RESUMO
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
BACKGROUND AND AIMS: Destroying visceral sensory nerves impacts pancreatic islet function, glucose metabolism, and diabetes onset, but how islet endocrine cells interact with sensory neurons has not been studied. METHODS: We characterized the anatomical pattern of pancreatic sensory innervation by combining viral tracing, immunohistochemistry, and reporter mouse models. To assess the functional interactions of ß-cells with vagal sensory neurons, we recorded Ca2+ responses in individual nodose neurons in vivo while selectively stimulating ß-cells with chemogenetic and pharmacologic approaches. RESULTS: We found that pancreatic islets are innervated by vagal sensory axons expressing Phox2b, substance P, calcitonin-gene related peptide, and the serotonin receptor 5-HT3R. Centrally, vagal neurons projecting to the pancreas terminate in the commissural nucleus of the solitary tract. Nodose neurons responded in vivo to chemogenetic stimulation of ß-cells and to pancreas infusion with serotonin, but were not sensitive to insulin. Responses to chemogenetic and pharmacologic stimulation of ß-cells were blocked by a 5-HT3R antagonist and were enhanced by increasing serotonin levels in ß-cells. We further confirmed directly in living pancreas slices that sensory terminals in the islet were sensitive to serotonin. CONCLUSIONS: Our study establishes that pancreatic ß-cells communicate with vagal sensory neurons, likely using serotonin signaling as a transduction mechanism. Serotonin is coreleased with insulin and may therefore convey information about the secretory state of ß-cells via vagal afferent nerves.
Assuntos
Vias Aferentes/fisiologia , Comunicação Celular , Células Secretoras de Insulina/fisiologia , Gânglio Nodoso/fisiologia , Células Receptoras Sensoriais/fisiologia , Animais , Feminino , Insulina/metabolismo , Microscopia Intravital , Masculino , Camundongos , Camundongos Transgênicos , Microscopia Confocal , Modelos Animais , Gânglio Nodoso/citologia , Serotonina/metabolismo , Transdução de Sinais/fisiologiaRESUMO
Cells in different tissues, including endocrine cells in the pancreas, live in complex microenvironments that are rich in cellular and acellular components. Intricate interactions with their microenvironment dictate most cellular properties, such as their function, structure and size, and maintain tissue homeostasis. Pancreatic islets are populated by endocrine, vascular and immune cells that are immersed in the extracellular matrix. While the intrinsic properties of beta cells have been vastly investigated, our understanding of their interactions with their surroundings has only recently begun to unveil. Here, we review current research on the interplay between the islet cellular and acellular components, and the role these components play in beta cell physiology and pathophysiology. Although beta cell failure is a key pathomechanism in diabetes, its causes are far from being fully elucidated. We, thus, propose deleterious alterations of the islet niche as potential underlying mechanisms contributing to beta cell failure. In sum, this review emphasises that the function of the pancreatic islet depends on all of its components. Graphical abstract.
Assuntos
Microambiente Celular , Diabetes Mellitus/metabolismo , Células Secretoras de Insulina/metabolismo , Animais , Membrana Basal/metabolismo , Membrana Basal/patologia , Células Endoteliais/metabolismo , Células Endoteliais/patologia , Matriz Extracelular/metabolismo , Matriz Extracelular/patologia , Humanos , Células Secretoras de Insulina/patologia , Ilhotas Pancreáticas/citologia , Ilhotas Pancreáticas/metabolismo , Ilhotas Pancreáticas/patologia , Comunicação Parácrina , Pericitos/metabolismo , Pericitos/patologiaRESUMO
AIMS/HYPOTHESIS: Autoimmune attack against the insulin-producing beta cells in the pancreatic islets results in type 1 diabetes. However, despite considerable research, details of the type 1 diabetes immunopathology in situ are not fully understood mainly because of difficult access to the pancreatic islets in vivo. METHODS: Here, we used direct non-invasive confocal imaging of islets transplanted in the anterior chamber of the eye (ACE) to investigate the anti-islet autoimmunity in NOD mice before, during and after diabetes onset. ACE-transplanted islets allowed longitudinal studies of the autoimmune attack against islets and revealed the infiltration kinetics and in situ motility dynamics of fluorescence-labelled autoreactive T cells during diabetes development. Ex vivo immunostaining was also used to compare immune cell infiltrations into islet grafts in the eye and kidney as well as in pancreatic islets of the same diabetic NOD mice. RESULTS: We found similar immune infiltration in native pancreatic and ACE-transplanted islets, which established the ACE-transplanted islets as reliable reporters of the autoimmune response. Longitudinal studies in ACE-transplanted islets identified in vivo hallmarks of islet inflammation that concurred with early immune infiltration of the islets and preceded their collapse and hyperglycaemia onset. A model incorporating data on ACE-transplanted islet degranulation and swelling allowed early prediction of the autoimmune attack in the pancreas and prompted treatments to intercept type 1 diabetes. CONCLUSIONS/INTERPRETATION: The current findings highlight the value of ACE-transplanted islets in studying early type 1 diabetes pathogenesis in vivo and underscore the need for timely intervention to halt disease progression.
