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1.
Methods Mol Biol ; 2128: 241-268, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32180198

RESUMO

Islets of Langerhans are clusters of endocrine cells embedded within the exocrine pancreas. Islets constitute only approximately 1-2% of the total pancreas mass in all species, so methods have been developed to digest the pancreas and purify islets from the surrounding acinar cells. This chapter provides detailed protocols for isolation of mouse islets and their in vitro functional characterization in terms of assessments of islet viability, hormone content and secretion, second messenger generation and ß-cell proliferation.

2.
Cell Mol Life Sci ; 2020 Jan 10.
Artigo em Inglês | MEDLINE | ID: mdl-31925452

RESUMO

AIMS: Endocannabinoids are lipid mediators involved in the regulation of glucose homeostasis. They interact with the canonical cannabinoid receptors CB1 and CB2, and it is now apparent that some cannabinoid receptor ligands are also agonists at GPR55. Thus, CB1 antagonists such as SR141716A, also known as rimonabant, and AM251 act as GPR55 agonists in some cell types. The complex pharmacological properties of cannabinoids make it difficult to fully identify the relative importance of CB1 and GPR55 in the functional effects of SR141716A, and AM251. Here, we determine whether SR141716A and AM251 regulation of mouse and human islet function is through their action as GPR55 agonists. METHODS: Islets isolated from Gpr55+/+ and Gpr55-/- mice and human donors were incubated in the absence or presence of 10 µM SR141716A or AM251, concentrations that are known to activate GPR55. Insulin secretion, cAMP, IP1, apoptosis and ß-cell proliferation were quantified by standard techniques. RESULTS: Our results provide the first evidence that SR141716A and AM251 are not GPR55 agonists in islets, as their effects are maintained in islets isolated from Gpr55-/- mice. Their signalling through Gq-coupled cascades to induce insulin secretion and human ß-cell proliferation, and protect against apoptosis in vitro, indicate that they have direct beneficial effects on islet function. CONCLUSION: These observations may be useful in directing development of peripherally restricted novel therapeutics that are structurally related to SR141716A and AM251, and which potentiate glucose-induced insulin secretion and stimulate ß-cell proliferation.

3.
Psychosom Med ; 81(7): 570-583, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31136376

RESUMO

OBJECTIVE: Individual studies have reported conflicting effects of selective serotonin reuptake inhibitors (SSRIs) on glycemia. We systematically reviewed the effects of SSRIs on glycemia and whether metabolic and psychological factors moderated these effects. METHODS: We systematically searched for placebo-controlled randomized controlled trials investigating the effect of SSRIs on glycemia (fasting blood glucose or HbA1c) as a primary or secondary outcome. Random effects meta-analysis was conducted to compute an overall treatment effect. Meta-regression tested whether depression, type 2 diabetes, insulin resistance, treatment duration, and weight loss moderated treatment effects. RESULTS: Sixteen randomized controlled trials (n = 835) were included and glycemia was usually a secondary outcome. Overall, SSRIs improved glycemia versus placebo (pooled effect size (ES) = -0.34, 95% confidence interval (CI) = -0.48 to -0.21; p < .001, I = 0%). Individually, fluoxetine (ES = -0.29, 95% CI = -0.54 to -0.05; p = .018) and escitalopram/citalopram (ES = -0.33, 95% CI = -0.59 to -0.07; p = .012) outperformed placebo, but paroxetine (ES = -0.19, 95% CI = -0.58 to 0.19; p = .33) did not. Results were similar in populations selected for depression as those not. Across studies, baseline insulin resistance (p = .46), treatment duration (p = .47), diabetes status (p = .41), and weight loss (p = .93) did not moderate changes. Heterogeneity for all analyses was nonsignificant. CONCLUSIONS: SSRIs seem to have an association with improvement in glycemia, which is not moderated by depression status, diabetes status, or change in weight across studies. Future powered trials with longer treatment duration are needed to confirm these findings. REGISTRATION: PROSPERO ID: CRD4201809239.

