Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 42
Filtrar
Más filtros

Banco de datos
País/Región como asunto
Tipo del documento
Intervalo de año de publicación
1.
Diabet Med ; 41(6): e15279, 2024 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-38185936

RESUMEN

AIMS: Evidence is accumulating of the therapeutic benefits of mesenchymal stromal cells (MSCs) in diabetes-related conditions. We have identified a novel population of stromal cells within islets of Langerhans - islet stellate cells (ISCs) - which have a similar morphology to MSCs. In this study we characterize mouse ISCs and compare their morphology and function to MSCs to determine whether ISCs may also have therapeutic potential in diabetes. METHODS: ISCs isolated from mouse islets were compared to mouse bone marrow MSCs by analysis of cell morphology; expression of cell-surface markers and extracellular matrix (ECM) components; proliferation; apoptosis; paracrine activity; and differentiation into adipocytes, chondrocytes and osteocytes. We also assessed the effects of co-culture with ISCs or MSCs on the insulin secretory capacity of islet beta cells. RESULTS: Although morphological similar, ISCs were functionally distinct from MSCs. Thus, ISCs were less proliferative and more apoptotic; they had different expression levels of important paracrine factors; and they were less efficient at differentiation down multiple lineages. Co-culture of mouse islets with ISCs enhanced glucose induced insulin secretion more effectively than co-culture with MSCs. CONCLUSIONS: ISCs are a specific sub-type of islet-derived stromal cells that possess biological behaviors distinct from MSCs. The enhanced beneficial effects of ISCs on islet beta cell function suggests that they may offer a therapeutic target for enhancing beta cell functional survival in diabetes.


Asunto(s)
Diferenciación Celular , Técnicas de Cocultivo , Células Secretoras de Insulina , Islotes Pancreáticos , Células Madre Mesenquimatosas , Animales , Ratones , Células Madre Mesenquimatosas/metabolismo , Células Madre Mesenquimatosas/fisiología , Células Secretoras de Insulina/metabolismo , Células Secretoras de Insulina/fisiología , Células Secretoras de Insulina/citología , Diferenciación Celular/fisiología , Islotes Pancreáticos/citología , Islotes Pancreáticos/metabolismo , Células Estrelladas Pancreáticas/metabolismo , Células Estrelladas Pancreáticas/fisiología , Proliferación Celular/fisiología , Insulina/metabolismo , Células Cultivadas , Secreción de Insulina/fisiología , Ratones Endogámicos C57BL , Masculino , Apoptosis/fisiología
2.
Diabet Med ; 40(12): e15227, 2023 12.
Artículo en Inglés | MEDLINE | ID: mdl-37728506

RESUMEN

AIMS: Human islet transplantation as a therapy for type 1 diabetes is compromised by the loss of functional beta cells in the immediate post-transplantation period. Mesenchymal stromal cells (MSCs) and MSC-derived secretory peptides improve the outcomes of islet transplantation in rodent models of diabetes. Here, we utilized a mouse model for human islet transplantation and assessed the effects of a cocktail of MSC-secreted peptides (screened by MSC-secretome for human islet GPCRs) on the functional survival of human islets. METHODS: Human islets from nine donors (Age: 36-57; BMI: 20-35) were treated with a cocktail of human recombinant annexin A1 (ANXA1), stromal cell-derived factor-1 (SDF-1/CXCL12) and complement component C3 (C3a). Glucose-stimulated insulin secretion (GSIS) was assessed in static incubation, and cytokine-induced apoptosis was assessed by measuring caspase 3/7 activity. mRNA expression levels were determined by qPCR. Human islet function in vivo was assessed using a novel model for human islet transplantation into a T1D mouse model. Human islet function in vivo was assessed using islet transplantation under the kidney capsule of immunodeficient mice prior to STZ destruction of endogenous mouse beta cells to model T1DM. RESULTS: Pretreatment with a cocktail of MSC-secreted peptides increased GSIS in vitro and protected against cytokine-induced apoptosis in human islets isolated from nine donors. Animals transplanted with either treated or untreated human islets remained normoglycaemic for up to 28 days after STZ-administration to ablate the endogenous mouse beta cells, whereas non-transplanted animals showed significantly increased blood glucose immediately after STZ administration. Removal of the human islet graft by nephrectomy resulted in rapid increases in blood glucose to similar levels as the non-transplanted controls. Pretreating human islets with the MSC-derived cocktail significantly improved glucose tolerance in graft recipients, consistent with enhanced functional survival of the treated islets in vivo. CONCLUSION: Pretreating human islets before transplantation with a defined cocktail of MSC-derived molecules could be employed to improve the quality of human islets for transplantation therapy for type 1 diabetes.