Assuntos
Diabetes Mellitus Tipo 1/diagnóstico por imagem , Animais , Autoimunidade/fisiologia , Diabetes Mellitus Tipo 1/imunologia , Diabetes Mellitus Tipo 1/cirurgia , Sobrevivência de Enxerto/fisiologia , Células Secretoras de Insulina/imunologia , Células Secretoras de Insulina/metabolismo , Ilhotas Pancreáticas/cirurgia , Transplante das Ilhotas Pancreáticas , Camundongos , Camundongos Endogâmicos NODRESUMO
Unfortunately there is a mistake in the presentation of the affiliations in this paper.
RESUMO
AIMS/HYPOTHESIS: Tissue-resident macrophages sense the microenvironment and respond by producing signals that act locally to maintain a stable tissue state. It is now known that pancreatic islets contain their own unique resident macrophages, which have been shown to promote proliferation of the insulin-secreting beta cell. However, it is unclear how beta cells communicate with islet-resident macrophages. Here we hypothesised that islet macrophages sense changes in islet activity by detecting signals derived from beta cells. METHODS: To investigate how islet-resident macrophages respond to cues from the microenvironment, we generated mice expressing a genetically encoded Ca2+ indicator in myeloid cells. We produced living pancreatic slices from these mice and used them to monitor macrophage responses to stimulation of acinar, neural and endocrine cells. RESULTS: Islet-resident macrophages expressed functional purinergic receptors, making them exquisite sensors of interstitial ATP levels. Indeed, islet-resident macrophages responded selectively to ATP released locally from beta cells that were physiologically activated with high levels of glucose. Because ATP is co-released with insulin and is exclusively secreted by beta cells, the activation of purinergic receptors on resident macrophages facilitates their awareness of beta cell secretory activity. CONCLUSIONS/INTERPRETATION: Our results indicate that islet macrophages detect ATP as a proxy signal for the activation state of beta cells. Sensing beta cell activity may allow macrophages to adjust the secretion of factors to promote a stable islet composition and size.
Assuntos
Trifosfato de Adenosina/metabolismo , Macrófagos/metabolismo , Pâncreas/citologia , Pâncreas/metabolismo , Animais , Insulina/metabolismo , Células Secretoras de Insulina/metabolismo , Ilhotas Pancreáticas/citologia , CamundongosRESUMO
In pancreatic ß cells, muscarinic cholinergic receptor M3 (M3R) stimulates glucose-induced secretion of insulin. Regulator of G-protein signaling (RGS) proteins are critical modulators of GPCR activity, yet their role in ß cells remains largely unknown. R7 subfamily RGS proteins are stabilized by the G-protein subunit Gß5, such that the knockout of the Gnb5 gene results in degradation of all R7 subunits. We found that Gnb5 knockout in mice or in the insulin-secreting MIN6 cell line almost completely eliminates insulinotropic activity of M3R. Moreover, overexpression of Gß5-RGS7 strongly promotes M3R-stimulated insulin secretion. Examination of this noncanonical mechanism in Gnb5-/- MIN6 cells showed that cAMP, diacylglycerol, or Ca2+ levels were not significantly affected. There was no reduction in the amplitude of free Ca2+ responses in islets from the Gnb5-/- mice, but the frequency of Ca2+ oscillations induced by cholinergic agonist was lowered by more than 30%. Ablation of Gnb5 impaired M3R-stimulated phosphorylation of ERK1/2. Stimulation of the ERK pathway in Gnb5-/- cells by epidermal growth factor restored M3R-stimulated insulin release to near normal levels. Identification of the novel role of Gß5-R7 in insulin secretion may lead to a new therapeutic approach for improving pancreatic ß-cell function.-Wang, Q., Pronin, A. N., Levay, K., Almaca, J., Fornoni, A., Caicedo, A., Slepak, V. Z. Regulator of G-protein signaling Gß5-R7 is a crucial activator of muscarinic M3 receptor-stimulated insulin secretion.