4.
Cell Physiol Biochem ; 52(4): 879-892, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30958662

RESUMO

BACKGROUND/AIMS: CXCL14, a secreted chemokine peptide that promotes obesity-induced insulin resistance, is expressed by islets, but its effects on islet function are unknown. The aim of this study was to determine the role of CXCL14 in ß-cells and investigate how it transduces these effects. METHODS: Cxcl14 and Cxc-receptor mRNA expression was quantified by qPCR and CXCL14 expression in the pancreas was determined by immunohistochemistry. The putative function of CXCL14 at CXCR4 and CXCR7 receptors was determined by ß-arrestin recruitment assays. The effects of CXCL14 on glucose-stimulated insulin secretion, cAMP production, glucose-6-phosphate accumulation, ATP generation, apoptosis and proliferation were determined using standard techniques. RESULTS: CXCL14 was present in mouse islets, where it was mainly localised to islet δ-cells. Cxc-receptor mRNA profiling indicated that Cxcr4 and Cxcr7 are the most abundant family members in islets, but CXCL14 did not promote ß-arrestin recruitment at CXCR4 or CXCR7 or antagonise CXCL12 activation of these receptors. CXCL14 induced a concentration-dependent inhibition of glucose-stimulated insulin secretion, which was not coupled to Gαi signalling. However, CXCL14 inhibited glucose-6-phosphate generation and ATP production in mouse islets. CONCLUSION: CXCL14 is expressed by islet δ-cells where it may have paracrine effects to inhibit insulin secretion in a CXCR4/CXCR7-independent manner through reductions in ß-cell ATP levels. These observations, together with the previously reported association of CXCL14 with obesity and impaired glucose homeostasis, suggest that inhibition of CXCL14 signalling could be explored to treat type 2 diabetes.


Assuntos
Quimiocinas CXC/metabolismo , AMP Cíclico/metabolismo , Secreção de Insulina , Células Secretoras de Insulina/metabolismo , Insulina/metabolismo , Receptores CXCR4/metabolismo , Receptores CXCR/metabolismo , Animais , Quimiocinas CXC/genética , AMP Cíclico/genética , Insulina/genética , Células Secretoras de Insulina/citologia , Masculino , Camundongos , Comunicação Parácrina , Receptores CXCR/genética , Receptores CXCR4/genética , Sistemas do Segundo Mensageiro
5.
Pharmacol Ther ; 198: 123-134, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-30825474

RESUMO

Adhesion G-protein coupled receptors (aGPCRs) are emerging as important actors in energy homeostasis. Recent biochemical and functional studies using transgenic mice indicate that aGPCRs play important roles in endocrine and metabolic functions including ß-cell differentiation, insulin secretion, adipogenesis and whole body fuel homeostasis. Most aGPCRs are orphans, for which endogenous ligands have not yet been identified, and many of the endogenous ligands of the already de-orphanised aGPCRs are components of the extracellular matrix (ECM). In this review we focus on aGPCR expression in metabolically active tissues, their activation by ECM proteins, and current knowledge of their potential roles in islet development, insulin secretion, adipogenesis and muscle function.


Assuntos
Receptores Acoplados a Proteínas-G/metabolismo , Animais , Metabolismo Energético , Humanos
6.
Diabetes Obes Metab ; 21(2): 330-339, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-30203438

RESUMO

AIMS: To evaluate the role of free fatty acid receptor 2 (FFAR2)/G-protein coupled receptor 43 in mediating the effects of the short chain fatty acids (SCFAs) sodium acetate (SA) and sodium propionate (SP) on islet function in vitro, and to identify the intracellular signalling pathways used in SCFA-induced potentiation of glucose-induced insulin secretion. MATERIALS AND METHODS: Islets of Langerhans were isolated from wild-type and FFAR2-/- mice and from human donors without diabetes. The effects of SA and SP on dynamic insulin secretion from perifused islets were quantified by radioimmunoassay, signalling downstream of SCFAs was profiled by single-cell calcium microfluorimetry, and measurement of cAMP was performed using a fluorescence assay. Islet apoptosis was induced by exposure to cytokines or sodium palmitate, and the effects of SA and SP in regulating islet apoptosis were assessed by quantification of caspase 3/7 activities. RESULTS: Deletion of FFAR2 did not affect islet morphology or insulin content. SA and SP reversibly potentiated insulin secretion from mouse islets in a FFAR2-dependent manner. SCFA-induced potentiation of insulin secretion was coupled to Gq activation of phospholipase C and protein kinase C, with no evidence of Gi-mediated signalling. SA and SP protected human and mouse islets from apoptosis, and these pro-survival properties were dependent on islet expression of FFAR2. CONCLUSION: Our results indicate that FFAR2 directly mediates both the stimulatory effects of SA and SP on insulin secretion and their protection against islet apoptosis. We have also shown that SCFA coupling in islets occurs via Gq-coupled intracellular signalling.