Asunto(s)
Diabetes Mellitus Experimental , Diabetes Mellitus Tipo 1 , Trasplante de Islotes Pancreáticos , Islotes Pancreáticos , Células Madre Mesenquimatosas , Humanos , Ratones , Animales , Adulto , Persona de Mediana Edad , Islotes Pancreáticos/metabolismo , Insulina/metabolismo , Glucemia/metabolismo , Diabetes Mellitus Tipo 1/metabolismo , Diabetes Mellitus Experimental/terapia , Trasplante de Islotes Pancreáticos/métodos , Células Madre Mesenquimatosas/metabolismo , Glucosa/farmacología , Glucosa/metabolismo , Citocinas/metabolismo , Modelos Animales de Enfermedad
3.
Diabet Med ; 39(12): e14962, 2022 12.
Artículo en Inglés | MEDLINE | ID: mdl-36151994

RESUMEN

AIMS: Beta cell endoplasmic reticulum (ER) stress can cause cellular death and dysfunction and has been implicated in the pathogenesis of diabetes. Animal models of beta cell ER stress are critical in further understanding this and for testing novel diabetes therapeutics. The KINGS mouse is a model of beta cell ER stress driven by a heterozygous mutation in Ins2. In this study, we investigated how beta cell ER stress in the KINGS mouse drives diabetes. METHODS: We investigated whether the unfolded protein response (UPR) was activated in islets isolated from male and female KINGS mice and whether this impacted beta cell mass and turnover. RESULTS: Whilst the UPR was up-regulated in KINGS islets, with increased protein expression of markers of all three UPR arms, this was not associated with a mass loss of beta cells; beta cell apoptosis rates did not increase until after the development of overt diabetes, and did not lead to substantial changes in beta cell mass. CONCLUSION: We propose that the KINGS mouse represents a model where beta cell maladaptive UPR signalling drives diabetes development without causing mass beta cell loss.


Asunto(s)
Diabetes Mellitus , Células Secretoras de Insulina , Femenino , Masculino , Ratones , Humanos , Animales , Estrés del Retículo Endoplásmico/fisiología , Células Secretoras de Insulina/metabolismo , Respuesta de Proteína Desplegada , Diabetes Mellitus/metabolismo , Apoptosis
4.
Diabetes Obes Metab ; 24(11): 2241-2252, 2022 11.
Artículo en Inglés | MEDLINE | ID: mdl-35815375

RESUMEN

AIM: To establish the impact of sex, dosing route, fasting duration and acute habituation stress on glucose tolerance test (GTT) measurements used in the preclinical evaluation of potential glucose-modulating therapeutics. METHODS: Adult male and female C57Bl/6J mice, implanted with HD-XG glucose telemetry devices, were fasted for 16 hours or 6 hours following acute habituation stress due to whole cage change, cage change with retention of used bedding or no cage change prior to intraperitoneal (IP) GTTs. To evaluate protocol refinement and sex on the ability of the GTT to detect drug effects, we administered 250 mg/kg oral metformin or 10 nmol/kg IP exendin-4 using optimized protocols. RESULTS: Female mice were less sensitive to human intervention when initiating fasting. Following a 6-hour fast, retention of bedding whilst changing the cage base promotes quicker stabilization of basal blood glucose in both sexes. Prolonged fasting for 16 hours resulted in an exaggerated GTT response but induced pronounced basal hypoglycaemia. Following GTT protocol optimization the effect of exendin-4 and metformin was equivalent in both sexes, with females showing a more modest but more reproducible GTT response. CONCLUSIONS: Variations in GTT protocol have profound effects on glucose homeostasis. Protocol refinement and/or the use of females still allows for detection of drug effects, providing evidence that more severe phenotypes are not an essential prerequisite when characterizing/validating new drugs.


Asunto(s)
Glucemia , Metformina , Adulto , Animales , Exenatida , Femenino , Glucosa , Prueba de Tolerancia a la Glucosa , Humanos , Masculino , Metformina/farmacología , Ratones , Ratones Endogámicos C57BL
5.
Stem Cells ; 38(4): 574-584, 2020 04.
Artículo en Inglés | MEDLINE | ID: mdl-31912945

RESUMEN

Pretransplant islet culture is associated with the loss of islet cell mass and insulin secretory function. Insulin secretion from islet ß-cells is primarily controlled by mitochondrial ATP generation in response to elevations in extracellular glucose. Coculture of islets with mesenchymal stromal cells (MSCs) improves islet insulin secretory function in vitro, which correlates with superior islet graft function in vivo. This study aimed to determine whether the improved islet function is associated with mitochondrial transfer from MSCs to cocultured islets. We have demonstrated mitochondrial transfer from human adipose MSCs to human islet ß-cells in coculture. Fluorescence imaging showed that mitochondrial transfer occurs, at least partially, through tunneling nanotube (TNT)-like structures. The extent of mitochondrial transfer to clinically relevant human islets was greater than that to experimental mouse islets. Human islets are subjected to more extreme cellular stressors than mouse islets, which may induce "danger signals" for MSCs, initiating the donation of MSC-derived mitochondria to human islet ß-cells. Our observations of increased MSC-mediated mitochondria transfer to hypoxia-exposed mouse islets are consistent with this and suggest that MSCs are most effective in supporting the secretory function of compromised ß-cells. Ensuring optimal MSC-derived mitochondria transfer in preculture and/or cotransplantation strategies could be used to maximize the therapeutic efficacy of MSCs, thus enabling the more widespread application of clinical islet transplantation.