Assuntos
Sinalização do Cálcio/fisiologia , Subunidades beta da Proteína de Ligação ao GTP/metabolismo , Células Secretoras de Insulina/metabolismo , Insulina/metabolismo , Sistema de Sinalização das MAP Quinases/fisiologia , Proteínas RGS/metabolismo , Receptor Muscarínico M3/metabolismo , Animais , Cálcio/metabolismo , Linhagem Celular , AMP Cíclico/genética , AMP Cíclico/metabolismo , Subunidades beta da Proteína de Ligação ao GTP/genética , Secreção de Insulina , Células Secretoras de Insulina/citologia , Camundongos , Camundongos Knockout , Proteína Quinase 3 Ativada por Mitógeno/genética , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Fosforilação/fisiologia , Proteínas RGS/genética , Receptor Muscarínico M3/genéticaRESUMO
Pancreatic islets secrete hormones that play a key role in regulating blood glucose levels (glycemia). Age-dependent impairment of islet function and concomitant dysregulation of glycemia are major health threats in aged populations. However, the major causes of the age-dependent decline of islet function are still disputed. Here we demonstrate that aging of pancreatic islets in mice and humans is notably associated with inflammation and fibrosis of islet blood vessels but does not affect glucose sensing and the insulin secretory capacity of islet beta cells. Accordingly, when transplanted into the anterior chamber of the eye of young mice with diabetes, islets from old mice are revascularized with healthy blood vessels, show strong islet cell proliferation, and fully restore control of glycemia. Our results indicate that beta cell function does not decline with age and suggest that islet function is threatened by an age-dependent impairment of islet vascular function. Strategies to mitigate age-dependent dysregulation in glycemia should therefore target systemic and/or local inflammation and fibrosis of the aged islet vasculature.
Assuntos
Envelhecimento , Glicemia/metabolismo , Capilares/fisiologia , Ilhotas Pancreáticas/fisiologia , Adolescente , Adulto , Idoso , Animais , Proliferação de Células , Fibrose , Glucose/metabolismo , Homeostase , Humanos , Inflamação , Insulina/metabolismo , Ilhotas Pancreáticas/citologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Pessoa de Meia-Idade , Perfusão , Fatores de Tempo , Adulto JovemRESUMO
Type 1 diabetes (T1D) patients who receive pancreatic islet transplant experience significant improvement in their quality-of-life. This comes primarily through improved control of blood sugar levels, restored awareness of hypoglycemia, and prevention of serious and potentially life-threatening diabetes-associated complications, such as kidney failure, heart and vascular disease, stroke, nerve damage, and blindness. Therefore, beta cell replacement through transplantation of isolated islets is an important option in the treatment of T1D. However, lasting success of this promising therapy depends on durable survival and efficacy of the transplanted islets, which are directly influenced by the islet isolation procedures. Thus, isolating pancreatic islets with consistent and reliable quality is critical in the clinical application of islet transplantation.Quality of isolated islets is important in pre-clinical studies as well, as efforts to advance and improve clinical outcomes of islet transplant therapy have relied heavily on animal models ranging from rodents, to pigs, to nonhuman primates. As a result, pancreatic islets have been isolated from these and other species and used in a variety of in vitro or in vivo applications for this and other research purposes. Protocols for islet isolation have been somewhat similar across species, especially, in mammals. However, given the increasing evidence about the distinct structural and functional features of human and mouse islets, using similar methods of islet isolation may contribute to inconsistencies in the islet quality, immunogenicity, and experimental outcomes. This may also contribute to the discrepancies commonly observed between pre-clinical findings and clinical outcomes. Therefore, it is prudent to consider the particular features of pancreatic islets from different species when optimizing islet isolation protocols.In this chapter, we explore the structural and functional features of pancreatic islets from mice, pigs, nonhuman primates, and humans because of their prevalent use in nonclinical, preclinical, and clinical applications.
