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1.
PLoS Biol ; 20(9): e3001761, 2022 09.
Artigo em Inglês | MEDLINE | ID: mdl-36099294

RESUMO

Insulin-secreting ß-cells are functionally heterogeneous. Whether there exist cells driving the first-phase calcium response in individual islets, has not been examined. We examine "first responder" cells, defined by the earliest [Ca2+] response during first-phase [Ca2+] elevation, distinct from previously identified "hub" and "leader" cells. We used islets isolated from Mip-CreER; Rosa-Stop-Lox-Stop-GCamP6s mice (ß-GCamP6s) that show ß-cell-specific GCamP6s expression following tamoxifen-induced CreER-mediated recombination. First responder cells showed characteristics of high membrane excitability and lower electrical coupling to their neighbors. The first-phase response time of ß-cells in the islet was spatially organized, dependent on the cell's distance to the first responder cell, and consistent over time up to approximately 24 h. When first responder cells were laser ablated, the first-phase [Ca2+] was slowed down, diminished, and discoordinated compared to random cell ablation. Cells that were next earliest to respond often took over the role of the first responder upon ablation. In summary, we discover and characterize a distinct first responder ß-cell state, critical for the islet first-phase response to glucose.


Assuntos
Células Secretoras de Insulina , Ilhotas Pancreáticas , Animais , Cálcio/metabolismo , Glucose/metabolismo , Glucose/farmacologia , Insulina/metabolismo , Células Secretoras de Insulina/metabolismo , Ilhotas Pancreáticas/metabolismo , Camundongos , Tamoxifeno/metabolismo
2.
Circ Res ; 130(5): 694-707, 2022 03 04.
Artigo em Inglês | MEDLINE | ID: mdl-35100822

RESUMO

BACKGROUND: Aberrant sympathetic nerve activity exacerbates cardiovascular risk in hypertension and diabetes, which are common comorbidities, yet clinically sympathetic nerve activity remains poorly controlled. The hypertensive diabetic state is associated with increased reflex sensitivity and tonic drive from the peripheral chemoreceptors, the cause of which is unknown. We have previously shown hypertension to be critically dependent on the carotid body (CB) input in spontaneously hypertensive rat, a model that also exhibits a number of diabetic traits. CB overstimulation by insulin and leptin has been similarly implicated in the development of increased sympathetic nerve activity in metabolic syndrome and obesity. Thus, we hypothesized that in hypertensive diabetic state (spontaneously hypertensive rat), the CB is sensitized by altered metabolic signaling causing excessive sympathetic activity levels and dysfunctional reflex regulation. METHODS: Using a hypothesis-free RNA-seq approach, we investigated potential molecular targets implicated in energy metabolism mediating CB sensitization and its regulation of sympathetic outflow in experimental hypertension. Identified targets were characterized using molecular and functional techniques assessing peripheral chemoreflex sensitivity in situ and in vivo. RESULTS: We discovered GLP1R (glucagon-like peptide-1 receptor) expression in the CBs of rat and human and showed that its decreased expression is linked to sympathetic hyperactivity in rats with cardiometabolic disease. We demonstrate GLP1R to be localized to CB chemosensory cells, while targeted administration of GLP1R agonist to the CB lowered its basal discharge and attenuated chemoreflex-evoked blood pressure and sympathetic responses. Importantly, hyperglycemia-induced peripheral chemoreflex sensitization and associated basal sympathetic overactivity were abolished by GLP1R activation in the CB suggesting a role in a homeostatic response to high blood glucose. CONCLUSIONS: We show that GLP1 (glucagon-like peptide-1) modulates the peripheral chemoreflex acting on the CB, supporting this organ as a multimodal receptor. Our findings pinpoint CBs as potential targets for ameliorating excessive sympathetic activity using GLP1R agonists in the hypertensive-diabetic condition.


