RESUMEN
AIMS/HYPOTHESIS: Apart from its fibrinolytic activity, the tissue plasminogen activator (tPA)/plasmin system has been reported to cleave the peptide amyloid beta, attenuating brain amyloid deposition in Alzheimer's disease. As aggregation of human islet amyloid polypeptide (hIAPP) is toxic to beta cells, we sought to determine whether activation of the fibrinolytic system can also reduce islet amyloid deposition and its cytotoxic effects, which are both observed in type 2 diabetes. METHODS: The expression of Plat (encoding tPA) and plasmin activity were measured in isolated islets from amyloid-prone hIAPP transgenic mice or non-transgenic control islets expressing non-amyloidogenic mouse islet amyloid polypeptide cultured in the absence or presence of the amyloid inhibitor Congo Red. Plat expression was also determined in hIAPP-treated primary islet endothelial cells, bone marrow-derived macrophages (BMDM) and INS-1 cells, in order to determine the islet cell type(s) producing tPA in response to hIAPP aggregation. Cell-free thioflavin-T assays and MS were used to respectively monitor hIAPP aggregation kinetics and investigate plasmin cleavage of hIAPP. Cell viability was assessed in INS-1 beta cells treated with hIAPP with or without plasmin. Finally, to confirm the findings in human samples, PLAT expression was measured in freshly isolated islets from donors with and without type 2 diabetes. RESULTS: In isolated islets from transgenic mice, islet Plat expression and plasmin activity increased significantly with the process of amyloid deposition (p≤0.01, n=5); these effects were not observed in islets from non-transgenic mice and were blocked by Congo Red (p≤0.01, n=4). In response to hIAPP exposure, Plat expression increased in BMDM and INS-1 cells vs vehicle-treated cells (p≤0.05, n=4), but not in islet endothelial cells. Plasmin reduced hIAPP fibril formation in a dose-dependent manner in a cell-free system, and restored hIAPP-induced loss of cell viability in INS-1 beta cells (p≤0.01, n=5). Plasmin cleaved monomeric hIAPP, inducing a rapid decrease in the abundance of full-length hIAPP and the appearance of hIAPP 1-11 and 12-37 fragments. hIAPP 12-37, which contains the critical amyloidogenic region, was not toxic to INS-1 cells. Finally, PLAT expression was significantly increased by 2.4-fold in islets from donors with type 2 diabetes (n=4) vs islets from donors without type 2 diabetes (n=7) (p≤0.05). CONCLUSIONS/INTERPRETATION: The fibrinolytic system is upregulated in islets with hIAPP aggregation. Plasmin rapidly degrades hIAPP, limiting its aggregation into amyloid and thus protecting beta cells from hIAPP-induced toxicity. Thus, increasing islet plasmin activity might be a strategy to limit beta cell loss in type 2 diabetes.
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BACKGROUND: Neutrophils are important in the pathophysiology of coronavirus disease 2019 (COVID-19), but the molecular changes contributing to altered neutrophil phenotypes following severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection are not fully understood. We used quantitative mass spectrometry-based proteomics to explore neutrophil phenotypes immediately following acute SARS-CoV-2 infection and during recovery. METHODS: Prospective observational study of hospitalised patients with PCR-confirmed SARS-CoV-2 infection (May to December 2020). Patients were enrolled within 96â h of admission, with longitudinal sampling up to 29â days. Control groups comprised non-COVID-19 acute lower respiratory tract infection (LRTI) and age-matched noninfected controls. Neutrophils were isolated from peripheral blood and analysed using mass spectrometry. COVID-19 severity and recovery were defined using the World Health Organization ordinal scale. RESULTS: Neutrophil proteomes from 84 COVID-19 patients were compared to those from 91 LRTI and 42 control participants. 5800 neutrophil proteins were identified, with >1700 proteins significantly changed in neutrophils from COVID-19 patients compared to noninfected controls. Neutrophils from COVID-19 patients initially all demonstrated a strong interferon signature, but this signature rapidly declined in patients with severe disease. Severe disease was associated with increased abundance of proteins involved in metabolism, immunosuppression and pattern recognition, while delayed recovery from COVID-19 was associated with decreased granule components and reduced abundance of metabolic proteins, chemokine and leukotriene receptors, integrins and inhibitory receptors. CONCLUSIONS: SARS-CoV-2 infection results in the sustained presence of circulating neutrophils with distinct proteomes suggesting altered metabolic and immunosuppressive profiles and altered capacities to respond to migratory signals and cues from other immune cells, pathogens or cytokines.