Assuntos
Ilhotas Pancreáticas/fisiologia , Animais , Humanos , Ilhotas Pancreáticas/irrigação sanguínea , Ilhotas Pancreáticas/citologia , Ilhotas Pancreáticas/inervação , Comunicação Parácrina , Transdução de SinaisRESUMO
The pancreatic islet secretes the hormones insulin and glucagon to regulate glucose metabolism. To generate an adequate secretory response, islet endocrine cells must receive multiple regulatory signals relaying information about changes in the internal and external environments. Islet cells also need to be made aware about the functional status of neighboring cells through paracrine interactions. All this information is used to orchestrate a hormonal response that contributes to glucose homeostasis. Several neurotransmitters have been proposed to work as paracrine signals in the islet. Most of these, however, have yet to meet the criteria to be considered bona fide paracrine signals, in particular in human islets. Here, we review recent findings describing autocrine and paracrine signaling mechanisms in human islets. These recent results are showing an increasingly complex picture of paracrine interactions in the human islet and emphasize that results from other species cannot be readily extrapolated to the human context. Investigators are unveiling new signaling mechanisms or finding new roles for known paracrine signals in human islets. While it is too early to provide a synthesis, the field of islet research is defining the paracrine and autocrine components that will be used to generate models about how islet function is regulated. Meanwhile, the identified signaling pathways can be proposed as therapeutic targets for treating diabetes, a devastating disease affecting millions worldwide.
Assuntos
Comunicação Autócrina , Ilhotas Pancreáticas/metabolismo , Comunicação Parácrina , Trifosfato de Adenosina/metabolismo , Animais , Ácido Glutâmico/metabolismo , Humanos , Ilhotas Pancreáticas/anatomia & histologia , Ácido gama-Aminobutírico/metabolismoRESUMO
AIMS/HYPOTHESIS: Insulin secretion is widely studied because it plays a central role in glucose homeostasis and diabetes. Processes from insulin granule fusion in beta cells to in vivo insulin secretion have been elucidated, but data at the cellular level do not fully account for several aspects of the macroscopic secretory pattern. Here we investigated how individual secretory events are coordinated spatially and temporally within intact human islets. METHODS: We used the fluorescent probe neuropeptide Y (NPY)-pHluorin to visualise insulin granule secretion in isolated intact human islets. RESULTS: We found that individual beta cells respond to increases in glucose concentration by releasing insulin granules in very discrete bursts with periods consistent with in vivo pulsatile insulin secretion. In successive secretory bursts during prolonged exposure to high glucose levels, secretory events progressively localised to preferential release sites, coinciding with the transition to second phase insulin secretion. Granule secretion was very synchronised in neighbouring beta cells, forming discrete regional clusters of activity. CONCLUSIONS/INTERPRETATION: These results reveal how individual secretory events are coordinated to produce pulsatile insulin secretion from human islets.
Assuntos
Exocitose/efeitos dos fármacos , Ilhotas Pancreáticas/metabolismo , Adulto , Células Cultivadas , Grânulos Citoplasmáticos/metabolismo , Feminino , Glucose/farmacologia , Proteínas de Fluorescência Verde , Humanos , Insulina/metabolismo , Secreção de Insulina , Células Secretoras de Insulina/metabolismo , Ilhotas Pancreáticas/anatomia & histologia , Ilhotas Pancreáticas/efeitos dos fármacos , Masculino , Pessoa de Meia-Idade , Neuropeptídeo Y/metabolismoRESUMO
The autonomic nervous system is thought to modulate blood glucose homeostasis by regulating endocrine cell activity in the pancreatic islets of Langerhans. The role of islet innervation, however, has remained elusive because the direct effects of autonomic nervous input on islet cell physiology cannot be studied in the pancreas. Here, we used an in vivo model to study the role of islet nervous input in glucose homeostasis. We transplanted islets into the anterior chamber of the eye and found that islet grafts became densely innervated by the rich parasympathetic and sympathetic nervous supply of the iris. Parasympathetic innervation was imaged intravitally by using transgenic mice expressing GFP in cholinergic axons. To manipulate selectively the islet nervous input, we increased the ambient illumination to increase the parasympathetic input to the islet grafts via the pupillary light reflex. This reduced fasting glycemia and improved glucose tolerance. These effects could be blocked by topical application of the muscarinic antagonist atropine to the eye, indicating that local cholinergic innervation had a direct effect on islet function in vivo. By using this approach, we found that parasympathetic innervation influences islet function in C57BL/6 mice but not in 129X1 mice, which reflected differences in innervation densities and may explain major strain differences in glucose homeostasis. This study directly demonstrates that autonomic axons innervating the islet modulate glucose homeostasis.