Assuntos
Corpo Carotídeo , Hipertensão , Animais , Pressão Sanguínea , Corpo Carotídeo/metabolismo , Glucose/metabolismo , Ratos , Ratos Endogâmicos SHR
3.
Clin Exp Immunol ; 212(1): 1-10, 2023 04 07.
Artigo em Inglês | MEDLINE | ID: mdl-36891817

RESUMO

Dysregulation of leukocyte trafficking, lipid metabolism, and other metabolic processes are the hallmarks that underpin and drive pathology in obesity. Current clinical management targets alternations in lifestyle choices (e.g. exercise, weight loss) to limit the impact of the disease. Crucially, re-gaining control over the pathogenic cellular and molecular processes may offer an alternative, complementary strategy for obese patients. Here we investigate the impact of the immunopeptide, PEPITEM, on pancreas homeostasis and leukocyte trafficking in mice on high-fed obesogenic diet (HFD). Both prophylactic and therapeutic treatment with PEPITEM alleviated the effects of HFD on the pancreas, reducing pancreatic beta cell size. Moreover, PEPITEM treatment also limited T-cell trafficking (CD4+ T-cells and KLRG1+ CD3+ T-cells) to obese visceral, but not subcutaneous, adipose tissue. Similarly, PEPITEM treatment reduced macrophage numbers within the peritoneal cavity of mice on HFD diet at both 6 and 12 weeks. By contrast, PEPITEM therapy elevated numbers of T and B cells were observed in the secondary lymphoid tissues (e.g. spleen and inguinal lymph node) when compared to the untreated HFD controls. Collectively our data highlights the potential for PEPITEM as a novel therapy to combat the systemic low-grade inflammation experienced in obesity and minimize the impact of obesity on pancreatic homeostasis. Thus, offering an alternative strategy to reduce the risk of developing obesity-related co-morbidities, such as type 2 diabetes mellitus, in individuals at high risk and struggling to control their weight through lifestyle modifications.


Assuntos
Diabetes Mellitus Tipo 2 , Camundongos , Animais , Diabetes Mellitus Tipo 2/metabolismo , Obesidade/complicações , Obesidade/metabolismo , Obesidade/patologia , Inflamação/patologia , Dieta , Linfócitos T CD4-Positivos/metabolismo , Camundongos Endogâmicos C57BL , Tecido Adiposo
4.
Diabet Med ; 40(12): e15220, 2023 12.
Artigo em Inglês | MEDLINE | ID: mdl-37669696

RESUMO

We previously developed, synthesized and tested light-activated sulfonylureas for optical control of KATP channels and pancreatic beta cell activity in vitro and in vivo. Such technology relies on installation of azobenzene photoswitches onto the sulfonylurea backbone, affording light-dependent isomerization, alteration in ligand affinity for SUR1 and hence KATP channel conductance. Inspired by molecular dynamics simulations and to further improve photoswitching characteristics, we set out to develop a novel push-pull closed ring azobenzene unit, before installing this on the sulfonylurea glimepiride as a small molecule recipient. Three fine-tuned, light-activated sulfonylureas were synthesized, encompassing azetidine, pyrrolidine and piperidine closed rings. Azetidine-, pyrrolidine- and piperidine-based sulfonylureas all increased beta cell Ca2+ -spiking activity upon continuous blue light illumination, similarly to first generation JB253. Notably, the pyrrolidine-based sulfonylurea showed superior switch OFF performance to JB253. As such, third generation sulfonylureas afford more precise optical control over primary pancreatic beta cells, and showcase the potential of pyrrolidine-azobenzenes as chemical photoswitches across drug classes.


Assuntos
Azetidinas , Células Secretoras de Insulina , Humanos , Compostos de Sulfonilureia/uso terapêutico , Trifosfato de Adenosina , Piperidinas , Pirrolidinas
5.
Angew Chem Int Ed Engl ; 62(20): e202302688, 2023 05 08.
Artigo em Inglês | MEDLINE | ID: mdl-36917014

RESUMO

The G protein-coupled kisspeptin receptor (GPR54 or KISS1R) is an important mediator in reproduction, metabolism and cancer biology; however, there are limited fluorescent probes or antibodies for direct imaging of these receptors in cells and intact tissues, which can help to interrogate their multiple biological roles. Herein, we describe the rational design and characterization of a new acid-resistant BODIPY-based amino acid (Trp-BODIPY PLUS), and its implementation for solid-phase synthesis of fluorescent bioactive peptides. Trp-BODIPY PLUS retains the binding capabilities of both short linear and cyclic peptides and displays notable turn-on fluorescence emission upon target binding for wash-free imaging. Finally, we employed Trp-BODIPY PLUS to prepare some of the first fluorogenic kisspeptin-based probes and visualized the expression and localization of GPR54 receptors in human cells and in whole mouse pancreatic islets by fluorescence imaging.