Assuntos
Apoptose/efeitos dos fármacos , Ácidos Graxos Voláteis/farmacologia , Secreção de Insulina/efeitos dos fármacos , Células Secretoras de Insulina/efeitos dos fármacos , Ilhotas Pancreáticas/efeitos dos fármacos , Receptores Acoplados a Proteínas-G/fisiologia , Adulto , Animais , Apoptose/genética , Células Cultivadas , Ácidos Graxos não Esterificados/farmacologia , Feminino , Humanos , Insulina/metabolismo , Células Secretoras de Insulina/metabolismo , Ilhotas Pancreáticas/patologia , Ilhotas Pancreáticas/fisiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Pessoa de Meia-Idade , Propionatos/farmacologia , Receptores de Superfície Celular/genética , Receptores de Superfície Celular/fisiologia , Receptores Acoplados a Proteínas-G/genética , Acetato de Sódio/farmacologia
7.
Cytotherapy ; 20(12): 1427-1436, 2018 12.
Artigo em Inglês | MEDLINE | ID: mdl-30377040

RESUMO

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.


Assuntos
Quimiocina CXCL12/farmacologia , Complemento C3a/farmacologia , Transplante das Ilhotas Pancreáticas/métodos , Células-Tronco Mesenquimais/metabolismo , Animais , Anexina A1/genética , Anexina A1/metabolismo , Anexina A1/farmacologia , Apoptose/fisiologia , Sobrevivência Celular/fisiologia , Células Cultivadas , Quimiocina CXCL12/genética , Quimiocina CXCL12/metabolismo , Complemento C3a/genética , Complemento C3a/metabolismo , Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Experimental/terapia , Glucose/metabolismo , Secreção de Insulina , Ilhotas Pancreáticas/citologia , Ilhotas Pancreáticas/efeitos dos fármacos , Masculino , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos , Receptores Acoplados a Proteínas-G/metabolismo
9.
Curr Opin Pharmacol ; 43: 27-33, 2018 12.
Artigo em Inglês | MEDLINE | ID: mdl-30086433

RESUMO

Adhesion receptors are transmembrane proteins that mediate cell-cell and cell-matrix communications. In addition to their adhesive role in maintaining islet architecture, they are also important for promoting islet cell survival, proliferation and secretory function. Their capacity for improving ß-cell mass and insulin secretion suggest that they may be suitable targets for pharmacological intervention, and their interactions with extracellular matrix proteins hold promise in improving islet transplantation outcomes. In this review, we have focused on integrins, cadherins and adhesion GPCRs, and highlight recent advances in their roles in islet function and discuss whether they could be targeted for diabetes therapy.


Assuntos
Glicemia/efeitos dos fármacos , Moléculas de Adesão Celular/metabolismo , Diabetes Mellitus/tratamento farmacológico , Desenho de Fármacos , Hipoglicemiantes/uso terapêutico , Ilhotas Pancreáticas/efeitos dos fármacos , Receptores Acoplados a Proteínas-G/efeitos dos fármacos , Animais , Biomarcadores/sangue , Glicemia/metabolismo , Diabetes Mellitus/sangue , Diabetes Mellitus/epidemiologia , Diabetes Mellitus/patologia , Difusão de Inovações , Humanos , Hipoglicemiantes/efeitos adversos , Ilhotas Pancreáticas/metabolismo , Ilhotas Pancreáticas/patologia , Terapia de Alvo Molecular , Receptores Acoplados a Proteínas-G/metabolismo , Transdução de Sinais/efeitos dos fármacos
10.
Cell Mol Life Sci ; 75(21): 4007-4019, 2018 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-29855662