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COVID-19 , Humanos , SARS-CoV-2 , Neutrófilos , Proteoma , CitocinasRESUMEN
AIMS/HYPOTHESIS: The islet vasculature, including its constituent islet endothelial cells, is a key contributor to the microenvironment necessary for normal beta cell health and function. In type 2 diabetes, islet amyloid polypeptide (IAPP) aggregates, forming amyloid deposits that accumulate between beta cells and islet capillaries. This process is known to be toxic to beta cells but its impact on the islet vasculature has not previously been studied. Here, we report the first characterisation of the effects of IAPP aggregation on islet endothelial cells/capillaries using cell-based and animal models. METHODS: Primary and immortalised islet endothelial cells were treated with amyloidogenic human IAPP (hIAPP) alone or in the presence of the amyloid blocker Congo Red or the Toll-like receptor (TLR) 2/4 antagonist OxPAPc. Cell viability was determined0 along with mRNA and protein levels of inflammatory markers. Islet capillary abundance, morphology and pericyte coverage were determined in pancreases from transgenic mice with beta cell expression of hIAPP using conventional and confocal microscopy. RESULTS: Aggregated hIAPP decreased endothelial cell viability in immortalised and primary islet endothelial cells (by 78% and 60%, respectively) and significantly increased expression of inflammatory markers Il6, Vcam1 and Edn1 mRNA relative to vehicle treatment in both cell types (p<0.05; n=4). Both cytotoxicity and the proinflammatory response were ameliorated by Congo Red (p<0.05; n=4); whereas TLR2/4-inhibition blocked inflammatory gene expression (p<0.05; n=6) without improving viability. Islets from high-fat-diet-fed amyloid-laden hIAPP transgenic mice also exhibited significantly increased expression of most markers of endothelial inflammation (p<0.05; n=5) along with decreased capillary density compared with non-transgenic littermates fed the same diet (p<0.01). Moreover, a 16% increase in capillary diameter was observed in amyloid-adjacent capillaries (p<0.01), accompanied by a doubling in pericyte structures positive for neuron-glial antigen 2 (p<0.001). CONCLUSIONS/INTERPRETATION: Islet endothelial cells are susceptible to hIAPP-induced cytotoxicity and exhibit a TLR2/4-dependent proinflammatory response to aggregated hIAPP. Additionally, we observed amyloid-selective effects that decreased islet capillary density, accompanied by increased capillary diameter and increased pericyte number. Together, these data demonstrate that the islet vasculature is a target of the cytotoxic and proinflammatory effects of aggregated hIAPP that likely contribute to the detrimental effects of hIAPP aggregation on beta cell function and survival in type 2 diabetes.
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Amiloidosis , Diabetes Mellitus Tipo 2 , Células Secretoras de Insulina , Islotes Pancreáticos , Amiloide/metabolismo , Amiloidosis/metabolismo , Animales , Rojo Congo/metabolismo , Rojo Congo/farmacología , Diabetes Mellitus Tipo 2/metabolismo , Células Endoteliales/metabolismo , Humanos , Células Secretoras de Insulina/metabolismo , Interleucina-6/metabolismo , Polipéptido Amiloide de los Islotes Pancreáticos/metabolismo , Islotes Pancreáticos/metabolismo , Ratones , Ratones Transgénicos , ARN Mensajero/metabolismo , Receptor Toll-Like 2/genética , Receptor Toll-Like 2/metabolismoRESUMEN
Type 2 diabetes is associated with the upregulation of neprilysin, a peptidase capable of cleaving glucoregulatory peptides such as glucagon-like peptide-1 (GLP-1). In humans, use of the neprilysin inhibitor sacubitril in combination with an angiotensin II receptor blocker was associated with increased plasma GLP-1 levels and improved glycemic control. Whether neprilysin inhibition per se is mediating these effects remains unknown. We sought to determine whether pharmacological neprilysin inhibition on its own confers beneficial effects on glycemic status and ß-cell function in a mouse model of reduced insulin secretion, and whether any such effects are dependent on GLP-1 receptor (GLP-1R) signaling. High-fat-fed male wild-type (Glp1r+/+) and GLP-1R knockout (Glp1r-/-) mice were treated with low-dose streptozotocin (STZ) to recapitulate type 2 diabetes-associated ß-cell dysfunction, or vehicle as control. Mice were continued on high-fat diet alone or supplemented with the neprilysin inhibitor sacubitril for 8 wk. At the end of the study period, ß-cell function was assessed by oral or intravenous glucose-tolerance test. Fasting and fed glucose were significantly lower in wild-type mice treated with sacubitril, although active GLP-1 levels and insulin secretion during oral glucose challenge were unchanged. In contrast, insulin secretion in response to intravenous glucose was significantly enhanced in sacubitril-treated wild-type mice, and this effect was blunted in Glp1r-/- mice. Similarly, sacubitril enhanced insulin secretion in vitro in islets from STZ-treated Glp1r+/+ but not Glp1r-/- mice. Together, our data suggest the insulinotropic effects of pharmacological neprilysin inhibition in a mouse model of ß-cell dysfunction are mediated via intra-islet GLP-1R signaling.NEW & NOTEWORTHY The neprilysin inhibitor, sacubitril, improves glycemic status in a mouse model of reduced insulin secretion. Sacubitril enhances intravenous but not oral glucose-mediated insulin secretion. The increased glucose-mediated insulin secretion is GLP-1 receptor-dependent. Neprilysin inhibition does not raise postprandial circulating active GLP-1 levels.
