited by the adverse effects of immunosuppressive therapy over the lifetime of the recipient and the limited supply of donor tissue. The latter concern may be addressed by recently described glucose-responsive mature beta cells that are derived from human embryonic stem cells (referred to as SC-ß cells), which may represent an unlimited source of human cells for pancreas replacement therapy. Strategies to address the immunosuppression concerns include immunoisolation of insulin-producing cells with porous biomaterials that function as an immune barrier. However, clinical implementation has been challenging because of host immune responses to the implant materials. Here we report the first long-term glycemic correction of a diabetic, immunocompetent animal model using human SC-ß cells. SC-ß cells were encapsulated with alginate derivatives capable of mitigating foreign-body responses in vivo and implanted into the intraperitoneal space of C57BL/6J mice treated with streptozotocin, which is an animal model for chemically induced type 1 diabetes. These implants induced glycemic correction without any immunosuppression until their removal at 174 d after implantation. Human C-peptide concentrations and in vivo glucose responsiveness demonstrated therapeutically relevant glycemic control. Implants retrieved after 174 d contained viable insulin-producing cells.