Assuntos
Sistema Nervoso Autônomo/fisiologia , Olho/inervação , Ilhotas Pancreáticas/fisiologia , Modelos Biológicos , Animais , Proteínas de Fluorescência Verde/metabolismo , Iris/inervação , Iris/fisiologia , Transplante das Ilhotas Pancreáticas , Camundongos , Camundongos da Linhagem 129 , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Fibras NervosasRESUMO
In this symposium review we discuss the role of neurotransmitters as paracrine signals that regulate pancreatic islet function. A large number of neurotransmitters and their receptors has been identified in the islet, but relatively little is known about their involvement in islet biology. Interestingly, neurotransmitters initially thought to be present in autonomic axons innervating the islet are also present in endocrine cells of the human islet. These neurotransmitters can thus be released as paracrine signals to help control hormone release. Here we propose that the role of neurotransmitters may extend beyond controlling endocrine cell function to work as signals modulating vascular flow and immune responses within the islet.
Assuntos
Insulina/metabolismo , Ilhotas Pancreáticas/metabolismo , Neurotransmissores/metabolismo , Comunicação Parácrina , Animais , Humanos , Secreção de Insulina , Ilhotas Pancreáticas/inervaçãoRESUMO
Intravital imaging emerged as an indispensible tool in biological research, and a variety of imaging techniques have been developed to noninvasively monitor tissues in vivo. However, most of the current techniques lack the resolution to study events at the single-cell level. Although intravital multiphoton microscopy has addressed this limitation, the need for repeated noninvasive access to the same tissue in longitudinal in vivo studies remains largely unmet. We now report on a previously unexplored approach to study immune responses after transplantation of pancreatic islets into the anterior chamber of the mouse eye. This approach enabled (i) longitudinal, noninvasive imaging of transplanted tissues in vivo; (ii) in vivo cytolabeling to assess cellular phenotype and viability in situ; (iii) local intervention by topical application or intraocular injection; and (iv) real-time tracking of infiltrating immune cells in the target tissue.
Assuntos
Câmara Anterior/citologia , Ilhotas Pancreáticas/citologia , Microscopia Confocal/métodos , Linfócitos T/citologia , Amidas/farmacologia , Animais , Câmara Anterior/metabolismo , Câmara Anterior/cirurgia , Antagonistas dos Receptores CCR5 , Quimiocinas/farmacologia , Diabetes Mellitus Experimental/terapia , Ensaio de Imunoadsorção Enzimática , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Interferon gama/metabolismo , Interleucina-2/metabolismo , Ilhotas Pancreáticas/metabolismo , Transplante das Ilhotas Pancreáticas/métodos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos CBA , Microscopia de Vídeo/métodos , Compostos de Amônio Quaternário/farmacologia , Receptores CCR5/metabolismo , Análise de Célula Única/métodos , Linfócitos T/efeitos dos fármacos , Linfócitos T/metabolismo , Fatores de TempoRESUMO
Long lagging behind insulin, glucagon research has caught up in large part, thanks to technological breakthroughs. Here we review how the field was propelled by the development of novel techniques and approaches. The glucagon radioimmunoassay and islet isolation are methods that now seem trivial, but for decades they were crucial in defining the biology of the pancreatic alpha cell and the role of glucagon in glucose homeostasis. More recently, mouse models have become the main workhorse of this research effort, if not of biomedical research in general. The mouse model allowed detailed mechanistic studies that are revealing alpha cell functions beyond its canonical glucoregulatory role. A recent profusion of gene expression and transcription regulation studies is providing new vistas into what constitutes alpha cell identity. In particular, the combination of transcriptomic techniques with functional recordings promises to move molecular guesswork into real-time physiology. The challenge right now is not to get enamored with these powerful techniques and to make sure that the research continues to be transformative and paradigm shifting. We should imagine a future in which the biology of the alpha cell will be studied at single-cell resolution, non-invasively, and in real time in the human body.
