RESUMO
Type 2 diabetes mellitus (T2D), a major cause of worldwide morbidity and mortality, is characterized by dysfunction of insulin-producing pancreatic islet ß cells1,2. T2D genome-wide association studies (GWAS) have identified hundreds of signals in non-coding and ß cell regulatory genomic regions, but deciphering their biological mechanisms remains challenging3-5. Here, to identify early disease-driving events, we performed traditional and multiplexed pancreatic tissue imaging, sorted-islet cell transcriptomics and islet functional analysis of early-stage T2D and control donors. By integrating diverse modalities, we show that early-stage T2D is characterized by ß cell-intrinsic defects that can be proportioned into gene regulatory modules with enrichment in signals of genetic risk. After identifying the ß cell hub gene and transcription factor RFX6 within one such module, we demonstrated multiple layers of genetic risk that converge on an RFX6-mediated network to reduce insulin secretion by ß cells. RFX6 perturbation in primary human islet cells alters ß cell chromatin architecture at regions enriched for T2D GWAS signals, and population-scale genetic analyses causally link genetically predicted reduced RFX6 expression with increased T2D risk. Understanding the molecular mechanisms of complex, systemic diseases necessitates integration of signals from multiple molecules, cells, organs and individuals, and thus we anticipate that this approach will be a useful template to identify and validate key regulatory networks and master hub genes for other diseases or traits using GWAS data.
Assuntos
Diabetes Mellitus Tipo 2 , Perfilação da Expressão Gênica , Redes Reguladoras de Genes , Predisposição Genética para Doença , Ilhotas Pancreáticas , Humanos , Estudos de Casos e Controles , Separação Celular , Cromatina/metabolismo , Diabetes Mellitus Tipo 2/genética , Diabetes Mellitus Tipo 2/metabolismo , Diabetes Mellitus Tipo 2/patologia , Diabetes Mellitus Tipo 2/fisiopatologia , Redes Reguladoras de Genes/genética , Estudo de Associação Genômica Ampla , Secreção de Insulina , Ilhotas Pancreáticas/metabolismo , Ilhotas Pancreáticas/patologia , Reprodutibilidade dos TestesRESUMO
Blood amino acid levels are maintained in a narrow physiological range. The pancreatic α cells have emerged as the primary aminoacidemia regulator through glucagon secretion to promote hepatic amino acid catabolism. Interruption of glucagon signaling disrupts the liver-α cells axis leading to hyperaminoacidemia, which triggers a compensatory rise in glucagon secretion and α cell hyperplasia. The mechanisms of hyperaminoacidemia-induced α cell hyperplasia remain incompletely understood. Using a mouse α cell line and in vivo studies in zebrafish and mice, we found that hyperaminoacidemia-induced α cell hyperplasia requires ErbB3 signaling. In addition to mechanistic target of rapamycin complex 1, another ErbB3 downstream effector signal transducer and activator of transcription 3 also plays a role in α cell hyperplasia. Mechanistically, ErbB3 may partner with ErbB2 to stimulate cyclin D2 and suppress p27 via mechanistic target of rapamycin complex 1 and signal transducer and activator of transcription 3. Our study identifies ErbB3 as a new regulator for hyperaminoacidemia-induced α cell proliferation and a critical component of the liver-α cells axis that regulates aminoacidemia.
RESUMO
Cytotoxic CD8 T lymphocytes play a central role in the tissue destruction of many autoimmune disorders. In type 1 diabetes (T1D), insulin and its precursor preproinsulin are major self-antigens targeted by T cells. We comprehensively examined preproinsulin specificity of CD8 T cells obtained from pancreatic islets of organ donors with and without T1D and identified epitopes throughout the entire preproinsulin protein and defective ribosomal products derived from preproinsulin messenger RNA. The frequency of preproinsulin-reactive T cells was significantly higher in T1D donors than nondiabetic donors and also differed by individual T1D donor, ranging from 3 to over 40%, with higher frequencies in T1D organ donors with HLA-A*02:01. Only T cells reactive to preproinsulin-related peptides isolated from T1D donors demonstrated potent autoreactivity. Reactivity to similar regions of preproinsulin was also observed in peripheral blood of a separate cohort of new-onset T1D patients. These findings have important implications for designing antigen-specific immunotherapies and identifying individuals that may benefit from such interventions.
Assuntos
Linfócitos T CD8-Positivos/imunologia , Diabetes Mellitus Tipo 1/imunologia , Insulina/imunologia , Ilhotas Pancreáticas/imunologia , Precursores de Proteínas/imunologia , Adolescente , Adulto , Autoantígenos/imunologia , Autoimunidade/imunologia , Criança , Diabetes Mellitus Tipo 1/patologia , Diabetes Mellitus Tipo 1/terapia , Feminino , Antígeno HLA-A2 , Humanos , Imunoterapia/métodos , Ilhotas Pancreáticas/citologia , Masculino , Adulto JovemRESUMO
We evaluated the potential for a monoclonal antibody antagonist of the glucagon receptor (Ab-4) to maintain glucose homeostasis in type 1 diabetic rodents. We noted durable and sustained improvements in glycemia which persist long after treatment withdrawal. Ab-4 promoted ß-cell survival and enhanced the recovery of insulin+ islet mass with concomitant increases in circulating insulin and C peptide. In PANIC-ATTAC mice, an inducible model of ß-cell apoptosis which allows for robust assessment of ß-cell regeneration following caspase-8-induced diabetes, Ab-4 drove a 6.7-fold increase in ß-cell mass. Lineage tracing suggests that this restoration of functional insulin-producing cells was at least partially driven by α-cell-to-ß-cell conversion. Following hyperglycemic onset in nonobese diabetic (NOD) mice, Ab-4 treatment promoted improvements in C-peptide levels and insulin+ islet mass was dramatically increased. Lastly, diabetic mice receiving human islet xenografts showed stable improvements in glycemic control and increased human insulin secretion.