Asunto(s)
Diabetes Mellitus Experimental/terapia , Células Secretoras de Insulina/metabolismo , Trasplante de Islotes Pancreáticos/métodos , Células Madre Mesenquimatosas/metabolismo , Mitocondrias/metabolismo , Animales , Células Cultivadas , Humanos , Ratones
6.
Diabet Med ; 38(12): e14711, 2021 12.
Artículo en Inglés | MEDLINE | ID: mdl-34614258

RESUMEN

Diabetes mellitus is characterised by hyperglycaemia, which results from an absolute or relative lack of insulin. Chronic and acute hyperglycaemia are associated with a range of health complications and an overall increased risk of mortality. Mouse models are vital in understanding the pathogenesis of this disease and its complications, as well as for developing new diabetes therapeutics. However, for experimental questions to be suitably tested, it is critical that factors inherent to the animal model are considered, as these can have profound impacts on experimental outcome, data reproducibility and robustness. In this review, we discuss key considerations relating to model choice, physiological characteristics (such as age, sex and genetic background) and husbandry practices and explore the impact of these on common experimental readouts used in preclinical diabetes research.


Asunto(s)
Investigación Biomédica/métodos , Diabetes Mellitus Experimental/terapia , Diabetes Mellitus Tipo 2/terapia , Manejo de la Enfermedad , Resistencia a la Insulina/fisiología , Animales , Diabetes Mellitus Experimental/genética , Diabetes Mellitus Tipo 2/genética , Ratones
7.
Diabet Med ; 38(12): e14705, 2021 12.
Artículo en Inglés | MEDLINE | ID: mdl-34596274

RESUMEN

Mice are used extensively in preclinical diabetes research to model various aspects of blood glucose homeostasis. Careful experimental design is vital for maximising welfare and improving reproducibility of data. Alongside decisions regarding physiological characteristics of the animal cohort (e.g., sex, strain and age), experimental protocols must also be carefully considered. This includes choosing relevant end points of interest and understanding what information they can provide and what their limitations are. Details of experimental protocols must, therefore, be carefully planned during the experimental design stage, especially considering the impact of researcher interventions on preclinical end points. Indeed, in line with the 3Rs of animal research, experiments should be refined where possible to maximise welfare. The role of welfare may be particularly pertinent in preclinical diabetes research as blood glucose concentrations are directly altered by physiological stress responses. Despite the potential impact of variations in experimental protocols, there is distinct lack of standardisation and consistency throughout the literature with regards to several experimental procedures including fasting, cage changing and glucose tolerance test protocol. This review firstly highlights practical considerations with regard to the choice of end points in preclinical diabetes research and the potential for novel technologies such as continuous glucose monitoring and glucose clamping techniques to improve data resolution. The potential influence of differing experimental protocols and in vivo procedures on both welfare and experimental outcomes is then discussed with focus on standardisation, consistency and full disclosure of methods.


Asunto(s)
Investigación Biomédica/métodos , Automonitorización de la Glucosa Sanguínea/métodos , Glucemia/metabolismo , Diabetes Mellitus Experimental/diagnóstico , Diabetes Mellitus/diagnóstico , Animales , Diabetes Mellitus/sangre , Diabetes Mellitus Experimental/sangre , Prueba de Tolerancia a la Glucosa , Ratones
8.
Cytotherapy ; 20(12): 1427-1436, 2018 12.
Artículo en Inglés | MEDLINE | ID: mdl-30377040

RESUMEN

BACKGROUND AIMS: Mesenchymal stromal cells (MSCs) enhance islet function both in vitro and in vivo, at least in part by secreting ligands that activate islet G-protein coupled receptors (GPCRs). We assessed whether pre-treatment with a defined "cocktail" of MSC-secreted GPCR ligands enhances islet functional survival in vitro and improves the outcomes of islet transplantation in an experimental model of diabetes. METHODS: Isolated islets were cultured for 48 h with ANXA1, SDF-1 or C3a, alone or in combination. Glucose-stimulated insulin secretion (GSIS) and cytokine-induced apoptosis were measured immediately after the 48 h culture period and at 24 h or 72 h following removal of the ligands from the culture media. Islets were syngeneically transplanted underneath the kidney capsule of streptozotocin-induced diabetic C57BL/6 mice and blood glucose levels monitored for 28 days. RESULTS: Pre-culturing islets with a cocktail of ANXA1/SDF-1/C3a potentiated GSIS and protected islet cells from cytokine-induced apoptosis in vitro. These effects were maintained for up to 72 h after the removal of the factors from the culture medium, suggesting a sustained protection of islet graft functional survival during the immediate post-transplantation period. Islets pre-treated with the cocktail of MSC secretory factors were more effective in reducing blood glucose in diabetic mice, consistent with their improved functional survival in vivo. DISCUSSION: Pre-culturing islets with a cocktail of MSC secretory products offers a well-defined, cell-free approach to improve clinical islet transplantation outcomes while avoiding many of the safety, regulatory and logistical hurdles of incorporating MSCs into transplantation protocols.