RESUMO

AIMS: G-protein coupled receptor 56 (GPR56) is the most abundant islet-expressed G-protein coupled receptor, suggesting a potential role in islet function. This study evaluated islet expression of GPR56 and its endogenous ligand collagen III, and their effects on ß-cell function. METHODS: GPR56 and collagen III expression in mouse and human pancreas sections was determined by fluorescence immunohistochemistry. Effects of collagen III on ß-cell proliferation, apoptosis, intracellular calcium ([Ca2+]i) and insulin secretion were determined by cellular BrdU incorporation, caspase 3/7 activities, microfluorimetry and radioimmunoassay, respectively. The role of GPR56 in islet vascularisation and innervation was evaluated by immunohistochemical staining for CD31 and TUJ1, respectively, in pancreases from wildtype (WT) and Gpr56-/- mice, and the requirement of GPR56 for normal glucose homeostasis was determined by glucose tolerance tests in WT and Gpr56-/- mice. RESULTS: Immunostaining of mouse and human pancreases revealed that GPR56 was expressed by islet ß-cells while collagen III was confined to the peri-islet basement membrane and islet capillaries. Collagen III protected ß-cells from cytokine-induced apoptosis, triggered increases in [Ca2+]i and potentiated glucose-induced insulin secretion from WT islets but not from Gpr56-/- islets. Deletion of GPR56 did not affect glucose-induced insulin secretion in vitro and it did not impair glucose tolerance in adult mice. GPR56 was not required for normal islet vascularisation or innervation. CONCLUSION: We have demonstrated that collagen III improves islet function by increasing insulin secretion and protecting against apoptosis. Our data suggest that collagen III may be effective in optimising islet function to improve islet transplantation outcomes, and GPR56 may be a target for the treatment of type 2 diabetes.


Assuntos
Colágeno/genética , Diabetes Mellitus Tipo 2/genética , Receptores Acoplados a Proteínas-G/genética , Animais , Apoptose/genética , Cálcio/metabolismo , Proliferação de Células/genética , Diabetes Mellitus Tipo 2/metabolismo , Diabetes Mellitus Tipo 2/patologia , Matriz Extracelular/genética , Matriz Extracelular/metabolismo , Regulação da Expressão Gênica , Humanos , Células Secretoras de Insulina/metabolismo , Células Secretoras de Insulina/patologia , Ilhotas Pancreáticas/metabolismo , Ilhotas Pancreáticas/patologia , Camundongos , Camundongos Knockout , Pâncreas/metabolismo , Pâncreas/patologia
11.
Cell Mol Life Sci ; 75(16): 3039-3050, 2018 08.
Artigo em Inglês | MEDLINE | ID: mdl-29455414

RESUMO

INTRODUCTION: Islets synthesise and secrete numerous peptides, some of which are known to be important regulators of islet function and glucose homeostasis. In this study, we quantified mRNAs encoding all peptide ligands of islet G protein-coupled receptors (GPCRs) in isolated human and mouse islets and carried out in vitro islet hormone secretion studies to provide functional confirmation for the species-specific role of peptide YY (PYY) in mouse islets. MATERIALS AND METHODS: GPCR peptide ligand mRNAs in human and mouse islets were quantified by quantitative real-time PCR relative to the reference genes ACTB, GAPDH, PPIA, TBP and TFRC. The pathways connecting GPCR peptide ligands with their receptors were identified by manual searches in the PubMed, IUPHAR and Ingenuity databases. Distribution of PYY protein in mouse and human islets was determined by immunohistochemistry. Insulin, glucagon and somatostatin secretion from islets was measured by radioimmunoassay. RESULTS: We have quantified GPCR peptide ligand mRNA expression in human and mouse islets and created specific signalomes mapping the pathways by which islet peptide ligands regulate human and mouse GPCR signalling. We also identified species-specific islet expression of several GPCR ligands. In particular, PYY mRNA levels were ~ 40,000-fold higher in mouse than human islets, suggesting a more important role of locally secreted Pyy in mouse islets. This was confirmed by IHC and functional experiments measuring insulin, glucagon and somatostatin secretion. DISCUSSION: The detailed human and mouse islet GPCR peptide ligand atlases will allow accurate translation of mouse islet functional studies for the identification of GPCR/peptide signalling pathways relevant for human physiology, which may lead to novel treatment modalities of diabetes and metabolic disease.