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Diabetes Mellitus Tipo 2 , Receptor del Péptido 1 Similar al Glucagón , Secreción de Insulina , Neprilisina , Aminobutiratos , Animales , Compuestos de Bifenilo , Diabetes Mellitus Tipo 2/tratamiento farmacológico , Péptido 1 Similar al Glucagón , Receptor del Péptido 1 Similar al Glucagón/metabolismo , Glucosa , Insulina/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Neprilisina/antagonistas & inhibidores , Neprilisina/metabolismoRESUMEN
Bacterial pathogens are confronted with a range of challenges at the site of infection, including exposure to antibiotic treatment and harsh physiological conditions, that can alter the fitness benefits and costs of acquiring antibiotic resistance. Here, we develop an experimental system to recapitulate resistance gene acquisition by Staphylococcus aureus and test how the subsequent evolution of the resistant bacterium is modulated by antibiotic treatment and oxygen levels, both of which are known to vary extensively at sites of infection. We show that acquiring tetracycline resistance was costly, reducing competitive growth against the isogenic strain without the resistance gene in the absence of the antibiotic, for S. aureus under hypoxic but not normoxic conditions. Treatment with tetracycline or doxycycline drove the emergence of enhanced resistance through mutations in an RluD-like protein-encoding gene and duplications of tetL, encoding the acquired tetracycline-specific efflux pump. In contrast, evolutionary adaptation by S. aureus to hypoxic conditions, which evolved in the absence of antibiotics through mutations affecting gyrB, was impeded by antibiotic treatment. Together, these data suggest that the horizontal acquisition of a new resistance mechanism is merely a starting point for the emergence of high-level resistance under antibiotic selection but that antibiotic treatment constrains pathogen adaptation to other important environmental selective forces such as hypoxia, which in turn could limit the survival of these highly resistant but poorly adapted genotypes after antibiotic treatment is ended.
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Staphylococcus aureus Resistente a Meticilina , Infecciones Estafilocócicas , Humanos , Antibacterianos/farmacología , Staphylococcus aureus/genética , Staphylococcus aureus Resistente a Meticilina/genética , Infecciones Estafilocócicas/tratamiento farmacológico , Infecciones Estafilocócicas/microbiología , Tetraciclina/farmacología , Hipoxia , Proteínas Bacterianas/genéticaRESUMEN
Interleukin 1ß (IL-1ß) is an important inflammatory mediator of type 2 diabetes. Here we show that oligomers of islet amyloid polypeptide (IAPP), a protein that forms amyloid deposits in the pancreas during type 2 diabetes, triggered the NLRP3 inflammasome and generated mature IL-1ß. One therapy for type 2 diabetes, glyburide, suppressed IAPP-mediated IL-1ß production in vitro. Processing of IL-1ß initiated by IAPP first required priming, a process that involved glucose metabolism and was facilitated by minimally oxidized low-density lipoprotein. Finally, mice transgenic for human IAPP had more IL-1ß in pancreatic islets, which localized together with amyloid and macrophages. Our findings identify previously unknown mechanisms in the pathogenesis of type 2 diabetes and treatment of pathology caused by IAPP.
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Amiloide/metabolismo , Proteínas Portadoras/metabolismo , Diabetes Mellitus Tipo 2/inmunología , Interleucina-1beta/metabolismo , Receptores Citoplasmáticos y Nucleares/metabolismo , Animales , Proteínas Portadoras/antagonistas & inhibidores , Proteínas Portadoras/genética , Células Cultivadas , Células Dendríticas/inmunología , Células Dendríticas/metabolismo , Diabetes Mellitus Tipo 2/metabolismo , Gliburida/farmacología , Humanos , Hipoglucemiantes/farmacología , Polipéptido Amiloide de los Islotes Pancreáticos , Islotes Pancreáticos/metabolismo , Macrófagos/inmunología , Macrófagos/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Proteína con Dominio Pirina 3 de la Familia NLR , Ratas , Receptores Citoplasmáticos y Nucleares/antagonistas & inhibidores , Receptores Citoplasmáticos y Nucleares/genéticaRESUMEN
The islets of Langerhans are well embedded within the exocrine pancreas (the latter comprised of ducts and acini), but the nature of interactions between these pancreatic compartments and their role in determining normal islet function and survival are poorly understood. However, these interactions appear to be critical, as when pancreatic exocrine disease occurs, islet function and insulin secretion frequently decline to the point that diabetes ensues, termed pancreatogenic diabetes. The most common forms of pancreatogenic diabetes involve sustained exocrine disease leading to ductal obstruction, acinar inflammation, and fibro-fatty replacement of the exocrine pancreas that predates the development of dysfunction of the endocrine pancreas, as seen in chronic pancreatitis-associated diabetes and cystic fibrosis-related diabetes and, more rarely, MODY type 8. Intriguingly, a form of tumour-induced diabetes has been described that is associated with pancreatic ductal adenocarcinoma. Here, we review the similarities and differences among these forms of pancreatogenic diabetes, with the goal of highlighting the importance of exocrine/ductal homeostasis for the maintenance of pancreatic islet function and survival and to highlight the need for a better understanding of the mechanisms underlying these diverse conditions. Graphical abstract.