Assuntos
Anticorpos Monoclonais/farmacologia , Diabetes Mellitus Experimental/terapia , Células Secretoras de Glucagon/efeitos dos fármacos , Hipoglicemiantes/farmacologia , Células Secretoras de Insulina/efeitos dos fármacos , Receptores de Glucagon/antagonistas & inibidores , Animais , Glicemia/metabolismo , Peptídeo C/metabolismo , Linhagem da Célula/efeitos dos fármacos , Transdiferenciação Celular/efeitos dos fármacos , Diabetes Mellitus Experimental/genética , Diabetes Mellitus Experimental/imunologia , Diabetes Mellitus Experimental/patologia , Diabetes Mellitus Tipo 1/genética , Diabetes Mellitus Tipo 1/imunologia , Diabetes Mellitus Tipo 1/patologia , Diabetes Mellitus Tipo 1/terapia , Expressão Gênica , Glucagon/antagonistas & inibidores , Glucagon/metabolismo , Células Secretoras de Glucagon/metabolismo , Células Secretoras de Glucagon/patologia , Humanos , Insulina/metabolismo , Células Secretoras de Insulina/metabolismo , Células Secretoras de Insulina/patologia , Ilhotas Pancreáticas/metabolismo , Ilhotas Pancreáticas/fisiologia , Transplante das Ilhotas Pancreáticas , Camundongos , Camundongos Endogâmicos NOD , Tamanho do Órgão/efeitos dos fármacos , Receptores de Glucagon/genética , Receptores de Glucagon/metabolismo , Resultado do TratamentoRESUMO
The autonomic nervous system regulates pancreatic function. Islet capillaries are essential for the extension of axonal projections into islets, and both of these structures are important for appropriate islet hormone secretion. Because beta cells provide important paracrine cues for islet glucagon secretion and neurovascular development, we postulated that beta cell loss in type 1 diabetes (T1D) would lead to a decline in intraislet capillaries and reduction of islet innervation, possibly contributing to abnormal glucagon secretion. To define morphological characteristics of capillaries and nerve fibers in islets and acinar tissue compartments, we analyzed neurovascular assembly across the largest cohort of T1D and normal individuals studied thus far. Because innervation has been studied extensively in rodent models of T1D, we also compared the neurovascular architecture between mouse and human pancreas and assembled transcriptomic profiles of molecules guiding islet angiogenesis and neuronal development. We found striking interspecies differences in islet neurovascular assembly but relatively modest differences at transcriptome level, suggesting that posttranscriptional regulation may be involved in this process. To determine whether islet neurovascular arrangement is altered after beta cell loss in T1D, we compared pancreatic tissues from non-diabetic, recent-onset T1D (<10-yr duration), and longstanding T1D (>10-yr duration) donors. Recent-onset T1D showed greater islet and acinar capillary density compared to non-diabetic and longstanding T1D donors. Both recent-onset and longstanding T1D had greater islet nerve fiber density compared to non-diabetic donors. We did not detect changes in sympathetic axons in either T1D cohort. Additionally, nerve fibers overlapped with extracellular matrix (ECM), supporting its role in the formation and function of axonal processes. These results indicate that pancreatic capillaries and nerve fibers persist in T1D despite beta cell loss, suggesting that alpha cell secretory changes may be decoupled from neurovascular components.NEW & NOTEWORTHY Defining the neurovascular architecture in the pancreas of individuals with type 1 diabetes (T1D) is crucial to understanding the mechanisms of dysregulated glucagon secretion. In the largest T1D cohort of biobanked tissues analyzed to date, we found that pancreatic capillaries and nerve fibers persist in human T1D despite beta cell loss, suggesting that alpha cell secretory changes may be decoupled from neurovascular components. Because innervation has been studied extensively in rodent T1D models, our studies also provide the first rigorous direct comparisons of neurovascular assembly in mouse and human, indicating dramatic interspecies differences.