Assuntos
Ilhotas Pancreáticas/metabolismo , Peptídeo YY/metabolismo , RNA Mensageiro/metabolismo , Receptores Acoplados a Proteínas-G/metabolismo , Transdução de Sinais , Animais , Expressão Gênica , Humanos , Imuno-Histoquímica , Ligantes , Masculino , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos ICR , Peptídeo YY/genética , RNA Mensageiro/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa
12.
Cell Physiol Biochem ; 45(2): 656-666, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29408822

RESUMO

BACKGROUND/AIMS: CRISPR-Cas9, a RNA-guided targeted genome editing tool, has revolutionized genetic engineering by offering the ability to precisely modify DNA. GPRC5B is an orphan receptor belonging to the group C family of G protein-coupled receptors (GPCRs). In this study, we analysed the functional roles of the Gprc5b receptor in MIN6 ß-cells using CRISPR-Cas9 and transient over-expression of Gprc5b. METHODS: The optimal transfection reagent for use in MIN6 ß-cells was determined by analysing efficiency of GFP plasmid delivery by cell sorting. A MIN6 ß-cell line in which Gprc5b expression was knocked down (Gprc5b KD) was generated using CRISPR-Cas9 technology. Gprc5b receptor mRNA expression, proliferation, apoptosis, Cignal 45-Pathway Reporter Array signalling and western blot assays were carried out using Gpcr5b KD MIN6 ß-cells that had been transiently transfected with different concentrations of mouse Gprc5b plasmid to over-express Gprc5b. RESULTS: JetPRIME® was the best candidate for MIN6 ß-cell transfection, providing approximately 30% transfection efficiency. CRISPR-Cas9 technology targeting Gprc5b led to stable knock-down of this receptor in MIN6 ß-cells and its re-expression induced proliferation and potentiated cytokine- and palmitate-induced apoptosis. The Cignal 45 Reporter analysis indicated Gprc5b-dependent regulation of apoptotic and proliferative pathways, and western blotting confirmed activation of signalling via TGF-ß and IFNγ. CONCLUSION: This study provides evidence of CRISPR-Cas9 technology being used to down-regulate Gprc5b expression in MIN6 ß-cells. This strategy allowed us to identify signalling pathways linking GPRC5B receptor expression to ß-cell proliferation and apoptosis.


Assuntos
Sistemas CRISPR-Cas/genética , Edição de Genes , Receptores Acoplados a Proteínas-G/metabolismo , Animais , Apoptose/efeitos dos fármacos , Proteínas Reguladoras de Apoptose/metabolismo , Sequência de Bases , Linhagem Celular Tumoral , Proliferação de Células , Células Secretoras de Insulina/citologia , Células Secretoras de Insulina/metabolismo , Interferon gama/metabolismo , Camundongos , Neuropeptídeos/metabolismo , Ácido Palmítico/toxicidade , Fosforilação , Plasmídeos/genética , Plasmídeos/metabolismo , Receptores Acoplados a Proteínas-G/antagonistas & inibidores , Receptores Acoplados a Proteínas-G/genética , Fator de Transcrição STAT1/metabolismo , Transdução de Sinais , Proteína Smad2/metabolismo , Proteína Smad3/metabolismo , Fator de Crescimento Transformador beta/metabolismo
13.
Cell Physiol Biochem ; 45(3): 1165-1171, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29448249

RESUMO

BACKGROUND/AIMS: Insulin-secreting islet ß-cells adapt to the insulin resistance associated with pregnancy by increasing functional ß-cell mass, but the placental signals involved in this process are not well defined. In the current study, we analysed expression of G-protein coupled receptor (GPCR) mRNAs in mouse islets and islet GPCR ligand mRNAs in placenta during pregnancy to generate an atlas of potential interactions between the placenta and ß-cells to inform future functional studies of islet adaptive responses to pregnancy. METHODS: Quantative RT-PCR arrays were used to measure mRNA expression levels of: (i) 342 GPCRs in islets from non-pregnant mice, and in islets isolated from mice on gestational days 12 and 18; (ii) 126 islet GPCR ligands in mouse placenta at gestational days 12 and 18. RESULTS: At gestational day 12, a time of rapid expansion of the ß-cell mass, 189 islet GPCR mRNAs were quantifiable, while 79 of the 126 known islet GPCR ligand mRNAs were detectable in placental extracts. Approximately half of the quantifiable placental GPCR ligand genes were of unknown function in ß-cells. The expression of some islet GPCR and placental ligand mRNAs varied during pregnancy, with altered expression of both GPCR and ligand mRNAs by gestational day 18. CONCLUSION: The current study has revealed numerous potential routes for interaction between the placenta and islets, and offers an atlas to inform further functional studies of their roles in adaptive responses to pregnancy, and in the regulation of the ß-cell mass.