Asunto(s)
Quimiocina CXCL12/farmacología , Complemento C3a/farmacología , Trasplante de Islotes Pancreáticos/métodos , Células Madre Mesenquimatosas/metabolismo , Animales , Anexina A1/genética , Anexina A1/metabolismo , Anexina A1/farmacología , Apoptosis/fisiología , Supervivencia Celular/fisiología , Células Cultivadas , Quimiocina CXCL12/genética , Quimiocina CXCL12/metabolismo , Complemento C3a/genética , Complemento C3a/metabolismo , Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Experimental/terapia , Glucosa/metabolismo , Secreción de Insulina , Islotes Pancreáticos/citología , Islotes Pancreáticos/efectos de los fármacos , Masculino , Ratones Endogámicos C57BL , Ratones Endogámicos , Receptores Acoplados a Proteínas G/metabolismo
9.
Diabetes Obes Metab ; 20(3): 599-609, 2018 03.
Artículo en Inglés | MEDLINE | ID: mdl-28940946

RESUMEN

AIMS: Two unmet therapeutic strategies for diabetes treatment are prevention of beta-cell death and stimulation of beta-cell replication. Our aim was to characterize the role of neuropeptide Y receptors in the control of beta-cell mass. MATERIALS AND METHODS: We used endogenous and selective agonists of the NPY receptor system to explore its role in the prevention of beta-cell apoptosis and proliferation in islets isolated from both mouse and human donors. We further explored the intra-cellular signalling cascades involved, using chemical inhibitors of key signalling pathways. As proof of principle we designed a long-acting analogue of [Leu31 Pro34 ]-NPY, an agonist of the islet-expressed Y receptors, to determine if targeting this system could preserve beta-cell mass in vivo. RESULTS: Our data reveal that NPY Y1, 4 and 5 receptor activation engages a generalized and powerful anti-apoptotic pathway that protects mouse and human islets from damage. These anti-apoptotic effects were dependent on stimulating a Gαi-PLC-PKC signalling cascade, which prevented cytokine-induced NFkB signalling. NPY receptor activation functionally protected islets by restoring glucose responsiveness following chemically induced injury in both species. NPY receptor activation attenuated beta-cell apoptosis, preserved functional beta-cell mass and attenuated the hyperglycaemic phenotype in a low-dose streptozotocin model of diabetes. CONCLUSION: Taken together, our observations identify the islet Y receptors as promising targets for the preservation of beta-cell mass. As such, targeting these receptors could help to maintain beta-cell mass in both type 1 and type 2 diabetes, and may also be useful for improving islet transplantation outcomes.


Asunto(s)
Células Secretoras de Insulina/citología , Receptores de Neuropéptido Y/fisiología , Análisis de Varianza , Animales , Apoptosis/fisiología , Proliferación Celular/fisiología , Humanos , Secreción de Insulina/fisiología , Células Secretoras de Insulina/metabolismo , Masculino , Ratones , Receptores de Neuropéptido Y/antagonistas & inhibidores , Receptores de Neuropéptido Y/metabolismo , Transducción de Señal/fisiología
10.
Clin Sci (Lond) ; 131(23): 2835-2845, 2017 Dec 01.
Artículo en Inglés | MEDLINE | ID: mdl-29101297

RESUMEN

AIMS: The aims of the present study were (i) to determine whether the reported beneficial effects of mesenchymal stromal cells (MSCs) on mouse islet function extend to clinically relevant human tissues (islets and MSCs), enabling translation into improved protocols for clinical human islet transplantation; and (ii) to identify possible mechanisms through which human MSCs influence human islet function. MATERIALS AND METHODS: Human islets were co-cultured with human adipose tissue-derived MSCs (hASCs) or pre-treated with its products - extracellular matrix (ECM) and annexin A1 (ANXA1). Mouse islets were pre-treated with mouse MSC-derived ECM. Islet insulin secretory function was assessed in vitro by radioimmunoassay. Quantitative RT-PCR was used to screen human adipMSCs for potential ligands of human islet G-protein-coupled receptors. RESULTS: We show that co-culture with hASCs improves human islet secretory function in vitro, as measured by glucose-stimulated insulin secretion, confirming previous reports using rodent tissues. Furthermore, we demonstrate that these beneficial effects on islet function can be partly attributed to the MSC-derived products ECM and ANXA1. CONCLUSIONS: Our results suggest that hASCs have the potential to improve the quality of human islets isolated for transplantation therapy of Type 1 diabetes. Furthermore, it may be possible to achieve improvements in human islet quality in a cell-free culture system by using the MSC-derived products ANXA1 and ECM.


Asunto(s)
Matriz Extracelular/fisiología , Islotes Pancreáticos/fisiología , Células Madre Mesenquimatosas/fisiología , Tejido Adiposo/citología , Animales , Anexina A1/metabolismo , Anexina A1/farmacología , Técnicas de Cocultivo , Humanos , Insulina/metabolismo , Secreción de Insulina , Islotes Pancreáticos/efectos de los fármacos , Islotes Pancreáticos/metabolismo , Ligandos , Células Madre Mesenquimatosas/metabolismo , Ratones Endogámicos C57BL , Receptores Odorantes/metabolismo
11.
Stem Cells ; 31(3): 547-59, 2013 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-23255220