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Fibrosis Quística/metabolismo , Diabetes Mellitus/metabolismo , Islotes Pancreáticos/metabolismo , Páncreas Exocrino/metabolismo , Pancreatitis Crónica/metabolismo , Animales , Carcinoma Ductal Pancreático/complicaciones , Carcinoma Ductal Pancreático/metabolismo , Carcinoma Ductal Pancreático/patología , Fibrosis Quística/complicaciones , Fibrosis Quística/patología , Diabetes Mellitus/etiología , Humanos , Islotes Pancreáticos/patología , Islotes Pancreáticos/fisiopatología , Páncreas Exocrino/patología , Páncreas Exocrino/fisiopatología , Enfermedades Pancreáticas/complicaciones , Enfermedades Pancreáticas/metabolismo , Enfermedades Pancreáticas/patología , Neoplasias Pancreáticas/complicaciones , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/patología , Pancreatitis Crónica/complicaciones , Pancreatitis Crónica/patologíaRESUMEN
AIMS/HYPOTHESIS: Aggregation of the beta cell secretory product human islet amyloid polypeptide (hIAPP) results in islet amyloid deposition, a pathological feature of type 2 diabetes. Amyloid formation is associated with increased levels of islet IL-1ß as well as beta cell dysfunction and death, but the mechanisms that promote amyloid deposition in situ remain unclear. We hypothesised that physiologically relevant concentrations of IL-1ß stimulate beta cell islet amyloid polypeptide (IAPP) release and promote amyloid formation. METHODS: We used a humanised mouse model of endogenous beta cell hIAPP expression to examine whether low (pg/ml) concentrations of IL-1ß promote islet amyloid formation in vitro. Amyloid-forming islets were cultured for 48 h in the presence or absence of IL-1ß with or without an IL-1ß neutralising antibody. Islet morphology was assessed by immunohistochemistry and islet mRNA expression, hormone content and release were also quantified. Cell-free thioflavin T assays were used to monitor hIAPP aggregation kinetics in the presence and absence of IL-1ß. RESULTS: Treatment with a low concentration of IL-1ß (4 pg/ml) for 48 h increased islet amyloid prevalence (93.52 ± 3.89% vs 43.83 ± 9.67% amyloid-containing islets) and amyloid severity (4.45 ± 0.82% vs 2.16 ± 0.50% amyloid area/islet area) in hIAPP-expressing mouse islets in vitro. This effect of IL-1ß was reduced when hIAPP-expressing islets were co-treated with an IL-1ß neutralising antibody. Cell-free hIAPP aggregation assays showed no effect of IL-1ß on hIAPP aggregation in vitro. Low concentration IL-1ß did not increase markers of the unfolded protein response (Atf4, Ddit3) or alter proIAPP processing enzyme gene expression (Pcsk1, Pcsk2, Cpe) in hIAPP-expressing islets. However, release of IAPP and insulin were increased over 48 h in IL-1ß-treated vs control islets (IAPP 0.409 ± 0.082 vs 0.165 ± 0.051 pmol/5 islets; insulin 87.5 ± 8.81 vs 48.3 ± 17.3 pmol/5 islets), and this effect was blocked by co-treatment with IL-1ß neutralising antibody. CONCLUSIONS/INTERPRETATION: Under amyloidogenic conditions, physiologically relevant levels of IL-1ß promote islet amyloid formation by increasing beta cell release of IAPP. Neutralisation of this effect of IL-1ß may decrease the deleterious effects of islet amyloid formation on beta cell function and survival.
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Interleucina-1beta/farmacología , Amiloidosis/tratamiento farmacológico , Animales , Humanos , Insulina/metabolismo , Células Secretoras de Insulina/efectos de los fármacos , Células Secretoras de Insulina/metabolismo , Polipéptido Amiloide de los Islotes Pancreáticos/metabolismo , RatonesRESUMEN
Neutrophils are key to host defence, and impaired neutrophil function predisposes to infection with an array of pathogens, with Staphylococcus aureus a common and sometimes life-threatening problem in this setting. Both infiltrating immune cells and replicating bacteria consume oxygen, contributing to the profound tissue hypoxia that characterises sites of infection. Hypoxia in turn has a dramatic effect on both neutrophil bactericidal function and the properties of S. aureus, including the production of virulence factors. Hypoxia thereby shapes the host-pathogen interaction and the progression of infection, for example promoting intracellular bacterial persistence, enabling local tissue destruction with the formation of an encaging abscess capsule, and facilitating the establishment and propagation of bacterial biofilms which block the access of host immune cells. Elucidating the molecular mechanisms underlying host-pathogen interactions in the setting of hypoxia will enable better understanding of persistent and recalcitrant infections due to S. aureus and may uncover novel therapeutic targets and strategies.
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Biopelículas/crecimiento & desarrollo , Interacciones Huésped-Patógeno , Neutrófilos , Infecciones Estafilocócicas/metabolismo , Staphylococcus aureus/fisiología , Factores de Virulencia/metabolismo , Animales , Hipoxia de la Célula , Humanos , Neutrófilos/metabolismo , Neutrófilos/microbiología , Neutrófilos/patología , Infecciones Estafilocócicas/patología , Infecciones Estafilocócicas/terapiaRESUMEN
AIMS/HYPOTHESIS: Islet amyloid deposits contribute to beta cell dysfunction and death in most individuals with type 2 diabetes but non-invasive methods to determine the presence of these pathological protein aggregates are currently not available. Therefore, we examined whether florbetapir, a radiopharmaceutical agent used for detection of amyloid-ß deposits in the brain, also allows identification of islet amyloid in the pancreas. METHODS: Saturation binding assays were used to determine the affinity of florbetapir for human islet amyloid polypeptide (hIAPP) aggregates in vitro. Islet amyloid-prone transgenic mice that express hIAPP in their beta cells and amyloid-free non-transgenic control mice were used to examine the ability of florbetapir to detect islet amyloid deposits in vitro, in vivo and ex vivo. Mice or mouse pancreases were subjected to autoradiographic, histochemical and/or positron emission tomography (PET) analyses to assess the utility of florbetapir in identifying islet amyloid. RESULTS: In vitro, florbetapir bound synthetic hIAPP fibrils with a dissociation constant of 7.9 nmol/l. Additionally, florbetapir bound preferentially to amyloid-containing hIAPP transgenic vs amyloid-free non-transgenic mouse pancreas sections in vitro, as determined by autoradiography (16,475 ± 5581 vs 5762 ± 575 density/unit area, p < 0.05). In hIAPP transgenic and non-transgenic mice fed a high-fat diet for 1 year, intravenous administration of florbetapir followed by PET scanning showed that the florbetapir signal was significantly higher in amyloid-laden hIAPP transgenic vs amyloid-free non-transgenic pancreases in vivo during the first 5 min of the scan (36.83 ± 2.22 vs 29.34 ± 2.03 standardised uptake value × min, p < 0.05). Following PET, pancreases were excised and florbetapir uptake was determined ex vivo by γ counting. Pancreatic uptake of florbetapir was significantly correlated with the degree of islet amyloid deposition, the latter assessed by histochemistry (r = 0.74, p < 0.001). CONCLUSIONS/INTERPRETATION: Florbetapir binds to islet amyloid deposits in a specific and quantitative manner. In the future, florbetapir may be useful as a non-invasive tool to identify islet amyloid deposits in humans.