Assuntos
Diabetes Mellitus Tipo 1 , Diabetes Mellitus Tipo 2 , Células Secretoras de Glucagon , Ilhotas Pancreáticas , Humanos , Camundongos , Animais , Diabetes Mellitus Tipo 1/metabolismo , Ilhotas Pancreáticas/metabolismo , Glucagon/metabolismo , Capilares/metabolismo , Células Secretoras de Glucagon/metabolismo , Diabetes Mellitus Tipo 2/metabolismo , Fibras Nervosas/metabolismoRESUMO
Pancreas volume is reduced in individuals with diabetes and in autoantibody positive individuals at high risk for developing type 1 diabetes (T1D). Studies investigating pancreas volume are underway to assess pancreas volume in large clinical databases and studies, but manual pancreas annotation is time-consuming and subjective, preventing extension to large studies and databases. This study develops deep learning for automated pancreas volume measurement in individuals with diabetes. A convolutional neural network was trained using manual pancreas annotation on 160 abdominal magnetic resonance imaging (MRI) scans from individuals with T1D, controls, or a combination thereof. Models trained using each cohort were then tested on scans of 25 individuals with T1D. Deep learning and manual segmentations of the pancreas displayed high overlap (Dice coefficient = 0.81) and excellent correlation of pancreas volume measurements (R2 = 0.94). Correlation was highest when training data included individuals both with and without T1D. The pancreas of individuals with T1D can be automatically segmented to measure pancreas volume. This algorithm can be applied to large imaging datasets to quantify the spectrum of human pancreas volume.
Assuntos
Aprendizado Profundo , Diabetes Mellitus Tipo 1/diagnóstico por imagem , Processamento de Imagem Assistida por Computador/métodos , Imageamento por Ressonância Magnética/métodos , Pâncreas/diagnóstico por imagem , Adolescente , Algoritmos , Diabetes Mellitus Tipo 1/patologia , Humanos , Imageamento Tridimensional/métodos , Masculino , Tamanho do Órgão , Pâncreas/patologia , Estudos RetrospectivosRESUMO
AIMS/HYPOTHESIS: Individuals with longstanding and recent-onset type 1 diabetes have a smaller pancreas. Since beta cells represent a very small portion of the pancreas, the loss of pancreas volume in diabetes is primarily due to the loss of pancreatic exocrine mass. However, the structural changes in the exocrine pancreas in diabetes are not well understood. METHODS: To characterise the pancreatic endocrine and exocrine compartments in diabetes, we studied pancreases from adult donors with type 1 diabetes compared with similarly aged donors without diabetes. Islet cell mass, islet morphometry, exocrine mass, acinar cell size and number and pancreas fibrosis were assessed by immunohistochemical staining. To better understand possible mechanisms of altered pancreas size, we measured pancreas size in three mouse models of insulin deficiency. RESULTS: Pancreases from donors with type 1 diabetes were approximately 45% smaller than those from donors without diabetes (47.4 ± 2.6 vs 85.7 ± 3.7 g), independent of diabetes duration or age of onset. Diabetic donor pancreases had decreased beta cell mass (0.061 ± 0.025 vs 0.94 ± 0.21 g) and reduced total exocrine mass (42.0 ± 4.9 vs 96.1 ± 6.5 g). Diabetic acinar cells were similar in size but fewer in number compared with those in pancreases from non-diabetic donors (63.7 ± 8.1 × 109 vs 121.6 ± 12.2 × 109 cells/pancreas), likely accounting for the difference in pancreas size. Within the type 1 diabetes exocrine tissue, there was a greater degree of fibrosis. The pancreases in three mouse models of insulin deficiency were similar in size to those in control mice. CONCLUSIONS/INTERPRETATION: Pancreases from donors with type 1 diabetes are smaller than normal donor pancreases because exocrine cells are fewer in number rather than smaller in size; these changes occur early in the disease process. Our mouse data suggest that decreased pancreas size in type 1 diabetes is not directly caused by insulin deficiency, but the precise mechanism responsible remains unclear.
Assuntos
Diabetes Mellitus Tipo 1/metabolismo , Pâncreas Exócrino/metabolismo , Células Acinares/metabolismo , Animais , Feminino , Células Secretoras de Insulina/metabolismo , Ilhotas Pancreáticas/metabolismo , Camundongos , Pâncreas/metabolismoRESUMO
Over the last two decades, improved access to human islets and the development of human islet distribution networks have enabled the use of millions of human islets in hundreds of scientific research projects, leading to a dramatic increase in our understanding of human islet biology. Here we discuss recent scientific advances as well as methodological and experimental challenges that impact human islet quality, experimental outcomes and the reporting of human islets used in scientific publications. In a survey of over 200 scientific publications with human islet experimentation, we found that the reporting of critical information was quite variable, sometimes obscure, and often failed to adequately outline the experiments and results using human islets. As the complexity of human islet research grows, we propose that members of the human islet research ecosystem work together to develop procedures and approaches for accessible and transparent collecting and reporting of crucial human islet characteristics and, through this, enhance collaboration, reproducibility and rigour, leading to further advances in our understanding of human islet biology.