RESUMEN

Following islet transplantation, islet graft revascularization is compromised due to loss of endothelial cells (ECs) during islet culture. TGF-ß signaling pathways are essential for vascular homeostasis but their importance for islet EC function is unclear. We have identified a population of multipotent mesenchymal stromal cells (MSCs) within islets and investigated how modulation of TGF-ß signaling by these cells influences islet EC viability. Cultured islets exhibited reduced expression of EC markers (VEGFR2, VE-cadherin and CD31), which was associated with diminished but sustained expression of endoglin a marker of both ECs and MSCs. Double fluorescent labeling of islets in situ with the EC marker CD31 disclosed a population of CD31-negative cells which were positive for endoglin. In vitro coculture of microvascular ECs with endoglin-positive, CD31-negative islet MSCs reduced VEGFR2 protein expression, disrupted EC angiogenic behavior, and increased EC detachment. Medium conditioned by islet MSCs significantly decreased EC viability and increased EC caspase 3/7 activity. EC:MSC cocultures showed enhanced Smad2 phosphorylation consistent with altered ALK5 signaling. Pharmacological inhibition of ALK5 activity with SB431542 (SB) improved EC survival upon contact with MSCs, and SB-treated cultured islets retained EC marker expression and sensitivity to exogenous VEGF164 . Thus, endoglin-expressing islet MSCs influence EC ALK5 signaling in vitro, which decreases EC viability, and changes in ALK5 activity in whole cultured islets contribute to islet EC loss. Modifying TGF-ß signaling may enable maintenance of islet ECs during islet isolation and thus improve islet graft revascularization post-transplantation.


Asunto(s)
Células Endoteliales/efectos de los fármacos , Trasplante de Islotes Pancreáticos/métodos , Islotes Pancreáticos/efectos de los fármacos , Células Madre Mesenquimatosas/efectos de los fármacos , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Receptores de Factores de Crecimiento Transformadores beta/antagonistas & inhibidores , Animales , Antígenos CD/biosíntesis , Benzamidas/farmacología , Supervivencia Celular/efectos de los fármacos , Técnicas de Cocultivo , Dioxoles/farmacología , Endoglina , Células Endoteliales/citología , Células Endoteliales/enzimología , Humanos , Péptidos y Proteínas de Señalización Intracelular/biosíntesis , Islotes Pancreáticos/irrigación sanguínea , Islotes Pancreáticos/citología , Islotes Pancreáticos/metabolismo , Masculino , Células Madre Mesenquimatosas/citología , Células Madre Mesenquimatosas/enzimología , Ratones , Ratones Endogámicos ICR , Neovascularización Fisiológica/efectos de los fármacos , Fosforilación , Proteínas Serina-Treonina Quinasas/metabolismo , Receptor Tipo I de Factor de Crecimiento Transformador beta , Receptores de Superficie Celular/biosíntesis , Receptores de Factores de Crecimiento Transformadores beta/metabolismo , Transducción de Señal , Factor de Crecimiento Transformador beta1/farmacología
12.
Diabetologia ; 56(11): 2467-76, 2013 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-23979485

RESUMEN

AIMS/HYPOTHESIS: Chemokine (C-C motif) ligand 5 (CCL5) acts at C-C chemokine receptors (CCRs) to promote immune cell recruitment to sites of inflammation, but is also an agonist at G-protein-coupled receptor 75 (GPR75), which has very limited homology with CCRs. GPR75 is coupled to Gq to elevate intracellular calcium, so we investigated whether islets express this receptor and whether its activation by CCL5 increases beta cell calcium levels and insulin secretion. METHODS: Islet CCL5 receptor mRNA expression was measured by quantitative RT-PCR and GPR75 was detected in islets by western blotting and immunohistochemistry. In some experiments GPR75 was downregulated by transient transfection with small interfering RNA. Real-time changes in intracellular calcium were determined by single-cell microfluorimetry. Dynamic insulin secretion from perifused islets was quantified by radioimmunoassay. Glucose homeostasis in lean and obese mice was determined by measuring glucose and insulin tolerance, and insulin secretion in vivo. RESULTS: Mouse and human islets express GPR75 and its ligand CCL5. Exogenous CCL5 reversibly increased intracellular calcium in beta cells via GPR75, this phenomenon being dependent on phospholipase C activation and calcium influx. CCL5 also stimulated insulin secretion from mouse and human islets in vitro, and improved glucose tolerance in lean mice and in a mouse model of hyperglycaemia and insulin resistance (ob/ob). The improvement in glucose tolerance was associated with enhanced insulin secretion in vivo, without changes in insulin sensitivity. CONCLUSIONS/INTERPRETATION: Although CCL5 is implicated in the pathogenesis of diabetes through activation of CCRs, it has beneficial effects on beta cells through GPR75 activation.