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Amiloide/química , Compuestos de Anilina/farmacología , Glicoles de Etileno/farmacología , Islotes Pancreáticos/diagnóstico por imagen , Tomografía de Emisión de Positrones , Animales , Composición Corporal , Calorimetría Indirecta , Radioisótopos de Flúor/farmacología , Regulación de la Expresión Génica , Técnica de Clampeo de la Glucosa , Prueba de Tolerancia a la Glucosa , Hipotálamo/metabolismo , Insulina/metabolismo , Resistencia a la Insulina , Ligandos , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Reacción en Cadena de la Polimerasa , Proteína Tirosina Fosfatasa no Receptora Tipo 1/metabolismo , Transducción de SeñalRESUMEN
The pancreatic islet is highly vascularised, with an extensive capillary network. In addition to providing nutrients and oxygen to islet endocrine cells and transporting hormones to the peripheral circulation, islet capillaries (comprised primarily of islet endothelial cells) are an important source of signals that enhance survival and function of the islet beta cell. In type 2 diabetes, and animal models thereof, evidence exists of morphological and functional abnormalities in these islet endothelial cells. In diabetes, islet capillaries are thickened, dilated and fragmented, and islet endothelial cells express markers of inflammation and activation. In vitro data suggest that this dysfunctional islet endothelial phenotype may contribute to impaired insulin release from the beta cell. This review examines potential candidate molecules that may mediate the positive effects of islet endothelial cells on beta cell survival and function under normal conditions. Further, it explores possible mechanisms underlying the development of islet endothelial dysfunction in diabetes and reviews therapeutic options for ameliorating this aspect of the islet lesion in type 2 diabetes. Finally, considerations regarding differences between human and rodent islet vasculature and the potentially unforeseen negative consequences of strategies to expand the islet vasculature, particularly under diabetic conditions, are discussed.
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Diabetes Mellitus Tipo 2/sangre , Diabetes Mellitus Tipo 2/microbiología , Microbioma Gastrointestinal/fisiología , Islotes Pancreáticos/fisiopatología , Animales , Glucemia/metabolismo , Diabetes Mellitus Tipo 2/metabolismo , Heces/microbiología , Humanos , Islotes Pancreáticos/metabolismoRESUMEN
AIMS/HYPOTHESIS: The S20G human islet amyloid polypeptide (hIAPP) substitution is associated with an earlier onset of type 2 diabetes in humans. Studies of synthetic S20G hIAPP in cell-free systems and immortalised beta cells have suggested that this may be due to increased hIAPP amyloidogenicity and cytotoxicity. Thus, using primary islets from mice with endogenous S20G hIAPP expression, we sought to determine whether the S20G gene mutation leads to increased amyloid-induced toxicity, beta cell loss and reduced beta cell function. METHODS: Islets from mice in which mouse Iapp was replaced with human wild-type or S20G hIAPP were isolated and cultured in vitro under amyloid-forming conditions. Levels of insulin and hIAPP mRNA and protein, amyloid deposition and beta cell apoptosis and area, as well as glucose-stimulated insulin and hIAPP secretion, were quantified. RESULTS: Islets expressing S20G hIAPP cultured in 16.7 mmol/l glucose demonstrated increased amyloid deposition and beta cell apoptosis, reduced beta cell area, decreased insulin content and diminished glucose-stimulated insulin secretion, compared with islets expressing wild-type hIAPP. Amyloid deposition and beta cell apoptosis were also increased when S20G islets were cultured in 11.1 mmol/l glucose (the concentration that is thought to be physiological for mouse islets). CONCLUSIONS/INTERPRETATION: S20G hIAPP reduces beta cell number and function, thereby possibly explaining the earlier onset of type 2 diabetes in individuals carrying this gene mutation.