Assuntos
Células Secretoras de Glucagon , Células Secretoras de Insulina , Ilhotas Pancreáticas , Camundongos , Animais , Humanos , Glucagon/metabolismo , Células Secretoras de Glucagon/metabolismo , Glucose/metabolismo , Insulina/metabolismo , Células Secretoras de Insulina/metabolismo , Modelos Animais de Doenças , Ilhotas Pancreáticas/metabolismoRESUMO
Extracellular ATP has been proposed as a paracrine signal in rodent islets, but it is unclear what role ATP plays in human islets. We now show the presence of an ATP signaling pathway that enhances the human beta cell's sensitivity and responsiveness to glucose fluctuations. By using in situ hybridization, RT-PCR, immunohistochemistry, and Western blotting as well as recordings of cytoplasmic-free Ca(2+) concentration, [Ca(2+)](i), and hormone release in vitro, we show that human beta cells express ionotropic ATP receptors of the P2X(3) type and that activation of these receptors by ATP coreleased with insulin amplifies glucose-induced insulin secretion. Released ATP activates P2X(3) receptors in the beta-cell plasma membrane, resulting in increased [Ca(2+)](i) and enhanced insulin secretion. Therefore, in human islets, released ATP forms a positive autocrine feedback loop that sensitizes the beta cell's secretory machinery. This may explain how the human pancreatic beta cell can respond so effectively to relatively modest changes in glucose concentration under physiological conditions in vivo.
Assuntos
Trifosfato de Adenosina/metabolismo , Comunicação Autócrina , Células Secretoras de Insulina/metabolismo , Insulina/metabolismo , Receptores Purinérgicos P2/metabolismo , Cálcio/metabolismo , Humanos , Secreção de Insulina , Receptores Purinérgicos P2/genética , Receptores Purinérgicos P2X3 , Transdução de SinaisRESUMO
Pancreatic tissue slices allow functional investigations under close physiological conditions in situ. This approach is particularly advantageous for studying infiltrated and structurally damaged islets as found in T1D. More importantly, slices allow studying the interplay between endocrine and exocrine compartments. We here describe how to perform agarose injections, tissue preparation, and slice procedure for mouse and human tissue. We then describe in detail how to use the slices to perform functional studies using hormone secretion and calcium imaging as readouts. For complete details on the use and execution of this protocol, please refer to Panzer et al. (2022).1.
Assuntos
Pâncreas , Hormônios Pancreáticos , Humanos , Animais , Camundongos , Cálcio , Técnicas Histológicas , SefaroseRESUMO
Low-dose IL-2 is a promising immunotherapy in clinical trials for treating type 1 diabetes. A new IL-2 analog, IL-2/CD25 fusion protein, has been shown to more efficiently delay or prevent diabetes in NOD mice by expanding the population of activated regulatory T cells. This therapy is intended for use before clinical diagnosis, in the early stages of type 1 diabetes progression. During this prediabetic period, there is a chronic decline in ß-cell function that has long-term implications for disease pathogenesis. Yet, to date, the effects of IL-2/CD25 on ß-cell function have not been evaluated. In this study, we treated prediabetic NOD mice with low-dose mouse IL-2/CD25 over 5 weeks and determined its impact on ß-cell function. This treatment limited the progressive impairment of glucose tolerance and insulin secretion typical of the later stages of prediabetes. Intracellular Ca2+ responses to glucose in ß-cells became more robust and synchronous, indicating that changing the local immune cell infiltrate with IL-2/CD25 preserved ß-cell function even after treatment cessation. Our study thus provides mechanistic insight and serves as a steppingstone for future research using low-dose IL-2/CD25 immunotherapy in patients. ARTICLE HIGHLIGHTS: Immunotherapies such as IL-2/CD25 are known to prevent or delay diabetes. However, their impact on individual ß-cell function is not yet understood. Female NOD mice progress from stage 1 to 2 pre-type 1 diabetes between 12 and 17 weeks. Treatment with mouse IL-2 (mIL-2)/CD25 prevents this progression even after treatment cessation. Individual ß-cell function (measured via intracellular Ca2+ responses to glucose) declines during the pathogenesis of type 1 diabetes. Treatment with mIL-2/CD25 therapy limits ß-cell dysfunction, and function continues to improve after treatment cessation. Insulin secretion is improved with mIL-2/CD25 therapy.