Asunto(s)
Glucosa/metabolismo , Insulina/metabolismo , Islotes Pancreáticos/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Animales , Quimiocina CCL5/metabolismo , Homeostasis/genética , Homeostasis/fisiología , Humanos , Inmunohistoquímica , Secreción de Insulina , Masculino , Ratones , Ratones Endogámicos ICR , Receptores Acoplados a Proteínas G/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa
13.
Diabetologia ; 56(11): 2477-86, 2013 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-23900510

RESUMEN

AIMS/HYPOTHESIS: The stress-activated nuclear protein transcription regulator 1 (NUPR1) is induced in response to glucose and TNF-α, both of which are elevated in type 2 diabetes, and Nupr1 has been implicated in cell proliferation and apoptosis cascades. We used Nupr1(-/-) mice to study the role of Nupr1 in glucose homeostasis under normal conditions and following maintenance on a high-fat diet (HFD). METHODS: Glucose homeostasis in vivo was determined by measuring glucose tolerance, insulin sensitivity and insulin secretion. Islet number, morphology and beta cell area were assessed by immunofluorescence and morphometric analysis, and islet cell proliferation was quantified by analysis of BrdU incorporation. Islet gene expression was measured by gene arrays and quantitative RT-PCR, and gene promoter activities were monitored by measuring luciferase activity. RESULTS: Nupr1(-/-) mice had increased beta cell mass as a consequence of enhanced islet cell proliferation. Nupr1-dependent suppression of beta cell Ccna2 and Tcf19 promoter activities was identified as a mechanism through which Nupr1 may regulate beta cell cycle progression. Nupr1(-/-) mice maintained on a normal diet were mildly insulin resistant, but were normoglycaemic with normal glucose tolerance because of compensatory increases in basal and glucose-induced insulin secretion. Nupr1 deletion was protective against HFD-induced obesity, insulin resistance and glucose intolerance. CONCLUSIONS/INTERPRETATION: Inhibition of NUPR1 expression or activity has the potential to protect against the metabolic defects associated with obesity and type 2 diabetes.


Asunto(s)
Proteínas de Unión al ADN/metabolismo , Intolerancia a la Glucosa/metabolismo , Células Secretoras de Insulina/metabolismo , Proteínas de Neoplasias/metabolismo , Animales , Western Blotting , Proteínas de Unión al ADN/genética , Femenino , Intolerancia a la Glucosa/genética , Humanos , Inmunohistoquímica , Células Secretoras de Insulina/citología , Masculino , Ratones , Ratones Noqueados , Proteínas de Neoplasias/genética
14.
Cytotherapy ; 15(2): 192-200, 2013 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-23321331

RESUMEN

BACKGROUND AIMS: Co-transplantation of islets with mesenchymal stem cells (MSCs) has been shown to improve graft outcome in mice, which has been partially attributed to the effects of MSCs on revascularization and preservation of islet morphology. Microencapsulation of islets provides an isolated-graft model of islet transplantation that is non-vascularized and prevents islet aggregation to preserve islet morphology. The aim of this study was to investigate whether MSCs could improve graft outcome in a microencapsulated/isolated-graft model of islet transplantation. METHODS: Mouse islets and kidney MSCs were co-encapsulated in alginate, and their function was assessed in vitro. A minimal mass of 350 syngeneic islets encapsulated alone or co-encapsulated with MSCs (islet+MSC) were transplanted intraperitoneally into diabetic mice, and blood glucose concentrations were monitored. Capsules were recovered 6 weeks after transplantation, and islet function was assessed. RESULTS: Islets co-encapsulated with MSCs in vitro had increased glucose-stimulated insulin secretion and content. The average blood glucose concentration of transplanted mice was significantly lower by 3 weeks in the islet+MSC group. By week 6, 71% of the co-encapsulated group were cured compared with 16% of the islet-alone group. Capsules recovered at 6 weeks had greater glucose-stimulated insulin secretion and insulin content in the islet+MSC group. CONCLUSIONS: MSCs improved the efficacy of microencapsulated islet transplantation. Using an isolated-graft model, we were able to eliminate the impact of MSC-mediated enhancement of revascularization and preservation of islet morphology and demonstrate that the improvement in insulin secretion and content is sustained in vivo and can significantly improve graft outcome.


Asunto(s)
Diabetes Mellitus Experimental/terapia , Trasplante de Islotes Pancreáticos , Trasplante de Células Madre Mesenquimatosas , Células Madre Mesenquimatosas/citología , Neovascularización Fisiológica , Animales , Glucemia , Humanos , Insulina/metabolismo , Secreción de Insulina , Islotes Pancreáticos/patología , Masculino , Ratones
15.
Front Microbiol ; 13: 1004679, 2022.
Artículo en Inglés | MEDLINE | ID: mdl-36386661