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Polipéptido Amiloide de los Islotes Pancreáticos/metabolismo , Islotes Pancreáticos/metabolismo , Amiloide/metabolismo , Animales , Apoptosis/efectos de los fármacos , Apoptosis/genética , Femenino , Glucosa/farmacología , Humanos , Técnicas In Vitro , Insulina/metabolismo , Células Secretoras de Insulina/efectos de los fármacos , Células Secretoras de Insulina/metabolismo , Polipéptido Amiloide de los Islotes Pancreáticos/química , Polipéptido Amiloide de los Islotes Pancreáticos/genética , Masculino , Ratones , Ratones Endogámicos C57BL , Mutación/genéticaRESUMEN
Under fasting conditions, increases in circulating concentrations of glucagon maintain glucose homeostasis via the induction of hepatic gluconeogenesis. Triggering of the cAMP pathway in hepatocytes stimulates the gluconeogenic program via the PKA-mediated phosphorylation of CREB and dephosphorylation of the cAMP-regulated CREB coactivators CRTC2 and CRTC3. In parallel, decreases in circulating insulin also increase gluconeogenic gene expression via the de-phosphorylation and activation of the forkhead transcription factor FOXO1. Hepatic gluconeogenesis is increased in insulin resistance where it contributes to the attendant hyperglycemia. Whether selective activation of the hepatic CREB/CRTC pathway is sufficient to trigger metabolic changes in other tissues is unclear, however. Modest hepatic expression of a phosphorylation-defective and therefore constitutively active CRTC2S171,275A protein increased gluconeogenic gene expression under fasting as well as feeding conditions. Circulating glucose concentrations were constitutively elevated in CRTC2S171,275A-expressing mice, leading to compensatory increases in circulating insulin concentrations that enhance FOXO1 phosphorylation. Despite accompanying decreases in FOXO1 activity, hepatic gluconeogenic gene expression remained elevated in CRTC2S171,275A mice, demonstrating that chronic increases in CRTC2 activity in the liver are indeed sufficient to promote hepatic insulin resistance and to disrupt glucose homeostasis.
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Resistencia a la Insulina , Hígado/metabolismo , Factores de Transcripción/metabolismo , Animales , Glucemia/metabolismo , Células Cultivadas , Regulación hacia Abajo , Proteína Forkhead Box O1 , Factores de Transcripción Forkhead/metabolismo , Insulina/metabolismo , Ratones , Ratones Endogámicos C57BL , Fosforilación , Transducción de SeñalRESUMEN
Deposition of human islet amyloid polypeptide (hIAPP, also known as amylin) as islet amyloid is a characteristic feature of the pancreas in type 2 diabetes, contributing to increased ß-cell apoptosis and reduced ß-cell mass. Matrix metalloproteinase-9 (MMP-9) is active in islets and cleaves hIAPP. We investigated whether hIAPP fragments arising from MMP-9 cleavage retain the potential to aggregate and cause toxicity, and whether overexpressing MMP-9 in amyloid-prone islets reduces amyloid burden and the resulting ß-cell toxicity. Synthetic hIAPP was incubated with MMP-9 and the major hIAPP fragments observed by MS comprised residues 1-15, 1-25, 16-37, 16-25, and 26-37. The fragments 1-15, 1-25, and 26-37 did not form amyloid fibrils in vitro and they were not cytotoxic when incubated with ß cells. Mixtures of these fragments with full-length hIAPP did not modulate the kinetics of fibril formation by full-length hIAPP. In contrast, the 16-37 fragment formed fibrils more rapidly than full-length hIAPP but was less cytotoxic. Co-incubation of MMP-9 and fragment 16-37 ablated amyloidogenicity, suggesting that MMP-9 cleaves hIAPP 16-37 into non-amyloidogenic fragments. Consistent with MMP-9 cleavage resulting in largely non-amyloidogenic degradation products, adenoviral overexpression of MMP-9 in amyloid-prone islets reduced amyloid deposition and ß-cell apoptosis. These findings suggest that increasing islet MMP-9 activity might be a strategy to limit ß-cell loss in type 2 diabetes.
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Apoptosis/efectos de los fármacos , Diabetes Mellitus Tipo 2/metabolismo , Células Secretoras de Insulina/enzimología , Polipéptido Amiloide de los Islotes Pancreáticos/toxicidad , Metaloproteinasa 9 de la Matriz/metabolismo , Péptidos/toxicidad , Animales , Línea Celular , Supervivencia Celular/efectos de los fármacos , Diabetes Mellitus Tipo 2/genética , Diabetes Mellitus Tipo 2/patología , Humanos , Células Secretoras de Insulina/patología , Metaloproteinasa 9 de la Matriz/genética , Ratones , Ratones TransgénicosRESUMEN
AIMS/HYPOTHESIS: Amyloid deposition and inflammation are characteristic of islet pathology in type 2 diabetes. The aim of this study was to determine whether islet amyloid formation is required for the development of islet inflammation in vivo. METHODS: Human islet amyloid polypeptide transgenic mice and non-transgenic littermates (the latter incapable of forming islet amyloid) were fed a low-fat (10%) or high-fat (60%) diet for 12 months; high-fat feeding induces islet amyloid formation in transgenic mice. At the conclusion of the study, glycaemia, beta cell function, islet amyloid deposition, markers of islet inflammation and islet macrophage infiltration were measured. RESULTS: Fasting plasma glucose levels did not differ by diet or genotype. Insulin release in response to i.v. glucose was significantly greater in both high vs low fat groups, and significantly lower in both transgenic compared with non-transgenic groups. Only high-fat-fed transgenic mice developed islet amyloid and showed a trend towards reduced beta cell area. Compared with islets from low-fat-fed transgenic or high-fat-fed non-transgenic mice, islets of high-fat-fed transgenic mice displayed a significant increase in the expression of genes encoding chemokines (Ccl2, Cxcl1), macrophage/dendritic cell markers (Emr1, Itgax), NACHT, LRR and PYD domains-containing protein 3 (NLRP3) inflammasome components (Nlrp3, Pycard, Casp1) and proinflammatory cytokines (Il1b, Tnf, Il6), as well as increased F4/80 staining, consistent with increased islet inflammation and macrophage infiltration. CONCLUSIONS/INTERPRETATION: Our results indicate that islet amyloid formation is required for the induction of islet inflammation in this long-term high-fat-diet model, and thus could promote beta cell dysfunction in type 2 diabetes via islet inflammation.