Assuntos
Diabetes Mellitus/fisiopatologia , Diabetes Mellitus/cirurgia , Secreção de Insulina/fisiologia , Transplante das Ilhotas Pancreáticas , Ilhotas Pancreáticas/fisiopatologia , HumanosRESUMO
AIMS/HYPOTHESIS: The molecular response and function of pancreatic islet cells during metabolic stress is a complex process. The anatomical location and small size of pancreatic islets coupled with current methodological limitations have prevented the achievement of a complete, coherent picture of the role that lipids and proteins play in cellular processes under normal conditions and in diseased states. Herein, we describe the development of untargeted tissue imaging mass spectrometry (IMS) technologies for the study of in situ protein and, more specifically, lipid distributions in murine and human pancreases. METHODS: We developed matrix-assisted laser desorption/ionisation (MALDI) IMS protocols to study metabolite, lipid and protein distributions in mouse (wild-type and ob/ob mouse models) and human pancreases. IMS allows for the facile discrimination of chemically similar lipid and metabolite isoforms that cannot be distinguished using standard immunohistochemical techniques. Co-registration of MS images with immunofluorescence images acquired from serial tissue sections allowed accurate cross-registration of cell types. By acquiring immunofluorescence images first, this serial section approach guides targeted high spatial resolution IMS analyses (down to 15 µm) of regions of interest and leads to reduced time requirements for data acquisition. RESULTS: MALDI IMS enabled the molecular identification of specific phospholipid and glycolipid isoforms in pancreatic islets with intra-islet spatial resolution. This technology shows that subtle differences in the chemical structure of phospholipids can dramatically affect their distribution patterns and, presumably, cellular function within the islet and exocrine compartments of the pancreas (e.g. 18:1 vs 18:2 fatty acyl groups in phosphatidylcholine lipids). We also observed the localisation of specific GM3 ganglioside lipids [GM3(d34:1), GM3(d36:1), GM3(d38:1) and GM3(d40:1)] within murine islet cells that were correlated with a higher level of GM3 synthase as verified by immunostaining. However, in human pancreas, GM3 gangliosides were equally distributed in both the endocrine and exocrine tissue, with only one GM3 isoform showing islet-specific localisation. CONCLUSIONS/INTERPRETATION: The development of more complete molecular profiles of pancreatic tissue will provide important insight into the molecular state of the pancreas during islet development, normal function, and diseased states. For example, this study demonstrates that these results can provide novel insight into the potential signalling mechanisms involving phospholipids and glycolipids that would be difficult to detect by targeted methods, and can help raise new hypotheses about the types of physiological control exerted on endocrine hormone-producing cells in islets. Importantly, the in situ measurements afforded by IMS do not require a priori knowledge of molecules of interest and are not susceptible to the limitations of immunohistochemistry, providing the opportunity for novel biomarker discovery. Notably, the presence of multiple GM3 isoforms in mouse islets and the differential localisation of lipids in human tissue underscore the important role these molecules play in regulating insulin modulation and suggest species, organ, and cell specificity. This approach demonstrates the importance of both high spatial resolution and high molecular specificity to accurately survey the molecular composition of complex, multi-functional tissues such as the pancreas.
Assuntos
Ilhotas Pancreáticas/metabolismo , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Animais , Imunofluorescência , Gangliosídeos/análise , Humanos , Imuno-Histoquímica , Camundongos , PâncreasRESUMO
New-onset post-transplant diabetes mellitus (PTDM) occurs frequently after allogeneic hematopoietic cell transplant (HCT). Although calcineurin inhibitors and corticosteroids are assumed to be the cause for hyperglycemia, patients developing PTDM have elevated fasting C-peptide levels before HCT and before immunosuppressive medications. To determine if PTDM results from established insulin resistance present before transplant, we performed oral glucose tolerance tests (OGTTs) and measured whole body, peripheral, and hepatic insulin sensitivity with euglycemic hyperinsulinemic clamps before and 90 days after HLA-identical sibling donor HCT in 20 patients without pretransplant diabetes. HCT recipients were prospectively followed for the development of new-onset PTDM defined as a weekly fasting blood glucose ≥ 126 mg/dL or random blood glucose ≥ 200 mg/dL. During the first 100 days all patients received calcineurin inhibitors, and 11 individuals (55%) were prospectively diagnosed with new-onset PTDM. PTDM diagnosis preceded corticosteroid treatment. During the pretransplant OGTT, elevated fasting (87 mg/dL versus 101 mg/dL; Pâ¯=â¯.005) but not 2-hour postprandial glucose levels predicted PTDM diagnosis (P = .648). In response to insulin infusion during the euglycemic hyperinsulinemic clamp, patients developing PTDM had lower whole body glucose utilization (Pâ¯=â¯.047) and decreased peripheral/skeletal muscle uptake (Pâ¯=â¯.031) before and after transplant, respectively, when compared with non-PTDM patients. Hepatic insulin sensitivity did not differ. Survival was decreased in PTDM patients (2-year estimate, 55% versus 100%; P = .039). Insulin resistance before HCT is a risk factor for PTDM independent of immunosuppression. Fasting pretransplant glucose levels identified PTDM susceptibility, and peripheral insulin resistance could be targeted for prevention and treatment of PTDM after HCT.