RESUMEN

The intestinal microbiota plays an important role in host metabolism via production of dietary metabolites. Microbiota imbalances are linked to type 2 diabetes (T2D), but dietary modification of the microbiota may promote glycemic control. Using a rodent model of T2D and an in vitro gut model system, this study investigated whether differences in gut microbiota between control mice and mice fed a high-fat, high-fructose (HFHFr) diet influenced the production of phenolic acid metabolites following fermentation of wholegrain (WW) and control wheat (CW). In addition, the study assessed whether changes in metabolite profiles affected pancreatic beta cell function. Fecal samples from control or HFHFr-fed mice were fermented in vitro with 0.1% (w/v) WW or CW for 0, 6, and 24 h. Microbiota composition was determined by bacterial 16S rRNA sequencing and phenolic acid (PA) profiles by UPLC-MS/MS. Cell viability, apoptosis and insulin release from pancreatic MIN6 beta cells and primary mouse islets were assessed in response to fermentation supernatants and selected PAs. HFHFr mice exhibited an overall dysbiotic microbiota with an increase in abundance of proteobacterial taxa (particularly Oxalobacteraceae) and Lachnospiraceae, and a decrease in Lactobacillus. A trend toward restoration of diversity and compositional reorganization was observed following WW fermentation at 6 h, although after 24 h, the HFHFr microbiota was monodominated by Cupriavidus. In parallel, the PA profile was significantly altered in the HFHFr group compared to controls with decreased levels of 3-OH-benzoic acid, 4-OH-benzoic acid, isoferulic acid and ferulic acid at 6 h of WW fermentation. In pancreatic beta cells, exposure to pre-fermentation supernatants led to inhibition of insulin release, which was reversed over fermentation time. We conclude that HFHFr mice as a model of T2D are characterized by a dysbiotic microbiota, which is modulated by the in vitro fermentation of WW. The differences in microbiota composition have implications for PA profile dynamics and for the secretory capacity of pancreatic beta cells.

16.
Mol Metab ; 53: 101285, 2021 11.
Artículo en Inglés | MEDLINE | ID: mdl-34224919

RESUMEN

OBJECTIVE: Members of the adhesion G protein-coupled receptor (aGPCR) subfamily are important actors in metabolic processes, with GPR56 (ADGRG1) emerging as a possible target for type 2 diabetes therapy. GPR56 can be activated by collagen III, its endogenous ligand, and by a synthetic seven amino-acid peptide (TYFAVLM; P7) contained within the GPR56 Stachel sequence. However, the mechanisms regulating GPR56 trafficking dynamics and agonist activities are not yet clear. METHODS: Here, we introduced SNAPf-tag into the N-terminal segment of GPR56 to monitor GPR56 cellular activity in situ. Confocal and super-resolution microscopy were used to investigate the trafficking pattern of GPR56 in native MIN6 ß-cells and in MIN6 ß-cells where GPR56 had been deleted by CRISPR-Cas9 gene editing. Insulin secretion, changes in intracellular calcium, and ß-cell apoptosis were determined by radioimmunoassay, single-cell calcium microfluorimetry, and measuring caspase 3/7 activities, respectively, in MIN6 ß-cells and human islets. RESULTS: SNAP-tag labelling indicated that GPR56 predominantly underwent constitutive internalisation in the absence of an exogenous agonist, unlike GLP-1R. Collagen III further stimulated GPR56 internalisation, whereas P7 was without significant effect. The overexpression of GPR56 in MIN6 ß-cells did not affect insulin secretion. However, it was associated with reduced ß-cell apoptosis, while the deletion of GPR56 made MIN6 ß-cells more susceptible to cytokine-induced apoptosis. P7 induced a rapid increase in the intracellular calcium in MIN6 ß-cells (in a GPR56-dependent manner) and human islets, and it also caused a sustained and reversible increase in insulin secretion from human islets. Collagen III protected human islets from cytokine-induced apoptosis, while P7 was without significant effect. CONCLUSIONS: These data indicate that GPR56 exhibits both agonist-dependent and -independent trafficking in ß-cells and suggest that while GPR56 undergoes constitutive signalling, it can also respond to its ligands when required. We have also identified that constitutive and agonist-dependent GPR56 activation is coupled to protect ß-cells against apoptosis, offering a potential therapeutic target to maintain ß-cell mass in type 2 diabetes.


Asunto(s)
Células Secretoras de Insulina/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Células HEK293 , Humanos , Microscopía Confocal , Receptores Acoplados a Proteínas G/genética , Transducción de Señal/genética
17.
Mol Metab ; 43: 101112, 2021 01.
Artículo en Inglés | MEDLINE | ID: mdl-33157254

RESUMEN

OBJECTIVE: Metabolic diseases are an increasing problem in society with the brain-metabolic axis as a master regulator of the human body for sustaining homeostasis under metabolic stress. However, metabolic inflammation and disease will trigger sustained activation of the hypothalamic-pituitary-adrenal axis. In this study, we investigated the role of metabolic stress on progenitor cells in the hypothalamic-pituitary-adrenal axis. METHODS: In vitro, we applied insulin and leptin to murine progenitor cells isolated from the pituitary and adrenal cortex and examined the role of these hormones on proliferation and differentiation. In vivo, we investigated two different mouse models of metabolic disease, obesity in leptin-deficient ob/ob mice and obesity achieved via feeding with a high-fat diet. RESULTS: Insulin was shown to lead to enhanced proliferation and differentiation of both pituitary and adrenocortical progenitors. No alterations in the progenitors were noted in our chronic metabolic stress models. However, hyperactivation of the hypothalamic-pituitary-adrenal axis was observed and the expression of the appetite-regulating genes Npy and Agrp changed in both the hypothalamus and adrenal. CONCLUSIONS: It is well-known that chronic stress and stress hormones such as glucocorticoids can induce metabolic changes including obesity and diabetes. In this article, we show for the first time that this might be based on an early sensitization of stem cells of the hypothalamic-pituitary-adrenal axis. Thus, pituitary and adrenal progenitor cells exposed to high levels of insulin are metabolically primed to a hyper-functional state leading to enhanced hormone production. Likewise, obese animals exhibit a hyperactive hypothalamic-pituitary-adrenal axis leading to adrenal hyperplasia. This might explain how stress in early life can increase the risk for developing metabolic syndrome in adulthood.