Assuntos
Células Secretoras de Insulina/metabolismo , Placenta/metabolismo , Animais , Feminino , Idade Gestacional , Camundongos , Gravidez , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Receptores Acoplados a Proteínas-G/genética , Receptores Acoplados a Proteínas-G/metabolismo
14.
Diabetes Obes Metab ; 20(3): 599-609, 2018 03.
Artigo em Inglês | MEDLINE | ID: mdl-28940946

RESUMO

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.


Assuntos
Células Secretoras de Insulina/citologia , Receptores de Neuropeptídeo Y/fisiologia , Análise de Variância , Animais , Apoptose/fisiologia , Proliferação de Células/fisiologia , Humanos , Secreção de Insulina/fisiologia , Células Secretoras de Insulina/metabolismo , Masculino , Camundongos , Receptores de Neuropeptídeo Y/antagonistas & inibidores , Receptores de Neuropeptídeo Y/metabolismo , Transdução de Sinais/fisiologia
15.
Cell Mol Life Sci ; 75(4): 715-726, 2018 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-28921001

RESUMO

AIMS: Complement components 3 and 5 (C3 and C5) play essential roles in the complement system, generating C3a and C5a peptides that are best known as chemotactic and inflammatory factors. In this study we characterised islet expression of C3 and C5 complement components, and the impact of C3aR and C5aR1 activation on islet function and viability. MATERIALS AND METHODS: Human and mouse islet mRNAs encoding key elements of the complement system were quantified by qPCR and distribution of C3 and C5 proteins was determined by immunohistochemistry. Activation of C3aR and C5aR1 was determined using DiscoverX beta-arrestin assays. Insulin secretion from human and mouse islets was measured by radioimmunoassay, and intracellular calcium ([Ca2+]i), ATP generation and apoptosis were assessed by standard techniques. RESULTS: C3 and C5 proteins and C3aR and C5aR1 were expressed by human and mouse islets, and C3 and C5 were mainly localised to ß- and α-cells. Conditioned media from islets exposed for 1 h to 5.5 and 20 mM glucose stimulated C3aR and C5aR1-driven beta-arrestin recruitment. Activation of C3aR and C5aR1 potentiated glucose-induced insulin secretion from human and mouse islets, increased [Ca2+]i and ATP generation, and protected islets against apoptosis induced by a pro-apoptotic cytokine cocktail or palmitate. CONCLUSIONS: Our observations demonstrate a functional link between activation of components of the innate immune system and improved ß-cell function, suggesting that low-level chronic inflammation may improve glucose homeostasis through direct effects on ß-cells.


Assuntos
Células Secretoras de Insulina/metabolismo , Células Secretoras de Insulina/fisiologia , Receptor da Anafilatoxina C5a/metabolismo , Receptores de Complemento/metabolismo , Animais , Apoptose/fisiologia , Células Cultivadas , Complemento C3/metabolismo , Complemento C5/metabolismo , Citocinas/metabolismo , Glucose/metabolismo , Humanos , Masculino , Camundongos , Camundongos Endogâmicos ICR , beta-Arrestinas/metabolismo
16.
Diabetes Obes Metab ; 20(4): 930-942, 2018 04.
Artigo em Inglês | MEDLINE | ID: mdl-29205751