Assuntos
Diabetes Mellitus/etiologia , Transplante de Células-Tronco Hematopoéticas/efeitos adversos , Condicionamento Pré-Transplante/efeitos adversos , Adulto , Feminino , Transplante de Células-Tronco Hematopoéticas/métodos , Humanos , Resistência à Insulina , Masculino , Pessoa de Meia-Idade , Condicionamento Pré-Transplante/métodos , Transplante HomólogoRESUMO
Homozygous Mnx1 mutation causes permanent neonatal diabetes in humans, but via unknown mechanisms. Our systematic and longitudinal analysis of Mnx1 function during murine pancreas organogenesis and into the adult uncovered novel stage-specific roles for Mnx1 in endocrine lineage allocation and ß-cell fate maintenance. Inactivation in the endocrine-progenitor stage shows that Mnx1 promotes ß-cell while suppressing δ-cell differentiation programs, and is crucial for postnatal ß-cell fate maintenance. Inactivating Mnx1 in embryonic ß-cells (Mnx1(Δbeta)) caused ß-to-δ-like cell transdifferentiation, which was delayed until postnatal stages. In the latter context, ß-cells escaping Mnx1 inactivation unexpectedly upregulated Mnx1 expression and underwent an age-independent persistent proliferation. Escaper ß-cells restored, but then eventually surpassed, the normal pancreatic ß-cell mass, leading to islet hyperplasia in aged mice. In vitro analysis of islets isolated from Mnx1(Δbeta) mice showed higher insulin secretory activity and greater insulin mRNA content than in wild-type islets. Mnx1(Δbeta) mice also showed a much faster return to euglycemia after ß-cell ablation, suggesting that the new ß-cells derived from the escaper population are functional. Our findings identify Mnx1 as an important factor in ß-cell differentiation and proliferation, with the potential for targeting to increase the number of endogenous ß-cells for diabetes therapy.
Assuntos
Diabetes Mellitus/patologia , Proteínas de Homeodomínio/metabolismo , Células Secretoras de Insulina/metabolismo , Fatores de Transcrição/metabolismo , Animais , Transdiferenciação Celular , Senescência Celular , Proteínas do Olho/metabolismo , Proteínas de Homeodomínio/genética , Humanos , Hiperplasia/metabolismo , Células Secretoras de Insulina/citologia , Camundongos , Fator de Transcrição PAX6 , Fatores de Transcrição Box Pareados/metabolismo , Proteínas Repressoras/metabolismo , Células Secretoras de Somatostatina/metabolismo , Fatores de Transcrição/genéticaRESUMO
The asymptomatic phase of type 1 diabetes is recognised by the presence of beta cell autoantibodies in the absence of hyperglycaemia. We propose that an accurate description of this stage is provided by the name 'Autoimmune Beta Cell Disorder' (ABCD). Specifically, we suggest that this nomenclature and diagnosis will, in a proactive manner, shift the paradigm towards type 1 diabetes being first and foremost an immune-mediated disease and only later a metabolic disease, presaging more active therapeutic intervention in the asymptomatic stage of disease, before end-stage beta cell failure. Furthermore, we argue that accepting ABCD as a diagnosis will be critical in order to accelerate pharmaceutical, academic and public activities leading to clinical trials that could reverse beta cell autoimmunity and halt progression to symptomatic insulin-requiring type 1 diabetes. We recognize that there are both opportunities and challenges in the implementation of the ABCD concept but hope that the notion of 'asymptomatic autoimmune disease' as a disorder will be widely discussed and eventually accepted.
Assuntos
Autoimunidade/fisiologia , Diabetes Mellitus Tipo 1/complicações , Células Secretoras de Insulina/patologia , Diabetes Mellitus Tipo 1/imunologia , Humanos , Células Secretoras de Insulina/imunologiaRESUMO
Neurovascular alignment is a common anatomical feature of organs, but the mechanisms leading to this arrangement are incompletely understood. Here, we show that vascular endothelial growth factor (VEGF) signaling profoundly affects both vascularization and innervation of the pancreatic islet. In mature islets, nerves are closely associated with capillaries, but the islet vascularization process during embryonic organogenesis significantly precedes islet innervation. Although a simple neuronal meshwork interconnects the developing islet clusters as they begin to form at E14.5, the substantial ingrowth of nerve fibers into islets occurs postnatally, when islet vascularization is already complete. Using genetic mouse models, we demonstrate that VEGF regulates islet innervation indirectly through its effects on intra-islet endothelial cells. Our data indicate that formation of a VEGF-directed, intra-islet vascular plexus is required for development of islet innervation, and that VEGF-induced islet hypervascularization leads to increased nerve fiber ingrowth. Transcriptome analysis of hypervascularized islets revealed an increased expression of extracellular matrix components and axon guidance molecules, with these transcripts being enriched in the islet-derived endothelial cell population. We propose a mechanism for coordinated neurovascular development within pancreatic islets, in which endocrine cell-derived VEGF directs the patterning of intra-islet capillaries during embryogenesis, forming a scaffold for the postnatal ingrowth of essential autonomic nerve fibers.