Assuntos
Diabetes Mellitus Tipo 1 , Estado Pré-Diabético , Feminino , Animais , Camundongos , Camundongos Endogâmicos NOD , Interleucina-2/metabolismo , Cálcio/metabolismo , Linfócitos T Reguladores , Glucose/metabolismo , ImunoterapiaRESUMO
Mitochondrial metabolism and oxidative respiration are crucial for pancreatic ß-cell function and stimulus secretion coupling. Oxidative phosphorylation (OxPhos) produces ATP and other metabolites that potentiate insulin secretion. However, the contribution of individual OxPhos complexes to ß-cell function is unknown. We generated ß-cell-specific, inducible OxPhos complex knock-out (KO) mouse models to investigate the effects of disrupting complex I, complex III, or complex IV on ß-cell function. Although all KO models had similar mitochondrial respiratory defects, complex III caused early hyperglycemia, glucose intolerance, and loss of glucose-stimulated insulin secretion in vivo. However, ex vivo insulin secretion did not change. Complex I and IV KO models showed diabetic phenotypes much later. Mitochondrial Ca2+ responses to glucose stimulation 3 weeks after gene deletion ranged from not affected to severely disrupted, depending on the complex targeted, supporting the unique roles of each complex in ß-cell signaling. Mitochondrial antioxidant enzyme immunostaining increased in islets from complex III KO, but not from complex I or IV KO mice, indicating that severe diabetic phenotype in the complex III-deficient mice is causing alterations in cellular redox status. The present study highlights that defects in individual OxPhos complexes lead to different pathogenic outcomes. ARTICLE HIGHLIGHTS: Mitochondrial metabolism is critical for ß-cell insulin secretion, and mitochondrial dysfunction is involved in type 2 diabetes pathogenesis. We determined whether individual oxidative phosphorylation complexes contribute uniquely to ß-cell function. Compared with loss of complex I and IV, loss of complex III resulted in severe in vivo hyperglycemia and altered ß-cell redox status. Loss of complex III altered cytosolic and mitochondrial Ca2+ signaling and increased expression of glycolytic enzymes. Individual complexes contribute differently to ß-cell function. This underscores the role of mitochondrial oxidative phosphorylation complex defects in diabetes pathogenesis.
Assuntos
Diabetes Mellitus Tipo 2 , Hiperglicemia , Células Secretoras de Insulina , Camundongos , Animais , Complexo III da Cadeia de Transporte de Elétrons/metabolismo , Diabetes Mellitus Tipo 2/metabolismo , Cálcio/metabolismo , Hiperglicemia/metabolismo , Células Secretoras de Insulina/metabolismo , Glucose/metabolismo , Camundongos Knockout , Insulina/metabolismoRESUMO
The Integrated Physiology of the Exocrine and Endocrine Compartments in Pancreatic Diseases workshop was a 1.5-day scientific conference at the National Institutes of Health (Bethesda, MD) that engaged clinical and basic science investigators interested in diseases of the pancreas. This report provides a summary of the proceedings from the workshop. The goals of the workshop were to forge connections and identify gaps in knowledge that could guide future research directions. Presentations were segregated into six major theme areas, including 1) pancreas anatomy and physiology, 2) diabetes in the setting of exocrine disease, 3) metabolic influences on the exocrine pancreas, 4) genetic drivers of pancreatic diseases, 5) tools for integrated pancreatic analysis, and 6) implications of exocrine-endocrine cross talk. For each theme, multiple presentations were followed by panel discussions on specific topics relevant to each area of research; these are summarized here. Significantly, the discussions resulted in the identification of research gaps and opportunities for the field to address. In general, it was concluded that as a pancreas research community, we must more thoughtfully integrate our current knowledge of normal physiology as well as the disease mechanisms that underlie endocrine and exocrine disorders so that there is a better understanding of the interplay between these compartments.