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Amiloide/inmunología , Amiloide/metabolismo , Diabetes Mellitus Tipo 2/metabolismo , Polipéptido Amiloide de los Islotes Pancreáticos/metabolismo , Animales , Glucemia/metabolismo , Proteínas de Unión al Calcio , Diabetes Mellitus Tipo 2/sangre , Grasas de la Dieta/efectos adversos , Ayuno/sangre , Genotipo , Humanos , Polipéptido Amiloide de los Islotes Pancreáticos/genética , Masculino , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Ratones , Ratones Transgénicos , Mucinas/genética , Mucinas/metabolismo , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismoRESUMEN
Deposition of islet amyloid polypeptide (IAPP) as amyloid is a pathological hallmark of the islet in type 2 diabetes, which is toxic to ß-cells. We previously showed that the enzyme neprilysin reduces islet amyloid deposition and thereby reduces ß-cell apoptosis, by inhibiting fibril formation. Two other enzymes, matrix metalloproteinase (MMP)-2 and MMP-9, are extracellular gelatinases capable of degrading another amyloidogenic peptide, Aß, the constituent of amyloid deposits in Alzheimer disease. We therefore investigated whether MMP-2 and MMP-9 play a role in reducing islet amyloid deposition. MMP-2 and MMP-9 mRNA were present in mouse islets but only MMP-9 activity was detectable. In an islet culture model where human IAPP (hIAPP) transgenic mouse islets develop amyloid but nontransgenic islets do not, a broad spectrum MMP inhibitor (GM6001) and an MMP-2/9 inhibitor increased amyloid formation and the resultant ß-cell apoptosis. In contrast, a specific MMP-2 inhibitor had no effect on either amyloid deposition or ß-cell apoptosis. Mass spectrometry demonstrated that MMP-9 degraded amyloidogenic hIAPP but not nonamyloidogenic mouse IAPP. Thus, MMP-9 constitutes an endogenous islet protease that limits islet amyloid deposition and its toxic effects via degradation of hIAPP. Because islet MMP-9 mRNA levels are decreased in type 2 diabetic subjects, islet MMP-9 activity may also be decreased in human type 2 diabetes, thereby contributing to increased islet amyloid deposition and ß-cell loss. Approaches to increase islet MMP-9 activity could reduce or prevent amyloid deposition and its toxic effects in type 2 diabetes.
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Amiloide/metabolismo , Polipéptido Amiloide de los Islotes Pancreáticos/metabolismo , Islotes Pancreáticos/enzimología , Metaloproteinasa 9 de la Matriz/metabolismo , Proteolisis , Secuencia de Aminoácidos , Animales , Apoptosis/efectos de los fármacos , Diabetes Mellitus Tipo 2/enzimología , Diabetes Mellitus Tipo 2/genética , Diabetes Mellitus Tipo 2/patología , Femenino , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Humanos , Células Secretoras de Insulina/citología , Células Secretoras de Insulina/efectos de los fármacos , Células Secretoras de Insulina/metabolismo , Polipéptido Amiloide de los Islotes Pancreáticos/química , Islotes Pancreáticos/efectos de los fármacos , Islotes Pancreáticos/patología , Masculino , Espectrometría de Masas , Metaloproteinasa 2 de la Matriz/genética , Metaloproteinasa 2 de la Matriz/metabolismo , Metaloproteinasa 9 de la Matriz/genética , Inhibidores de la Metaloproteinasa de la Matriz/farmacología , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Datos de Secuencia Molecular , Proteolisis/efectos de los fármacos , ARN Mensajero/genética , ARN Mensajero/metabolismoRESUMEN
A rapid, quantitative serum S100A8/A9 (calprotectin) lateral flow test in combination with clinical status predicted outcomes in people hospitalised with COVID-19 and associated with a patient cluster driven by markers of neutrophil activation https://bit.ly/48e1BIv.