Assuntos
Vasos Sanguíneos/fisiologia , Comunicação Celular/genética , Ilhotas Pancreáticas/irrigação sanguínea , Ilhotas Pancreáticas/inervação , Neovascularização Fisiológica/fisiologia , Fator A de Crescimento do Endotélio Vascular/fisiologia , Animais , Vasos Sanguíneos/embriologia , Células Cultivadas , Embrião de Mamíferos , Endotélio Vascular/embriologia , Endotélio Vascular/metabolismo , Endotélio Vascular/fisiologia , Feminino , Ilhotas Pancreáticas/embriologia , Camundongos , Camundongos Transgênicos , Fator A de Crescimento do Endotélio Vascular/genéticaRESUMO
The progressive loss of pancreatic ß-cell mass that occurs in both type 1 and type 2 diabetes is a primary factor driving efforts to identify strategies for effectively increasing, enhancing or restoring ß-cell mass. While factors that seem to influence ß-cell proliferation in specific contexts have been described, reliable stimulation of human ß-cell proliferation has remained a challenge. Importantly, ß-cells exist in the context of a complex, integrated pancreatic islet microenvironment where they interact with other endocrine cells, vascular endothelial cells, extracellular matrix, neuronal projections and islet macrophages. This review highlights different components of the pancreatic microenvironment, and reviews what is known about how signaling that occurs between ß-cells and these other components influences ß-cell proliferation. Future efforts to further define the role of the pancreatic islet microenvironment on ß-cell proliferation may lead to the development of successful approaches to increase or restore ß-cell mass in diabetes.
Assuntos
Comunicação Celular , Proliferação de Células , Microambiente Celular , Células Secretoras de Insulina/metabolismo , Ilhotas Pancreáticas/citologia , Modelos Biológicos , Animais , Apoptose , Diabetes Mellitus Tipo 1/metabolismo , Diabetes Mellitus Tipo 1/patologia , Diabetes Mellitus Tipo 2/metabolismo , Diabetes Mellitus Tipo 2/patologia , Matriz Extracelular/imunologia , Matriz Extracelular/metabolismo , Matriz Extracelular/patologia , Células Secretoras de Glucagon/citologia , Células Secretoras de Glucagon/imunologia , Células Secretoras de Glucagon/metabolismo , Células Secretoras de Glucagon/patologia , Humanos , Células Secretoras de Insulina/citologia , Células Secretoras de Insulina/imunologia , Células Secretoras de Insulina/patologia , Ilhotas Pancreáticas/irrigação sanguínea , Ilhotas Pancreáticas/inervação , Ilhotas Pancreáticas/patologia , Macrófagos/citologia , Macrófagos/imunologia , Macrófagos/metabolismo , Macrófagos/patologia , Células Secretoras de Polipeptídeo Pancreático/citologia , Células Secretoras de Polipeptídeo Pancreático/imunologia , Células Secretoras de Polipeptídeo Pancreático/metabolismo , Células Secretoras de Polipeptídeo Pancreático/patologia , Células Secretoras de Somatostatina/citologia , Células Secretoras de Somatostatina/imunologia , Células Secretoras de Somatostatina/metabolismo , Células Secretoras de Somatostatina/patologia , Especificidade da EspécieRESUMO
Glucocorticoids signal through the glucocorticoid receptor (GR) and are administered clinically for a variety of situations, including inflammatory disorders, specific cancers, rheumatoid arthritis, and organ/tissue transplantation. However, glucocorticoid therapy is also associated with additional complications, including steroid-induced diabetes. We hypothesized that modification of the steroid backbone is one strategy to enhance the therapeutic potential of GR activation. Toward this goal, two commercially unavailable, thiobenzothiazole-containing derivatives of hydrocortisone (termed MS4 and MS6) were examined using 832/13 rat insulinoma cells as well as rodent and human islets. We found that MS4 had transrepression properties but lacked transactivation ability, whereas MS6 retained both transactivation and transrepression activities. In addition, MS4 and MS6 both displayed anti-inflammatory activity. Furthermore, MS4 displayed reduced impact on islet ß-cell function in both rodent and human islets. Similar to dexamethasone, MS6 promoted adipocyte development in vitro, whereas MS4 did not. Moreover, neither MS4 nor MS6 activated the Pck1 (Pepck) gene in primary rat hepatocytes. We conclude that modification of the functional groups attached to the D-ring of the hydrocortisone steroid molecule produces compounds with altered structure-function GR agonist activity with decreased impact on insulin secretion and reduced adipogenic potential but with preservation of anti-inflammatory activity.