Asunto(s)
Insulina/metabolismo , Obesidad/metabolismo , Células Madre/fisiología , Estrés Fisiológico/fisiología , Animales , Diferenciación Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Femenino , Sistema Hipotálamo-Hipofisario/metabolismo , Sistema Hipotálamo-Hipofisario/fisiología , Insulina/farmacología , Leptina/metabolismo , Leptina/farmacología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Obesos , Ratones Transgénicos , Sistema Hipófiso-Suprarrenal/metabolismo , Sistema Hipófiso-Suprarrenal/fisiología , Células Madre/efectos de los fármacos , Células Madre/metabolismo
18.
Methods Mol Biol ; 2076: 265-280, 2020.
Artículo en Inglés | MEDLINE | ID: mdl-31586334

RESUMEN

Islet transplantation is a potential treatment for Type 1 diabetes; however, improvements need to be made before it could become clinically widely available. In preclinical studies, the mouse is often used to model islet transplantation, with most studies aiming to improve transplantation outcome by manipulating the islets prior to transplantation or by treating the recipient mouse. Here, we describe the process of islet transplantation in the mouse, including how one can make the mouse diabetic, isolate donor islets, and transplant the islets into two different sites. Finally, we discuss how to assess the outcome of the transplantation in order to determine whether the experimental intervention has been beneficial.


Asunto(s)
Trasplante de Islotes Pancreáticos , Islotes Pancreáticos , Animales , Glucemia , Separación Celular/métodos , Diabetes Mellitus Experimental , Rechazo de Injerto , Supervivencia de Injerto , Xenoinjertos , Insulina/sangre , Islotes Pancreáticos/citología , Trasplante de Islotes Pancreáticos/efectos adversos , Trasplante de Islotes Pancreáticos/métodos , Ratones , Trasplante Homólogo , Resultado del Tratamiento
19.
Methods Mol Biol ; 2128: 135-147, 2020.
Artículo en Inglés | MEDLINE | ID: mdl-32180191

RESUMEN

Streptozotocin (STZ) selectively destroys beta cells and is widely used to induce experimental diabetes in rodents. Rodent beta cells are very sensitive to the toxic effects of STZ, while human beta cells are highly resistant to STZ. Taking advantage of this characteristic, here, we describe two protocols for the induction of STZ-diabetes. In the first model, hyperglycemia is induced prior to islet transplantation, whereas in the second model, STZ is injected after islet transplantation. The former model has many applications and thus is the most commonly used method. However, when implanting human islets into mice, there are clear benefits to administering STZ after the transplantation. It reduces the cost and burden of experiments and the number of human islets needed for transplantation and improves the welfare and survival of animals used in the experiments. In both methods, a key step in the experimental protocol is to remove the graft-bearing kidney at the end of the experiment and monitor onset of hyperglycemia. This can be used to demonstrate that the glycemic control of the animal is due to the engrafted islets and not regeneration of endogenous beta cells. This chapter outlines protocols of administering streptozotocin pre- and post-islet transplantation in mice as well as nephrectomy to remove the graft-bearing kidney.


Asunto(s)
Diabetes Mellitus Experimental/inducido químicamente , Trasplante de Islotes Pancreáticos/métodos , Nefrectomía/métodos , Animales , Glucemia/análisis , Femenino , Humanos , Hiperglucemia/sangre , Células Secretoras de Insulina/efectos de los fármacos , Riñón/cirugía , Masculino , Ratones , Estreptozocina/administración & dosificación
20.
Methods Mol Biol ; 2128: 225-239, 2020.
Artículo en Inglés | MEDLINE | ID: mdl-32180197

RESUMEN

Measurement of blood glucose concentration is a common end point in studies using animal models of diabetes. Usually a blood glucose meter is used to measure non-fasted blood glucose concentrations, typically at frequencies of between 1 and 7 times per week. This process involves pricking the tip of the tail to collect a small blood sample (0.5-5 µL), which could potentially cause a stress response and affect blood glucose concentrations. Moreover, with blood glucose concentrations constantly fluctuating in response to feeding and activity, a single-point measurement can easily misrepresent the actual glycemic control of the animal. In this chapter, we discuss the use of continuous glucose monitoring in mice by radio-telemetry which allows second-by-second changes in blood glucose to be captured without restraining the mouse. Glucose excursions rather than single-point measurements may prove more useful in detecting effects of treatments, and lack of handling may avoid stress responses causing artefacts. We outline what is involved in implanting such devices into mice including some practical tips to maximize success.


Asunto(s)
Automonitorización de la Glucosa Sanguínea/instrumentación , Glucemia/análisis , Monitoreo Fisiológico/métodos , Implantación de Prótesis/métodos , Telemetría/instrumentación , Telemetría/métodos , Animales , Estado de Conciencia/fisiología , Diabetes Mellitus Experimental , Locomoción/fisiología , Prótesis e Implantes
SELECCIÓN DE REFERENCIAS
DETALLE DE LA BÚSQUEDA