RESUMEN
Introduction: Sulforaphane can induce the transcription factor, Nrf2, promoting antioxidant and anti-inflammatory responses. In this study, hospitalised patients with community-acquired pneumonia (CAP) were treated with stabilised synthetic sulforaphane (SFX-01) to evaluate impact on clinical status and inflammation. Methods: Double-blind, randomised, placebo-controlled trial of SFX-01 (300â mg oral capsule, once daily for 14â days) conducted in Dundee, UK, between November 2020 and May 2021. Patients had radiologically confirmed CAP and CURB-65 (confusion, urea >7â mmol·L-1, respiratory rate ≥30â breaths·min-1, blood pressure <90â mmHg (systolic) or ≤60â mmHg (diastolic), age ≥65â years) score ≥1. The primary outcome was the seven-point World Health Organization clinical status scale at day 15. Secondary outcomes included time to clinical improvement, length of stay and mortality. Effects on Nrf2 activity and inflammation were evaluated on days 1, 8 and 15 by measurement of 45 serum cytokines and mRNA sequencing of peripheral blood leukocytes. Results: The trial was terminated prematurely due to futility with 133 patients enrolled. 65 patients were randomised to SFX-01 treatment and 68 patients to placebo. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection was the cause of CAP in 103 (77%) cases. SFX-01 treatment did not improve clinical status at day 15 (adjusted OR 0.87, 95% CI 0.41-1.83; p=0.71), time to clinical improvement (adjusted hazard ratio (aHR) 1.02, 95% CI 0.70-1.49), length of stay (aHR 0.84, 95% CI 0.56-1.26) or 28-day mortality (aHR 1.45, 95% CI 0.67-3.16). The expression of Nrf2 targets and pro-inflammatory genes, including interleukin (IL)-6, IL-1ß and tumour necrosis factor-α, was not significantly changed by SFX-01 treatment. At days 8 and 15, respectively, 310 and 42 significant differentially expressed genes were identified between groups (false discovery rate adjusted p<0.05, log2FC >1). Conclusion: SFX-01 treatment did not improve clinical status or modulate key Nrf2 targets in patients with CAP primarily due to SARS-CoV-2 infection.
RESUMEN
Islet amyloid, a pathologic feature of type 2 diabetes, contains the islet ß-cell peptide islet amyloid polypeptide (IAPP) as its unique amyloidogenic component. Islet amyloid also contains heparan sulfate proteoglycans (HSPGs) that may contribute to amyloid formation by binding IAPP via their heparan sulfate (HS) chains. We hypothesized that ß-cells produce HS that bind IAPP via regions of highly sulfated disaccharides. Unexpectedly, HS from the ß-cell line ß-TC3 contained fewer regions of highly sulfated disaccharides compared with control normal murine mammary gland (NMuMG) cells. The proportion of HS that bound IAPP was similar in both cell lines (â¼65%). The sulfation pattern of IAPP-bound versus non-bound HS from ß-TC3 cells was similar. In contrast, IAPP-bound HS from NMuMG cells contained frequent highly sulfated regions, whereas the non-bound material demonstrated fewer sulfated regions. Fibril formation from IAPP was stimulated equally by IAPP-bound ß-TC3 HS, non-bound ß-TC3 HS, and non-bound NMuMG HS but was stimulated to a greater extent by the highly sulfated IAPP-bound NMuMG HS. Desulfation of HS decreased the ability of both ß-TC3 and NMuMG HS to stimulate IAPP maximal fibril formation, but desulfated HS from both cell types still accelerated fibril formation relative to IAPP alone. In summary, neither binding to nor acceleration of fibril formation from the amyloidogenic peptide IAPP is dependent on overall sulfation in HS synthesized by ß-TC3 cells. This information will be important in determining approaches to reduce HS-IAPP interactions and ultimately prevent islet amyloid formation and its toxic effects in type 2 diabetes.
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Amiloide/química , Heparitina Sulfato/química , Células Secretoras de Insulina/metabolismo , Polipéptido Amiloide de los Islotes Pancreáticos/química , Animales , Benzotiazoles , Conformación de Carbohidratos , Línea Celular , Cromatografía en Gel , Medios de Cultivo Condicionados , Colorantes Fluorescentes/química , Liasa de Heparina/química , Heparitina Sulfato/metabolismo , Humanos , Proteínas Inmovilizadas/química , Ratones , Ácido Nitroso/química , Polisacárido Liasas/química , Unión Proteica , Multimerización de Proteína , Proteoglicanos/química , Proteoglicanos/aislamiento & purificación , Tiazoles/químicaRESUMEN
The dipeptidyl peptidase-4 (DPP-4) inhibitor sitagliptin is an attractive therapy for diabetes, as it increases insulin release and may preserve ß-cell mass. However, sitagliptin also increases ß-cell release of human islet amyloid polypeptide (hIAPP), the peptide component of islet amyloid, which is cosecreted with insulin. Thus, sitagliptin treatment may promote islet amyloid formation and its associated ß-cell toxicity. Conversely, metformin treatment decreases islet amyloid formation by decreasing ß-cell secretory demand and could therefore offset sitagliptin's potential proamyloidogenic effects. Sitagliptin treatment has also been reported to be detrimental to the exocrine pancreas. We investigated whether long-term sitagliptin treatment, alone or with metformin, increased islet amyloid deposition and ß-cell toxicity and induced pancreatic ductal proliferation, pancreatitis, and/or pancreatic metaplasia/neoplasia. hIAPP transgenic and nontransgenic littermates were followed for 1 yr on no treatment, sitagliptin, metformin, or the combination. Islet amyloid deposition, ß-cell mass, insulin release, and measures of exocrine pancreas pathology were determined. Relative to untreated mice, sitagliptin treatment did not increase amyloid deposition, despite increasing hIAPP release, and prevented amyloid-induced ß-cell loss. Metformin treatment alone or with sitagliptin decreased islet amyloid deposition to a similar extent vs untreated mice. Ductal proliferation was not altered among treatment groups, and no evidence of pancreatitis, ductal metaplasia, or neoplasia were observed. Therefore, long-term sitagliptin treatment stimulates ß-cell secretion without increasing amyloid formation and protects against amyloid-induced ß-cell loss. This suggests a novel effect of sitagliptin to protect the ß-cell in type 2 diabetes that appears to occur without adverse effects on the exocrine pancreas.