Assuntos
Anti-Inflamatórios/farmacologia , Benzimidazóis/farmacologia , Benzotiazóis/farmacologia , Hidrocortisona/análogos & derivados , Hidrocortisona/farmacologia , Inflamação/tratamento farmacológico , Insulina/metabolismo , Ilhotas Pancreáticas/efeitos dos fármacos , Ilhotas Pancreáticas/imunologia , Tiazóis/química , Células 3T3-L1 , Animais , Anti-Inflamatórios/síntese química , Apoptose/efeitos dos fármacos , Benzimidazóis/síntese química , Benzotiazóis/síntese química , Western Blotting , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Dexametasona/farmacologia , Perfilação da Expressão Gênica , Hepatócitos/efeitos dos fármacos , Hepatócitos/imunologia , Hepatócitos/metabolismo , Humanos , Hidrocortisona/síntese química , Técnicas Imunoenzimáticas , Inflamação/imunologia , Inflamação/metabolismo , Inflamação/patologia , Secreção de Insulina , Ilhotas Pancreáticas/metabolismo , Ilhotas Pancreáticas/patologia , Metabolômica , Camundongos , Camundongos Endogâmicos C57BL , Consumo de Oxigênio/efeitos dos fármacos , RNA Mensageiro/genética , Ratos , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Numerous compounds stimulate rodent ß-cell proliferation; however, translating these findings to human ß-cells remains a challenge. To examine human ß-cell proliferation in response to such compounds, we developed a medium-throughput in vitro method of quantifying adult human ß-cell proliferation markers. This method is based on high-content imaging of dispersed islet cells seeded in 384-well plates and automated cell counting that identifies fluorescently labeled ß-cells with high specificity using both nuclear and cytoplasmic markers. ß-Cells from each donor were assessed for their function and ability to enter the cell cycle by cotransduction with adenoviruses encoding cell cycle regulators cdk6 and cyclin D3. Using this approach, we tested 12 previously identified mitogens, including neurotransmitters, hormones, growth factors, and molecules, involved in adenosine and Tgf-1ß signaling. Each compound was tested in a wide concentration range either in the presence of basal (5 mM) or high (11 mM) glucose. Treatment with the control compound harmine, a Dyrk1a inhibitor, led to a significant increase in Ki-67+ ß-cells, whereas treatment with other compounds had limited to no effect on human ß-cell proliferation. This new scalable approach reduces the time and effort required for sensitive and specific evaluation of human ß-cell proliferation, thus allowing for increased testing of candidate human ß-cell mitogens.
Assuntos
Proliferação de Células/efeitos dos fármacos , Células Secretoras de Insulina/efeitos dos fármacos , Ativinas/farmacologia , Adenosina/análogos & derivados , Adenosina/farmacologia , Agonistas do Receptor A2 de Adenosina/farmacologia , Adenosina-5'-(N-etilcarboxamida)/farmacologia , Adulto , Automação , Técnicas de Cultura de Células , Avaliação Pré-Clínica de Medicamentos , Eritropoetina/farmacologia , Exenatida , Feminino , GABAérgicos/farmacologia , Harmina/farmacologia , Humanos , Incretinas/farmacologia , Masculino , Pessoa de Meia-Idade , Inibidores da Monoaminoxidase/farmacologia , Miostatina/farmacologia , Nucleosídeos/farmacologia , Peptídeos/farmacologia , Fator de Crescimento Derivado de Plaquetas/farmacologia , Prolactina/farmacologia , Regeneração/efeitos dos fármacos , Serotonina/farmacologia , Agonistas do Receptor de Serotonina/farmacologia , Vasodilatadores/farmacologia , Peçonhas/farmacologia , Adulto Jovem , Ácido gama-Aminobutírico/farmacologiaRESUMO
Analysis of MafB(-/-) mice has suggested that the MAFB transcription factor was essential to islet α- and ß-cell formation during development, although the postnatal physiological impact could not be studied here because these mutants died due to problems in neural development. Pancreas-wide mutant mice were generated to compare the postnatal significance of MafB (MafB(Δpanc)) and MafA/B (MafAB(Δpanc)) with deficiencies associated with the related ß-cell-enriched MafA mutant (MafA(Δpanc)). Insulin(+) cell production and ß-cell activity were merely delayed in MafB(Δpanc) islets until MafA was comprehensively expressed in this cell population. We propose that MafA compensates for the absence of MafB in MafB(Δpanc) mice, which is supported by the death of MafAB(Δpanc) mice soon after birth from hyperglycemia. However, glucose-induced glucagon secretion was compromised in adult MafB(Δpanc) islet α-cells. Based upon these results, we conclude that MafB is only essential to islet α-cell activity and not ß-cell. Interestingly, a notable difference between mice and humans is that MAFB is coexpressed with MAFA in adult human islet ß-cells. Here, we show that nonhuman primate (NHP) islet α- and ß-cells also produce MAFB, implying that MAFB represents a unique signature and likely important regulator of the primate islet ß-cell.
Assuntos
Células Secretoras de Insulina/metabolismo , Ilhotas Pancreáticas/fisiologia , Fator de Transcrição MafB/fisiologia , Adolescente , Adulto , Animais , Biomarcadores/metabolismo , Feminino , Humanos , Macaca mulatta , Fator de Transcrição MafB/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Pessoa de Meia-Idade , Primatas , Roedores , Adulto JovemRESUMO
Efforts to restore ß-cell number or mass in type 1 diabetes (T1D) must combine an intervention to stimulate proliferation of remaining ß-cells and an intervention to mitigate or control the ß-cell-directed autoimmunity. This review highlights features of the ß-cell, including it being part of a pancreatic islet, a mini-organ that is highly vascularized and highly innervated, and efforts to promote ß-cell proliferation. In addition, the ß-cell in T1D exists in a microenvironment with interactions and input from other islet cell types, extracellular matrix, vascular endothelial cells, neuronal projections, and immune cells, all of which likely influence the ß-cell's capacity for replication. Physiologic ß-cell proliferation occurs in human and rodents in the neonatal period and early in life, after which there is an age-dependent decline in ß-cell proliferation, and also as part of the ß-cell's compensatory response to the metabolic challenges of pregnancy and insulin resistance. This review reviews the molecular pathways involved in this ß-cell proliferation and highlights recent work in two areas: 1) Investigators, using high-throughput screening to discover small molecules that promote human ß-cell proliferation, are now focusing on the dual-specificity tyrosine-regulated kinase-1a and cell cycle-dependent kinase inhibitors CDKN2C/p18 or CDKN1A/p21as targets of compounds to stimulate adult human ß-cell proliferation. 2) Local inflammation, macrophages, and the local ß-cell microenvironment promote ß-cell proliferation. Future efforts to harness the responsible mechanisms may lead to new approaches to promote ß-cell proliferation in T1D.