RESUMO

AIMS: To examine the effects of Abn-CBD (GPR55 agonist) and LH-21 (CB1 antagonist) on human and mouse islet function, and to determine signalling via GPR55 using islets from GPR55-/- mice. MATERIALS AND METHODS: Islets isolated from human organ donors and mice were incubated in the absence or presence of Abn-CBD or LH-21, and insulin secretion, [Ca2+ ]i, cAMP, apoptosis, ß-cell proliferation and CREB and AKT phosphorylation were examined using standard techniques. RESULTS: Abn-CBD potentiated glucose-stimulated insulin secretion and elevated [Ca2+ ]i in human islets and islets from both GPR55+/+ and GPR55-/- mice. LH-21 also increased insulin secretion and [Ca2+ ]i in human islets and GPR55+/+ mouse islets, but concentrations of LH-21 up to 0.1 µM were ineffective in islets from GPR55-/- mice. Neither ligand affected basal insulin secretion or islet cAMP levels. Abn-CBD and LH-21 reduced cytokine-induced apoptosis in human islets and GPR55+/+ mouse islets, and these effects were suppressed after GPR55 deletion. They also increased ß-cell proliferation: the effects of Abn-CBD were preserved in islets from GPR55-/- mice, while those of LH-21 were abolished. Abn-CBD and LH-21 increased AKT phosphorylation in mouse and human islets. CONCLUSIONS: This study showed that Abn-CBD and LH-21 improve human and mouse islet ß-cell function and viability. Use of islets from GPR55-/- mice suggests that designation of Abn-CBD and LH-21 as a GPR55 agonist and a CB1 antagonist, should be revised.


Assuntos
Células Secretoras de Insulina/efeitos dos fármacos , Ilhotas Pancreáticas/efeitos dos fármacos , Resorcinóis/farmacologia , Triazóis/farmacologia , Adulto , Animais , Células Cultivadas , Feminino , Humanos , Células Secretoras de Insulina/fisiologia , Ilhotas Pancreáticas/fisiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Pessoa de Meia-Idade , Receptores de Canabinoides/metabolismo , Receptores Acoplados a Proteínas-G/metabolismo , Transdução de Sinais/efeitos dos fármacos
17.
Cell Physiol Biochem ; 44(4): 1352-1359, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-29186709

RESUMO

BACKGROUND/AIMS: Rodent islets are often used for basic science research but they do not always recapitulate signalling events in human islets. This study evaluated the glucose-dependent responses of human and mouse islets in terms of dynamic insulin secretion, metabolic coupling and the role of glucose transporters. METHODS: Glucose-induced insulin secretion from isolated mouse and human islets was profiled by perifusion and islet ATP levels were measured by chemoluminescence assay. Glucose transporter expression was determined by qPCR and western blotting. RESULTS: Human islets show a left-shifted glucose concentration-insulin secretion profile compared to mouse islets. These data are consistent with glucose transporter expression, with human islets expressing mainly GLUT1 and GLUT3, and GLUT2 being the predominant transporter in mouse islets. Using the GLUT1 inhibitor STF-31 we unveiled an important role for GLUT1 for differences in glucose-induced insulin secretion profiles observed between the two species. CONCLUSION: The high affinity of GLUT1/3 for glucose reflects the left-shifted glucose-induced insulin secretory response of human islets and the impairment of insulin secretion from human islets after STF-31 treatment indicates an important role for GLUT1 in human islet stimulus-secretion coupling. Our data provide further insight into key differences between insulin secretion regulation in mouse and human islets.


Assuntos
Trifosfato de Adenosina/metabolismo , Glucose/farmacologia , Insulina/metabolismo , Ilhotas Pancreáticas/efeitos dos fármacos , Adulto , Animais , Feminino , Transportador de Glucose Tipo 1/antagonistas & inibidores , Transportador de Glucose Tipo 1/genética , Transportador de Glucose Tipo 1/metabolismo , Transportador de Glucose Tipo 2/antagonistas & inibidores , Transportador de Glucose Tipo 2/genética , Transportador de Glucose Tipo 2/metabolismo , Transportador de Glucose Tipo 3/antagonistas & inibidores , Transportador de Glucose Tipo 3/genética , Transportador de Glucose Tipo 3/metabolismo , Humanos , Secreção de Insulina , Ilhotas Pancreáticas/citologia , Ilhotas Pancreáticas/metabolismo , Cinética , Masculino , Camundongos , Pessoa de Meia-Idade , Piridinas/farmacologia , RNA Mensageiro/metabolismo
18.
Curr Opin Pharmacol ; 37: 24-28, 2017 12.
Artigo em Inglês | MEDLINE | ID: mdl-28822846

RESUMO

The incidence of type 2 diabetes (T2D) is increasing at an alarming rate, which is imposing substantial healthcare and economic burdens worldwide. T2D can be treated by a range of drugs, but there is a need to identify additional therapeutic options. Human islets express nearly three hundred G-protein-coupled receptors (GPCRs), which could be targeted for the treatment of T2D. However, to date, the GLP-1 receptor is the only islet GPCR for which agonists are in current clinical use. This review explores pharmaceutical development of drugs that activate individual or multiple ß-cell GPCRs and explains how our knowledge of GPCR expression by human islets may inform direction on novel GPCR targets.


Assuntos
Diabetes Mellitus Tipo 2/tratamento farmacológico , Ilhotas Pancreáticas/metabolismo , Receptores Acoplados a Proteínas-G/metabolismo , Animais , Diabetes Mellitus Tipo 2/metabolismo , Humanos
19.
Sci Rep ; 7(1): 3946, 2017 06 21.
Artigo em Inglês | MEDLINE | ID: mdl-28638091

RESUMO

LH-21 is a triazol derivative that has been described as a low-permeant neutral CB1 antagonist, though its pharmacology is still unclear. It has been associated with anti-obesity actions in obese rats. However, its role in preventing type 2 diabetes (T2D) onset have not been studied yet. Given CB1 receptors remain as potential pharmacological targets to fight against obesity and T2D, we wanted to explore the metabolic impact of this compound in an animal model of obesity and pre-diabetes as well as the lack of relevant actions in related central processes such as anxiety. C57BL/6J mice were rendered obese and pre-diabetic by feeding a high-fat diet for 15 weeks and then treated with LH-21 or vehicle for two weeks. Food intake, body weight and glucose handling were assessed, together with other relevant parameters. Behavioural performance was evaluated by the open field test and the elevated plus maze. LH-21 did not affect food intake nor body weight but it improved glucose handling, displaying tissue-specific beneficial actions. Unexpectedly, LH-21 induced anxiolysis and reverted obesity-induced anxiety, apparently through GPR55 receptor. These results suggest that LH-21 can be a new candidate to fight against diabetes onset. Indeed, this compound shows potential in counteracting obesity-related anxiety.


Assuntos
Ansiedade/prevenção & controle , Glicemia/metabolismo , Obesidade/metabolismo , Estado Pré-Diabético/metabolismo , Receptor CB1 de Canabinoide/antagonistas & inibidores , Triazóis/administração & dosagem , Animais , Comportamento Animal , Dieta Hiperlipídica , Modelos Animais de Doenças , Mediadores da Inflamação/metabolismo , Insulina/metabolismo , Ilhotas Pancreáticas/efeitos dos fármacos , Ilhotas Pancreáticas/metabolismo , Fígado/efeitos dos fármacos , Fígado/metabolismo , Camundongos Endogâmicos C57BL , Obesidade/prevenção & controle , Estado Pré-Diabético/prevenção & controle
20.
Sci Rep ; 7: 46600, 2017 04 19.
Artigo em Inglês | MEDLINE | ID: mdl-28422162

RESUMO

G-protein coupled receptors (GPCRs) are essential for islet function, but most studies use rodent islets due to limited human islet availability. We have systematically compared the GPCR mRNA expression in human and mouse islets to determine to what extent mouse islets can be used as surrogates for human islets to study islet GPCR function, and we have identified species-specific expression of several GPCRs. The A3 receptor (ADORA3) was expressed only in mouse islets and the A3 agonist MRS 5698 inhibited glucose-induced insulin secretion from mouse islets, with no effect on human islets. Similarly, mRNAs encoding the galanin receptors GAL1 (GALR1), GAL2 (GALR2) and GAL3 GALR3) were abundantly expressed in mouse islets but present only at low levels in human islets, so that it reads (GALR3) and galanin inhibited insulin secretion only from mouse islets. Conversely, the sst1 receptor (SSTR1) was abundant only in human islets and its selective activation by CH 275 inhibited insulin secretion from human islets, with no effect on mouse islets. Our comprehensive human and mouse islet GPCR atlas has demonstrated that species differences do exist in islet GPCR expression and function, which are likely to impact on the translatability of mouse studies to the human context.


Assuntos
Regulação da Expressão Gênica , Secreção de Insulina , Insulina/metabolismo , Ilhotas Pancreáticas/metabolismo , Receptor A3 de Adenosina/metabolismo , Receptores de Galanina/biossíntese , Receptores de Somatostatina/biossíntese , Animais , Humanos , Ilhotas Pancreáticas/citologia , Masculino , Camundongos , Especificidade da Espécie
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