Assuntos
Diabetes Mellitus Tipo 1/etiologia , Diabetes Mellitus Tipo 1/metabolismo , Células Secretoras de Insulina/imunologia , Células Secretoras de Insulina/metabolismo , Animais , Autoimunidade , Proliferação de Células/efeitos dos fármacos , Microambiente Celular , Diabetes Mellitus Tipo 1/terapia , Metabolismo Energético/efeitos dos fármacos , Humanos , Células Secretoras de Insulina/efeitos dos fármacos , Ilhotas Pancreáticas/imunologia , Ilhotas Pancreáticas/metabolismo , Macrófagos/imunologia , Macrófagos/metabolismo , Terapia de Alvo Molecular , Regeneração , Transdução de Sinais/efeitos dos fármacosRESUMO
Human islet research is providing new insights into human islet biology and diabetes, using islets isolated at multiple US centers from donors with varying characteristics. This creates challenges for understanding, interpreting, and integrating research findings from the many laboratories that use these islets. In what is, to our knowledge, the first standardized assessment of human islet preparations from multiple isolation centers, we measured insulin secretion from 202 preparations isolated at 15 centers over 11 years and noted five distinct patterns of insulin secretion. Approximately three quarters were appropriately responsive to stimuli, but one quarter were dysfunctional, with unstable basal insulin secretion and/or an impairment in stimulated insulin secretion. Importantly, the patterns of insulin secretion by responsive human islet preparations (stable Baseline and Fold stimulation of insulin secretion) isolated at different centers were similar and improved slightly over the years studied. When all preparations studied were considered, basal and stimulated insulin secretion did not correlate with isolation center, biological differences of the islet donor, or differences in isolation, such as Cold Ischemia Time. Dysfunctional islet preparations could not be predicted from the information provided by the isolation center and had altered expression of genes encoding components of the glucose-sensing pathway, but not of insulin production or cell death. These results indicate that insulin secretion by most preparations from multiple centers is similar but that in vitro responsiveness of human islets cannot be predicted, necessitating preexperimental human islet assessment. These results should be considered when one is designing, interpreting, and integrating experiments using human islets.
Assuntos
Insulina/metabolismo , Ilhotas Pancreáticas/metabolismo , Pesquisa , Doadores de Tecidos , Obtenção de Tecidos e Órgãos , Adolescente , Adulto , Idoso , Criança , Feminino , Humanos , Secreção de Insulina , Masculino , Pessoa de Meia-Idade , Manejo de Espécimes , Doadores de Tecidos/estatística & dados numéricos , Doadores de Tecidos/provisão & distribuição , Obtenção de Tecidos e Órgãos/estatística & dados numéricos , Adulto JovemRESUMO
Quantitative prediction of in vivo behavior using an in vitro assay would dramatically accelerate pharmaceutical development. However, studies quantitatively correlating in vivo properties with in vitro assay results are rare because of the difficulty in quantitatively understanding the in vivo behavior of an agent. We now demonstrate such a correlation as a case study based on our quantitative understanding of the in vivo chemistry. In an ongoing pretargeting project, we designed a trifunctional antibody (Ab) that concomitantly carried a biotin and a DNA analogue (hereafter termed MORF). The biotin and the MORF were fused into one structure prior to conjugation to the Ab for the concomitant attachment. Because it was known that avidin-bound Ab molecules leave the circulation rapidly, this design would theoretically allow complete clearance by avidin. The clearability of the trifunctional Ab was determined by calculating the blood MORF concentration ratio of avidin-treated Ab to non-avidin-treated Ab using mice injected with these compounds. In theory, any compromised clearability should be due to the presence of impurities. In vitro, we measured the biotinylated percentage of the Ab-reacting (MORF-biotin)â-NH2 modifier, by addition of streptavidin to the radiolabeled (MORF-biotin)â-NH2 samples and subsequent high-performance liquid chromatography (HPLC) analysis. On the basis of our previous quantitative understanding, we predicted that the clearability of the Ab would be equal to the biotinylation percentage measured via HPLC. We validated this prediction within a 3% difference. In addition to the high avidin-induced clearability of the trifunctional Ab (up to â¼95%) achieved by the design, we were able to predict the required quality of the (MORF-biotin)â-NH2 modifier for any given in vivo clearability. This approach may greatly reduce the steps and time currently required in pharmaceutical development in the process of synthesis, chemical analysis, in vitro cell study, and in vivo validation.