Assuntos
Ilhotas Pancreáticas , Kisspeptinas , Camundongos , Animais , Humanos , Kisspeptinas/química , Kisspeptinas/metabolismo , Peptídeos/química , Ilhotas Pancreáticas/diagnóstico por imagem , Ilhotas Pancreáticas/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Imagem Óptica , Aminoácidos/metabolismo
6.
J Physiol ; 600(5): 1119-1133, 2022 03.
Artigo em Inglês | MEDLINE | ID: mdl-33719063

RESUMO

The contribution of glucagon to type 1 and type 2 diabetes has long been known, but the underlying defects in alpha cell function are not well-described. During both disease states, alpha cells respond inappropriately to stimuli, leading to dysregulated glucagon secretion, impaired glucose tolerance and hypoglycaemia. The mechanisms involved in this dysfunction are complex, but possibly include changes in alpha cell glucose-sensing, alpha cell de-differentiation, paracrine feedback, as well as alpha cell mass. However, the molecular underpinnings of alpha cell failure are still poorly understood. Recent transcriptomic analyses have identified vitamin D binding protein (DBP), encoded by GC/Gc, as an alpha cell signature gene. DBP is highly localized to the liver and alpha cells and is virtually absent from other tissues and cell types under non-pathological conditions. While the vitamin D transportation role of DBP is well characterized in the liver and circulation, its function in alpha cells remains more enigmatic. Recent work reveals that loss of DBP leads to smaller and hyperplastic alpha cells, which secrete less glucagon in response to low glucose concentration, despite vitamin D sufficiency. Alpha cells lacking DBP display impaired Ca2+ fluxes and Na+ conductance, as well as changes in glucagon granule distribution. Underlying these defects is an increase in the ratio of cytoskeletal F-actin to G-actin, highlighting a novel intracellular actin scavenging role for DBP in islets.


Assuntos
Diabetes Mellitus Tipo 2 , Globulinas , Actinas/metabolismo , Globulinas/metabolismo , Glucagon , Glucose , Humanos , Vitamina D/metabolismo , Proteína de Ligação a Vitamina D/genética , Proteína de Ligação a Vitamina D/metabolismo
7.
Org Biomol Chem ; 20(30): 5967-5980, 2022 08 03.
Artigo em Inglês | MEDLINE | ID: mdl-35188523

RESUMO

The (in)ability to permeate membranes is a key feature of chemical biology probes that defines their suitability for specific applications. Here we report sulfonated rhodamines that endow xanthene dyes with cellular impermeability for analysis of surface proteins. We fuse charged sulfonates to red and far-red dyes to obtain Sulfo549 and Sulfo646, respectively, and further link these to benzylguanine and choloralkane substrates for SNAP-tag and Halo-tag labelling. Sulfonated rhodamine-conjugated fluorophores maintain desirable photophysical properties, such as brightness and photostability. While transfected cells with a nuclear localized SNAP-tag remain unlabelled, extracellular exposed tags can be cleanly visualized. By multiplexing with a permeable rhodamine, we are able to differentiate extra- and intracellular SNAP- and Halo-tags, including those installed on the glucagon-like peptide-1 receptor, a prototypical class B G protein-coupled receptor. Sulfo549 and Sulfo646 also labelled transfected neurons derived from induced pluripotent stem cells (iPSCs), allowing STED nanoscopy of the axonal membrane. Together, this work provides a new avenue for rendering dyes impermeable for exclusive extracellular visualization via self-labelling protein tags. We anticipate that Sulfo549, Sulfo646 and their congeners will be useful for a number of cell biology applications where labelling of intracellular sites interferes with accurate surface protein analysis.


Assuntos
Corantes Fluorescentes , Proteínas de Membrana , Corantes Fluorescentes/química , Rodaminas/química
8.
Mol Pharmacol ; 100(4): 319-334, 2021 10.
Artigo em Inglês | MEDLINE | ID: mdl-34315812

RESUMO

The glucagon-like peptide-1 receptor (GLP-1R) is a class B G protein-coupled receptor and mainstay therapeutic target for the treatment of type 2 diabetes and obesity. Recent reports have highlighted how biased agonism at the GLP-1R affects sustained glucose-stimulated insulin secretion through avoidance of desensitization and downregulation. A number of GLP-1R agonists (GLP-1RAs) feature a fatty acid moiety to prolong their pharmacokinetics via increased albumin binding, but the potential for these chemical changes to influence GLP-1R function has rarely been investigated beyond potency assessments for cAMP. Here, we directly compare the prototypical GLP-1RA exendin-4 with its C-terminally acylated analog, exendin-4-C16. We examine relative propensities of each ligand to recruit and activate G proteins and ß-arrestins, endocytic and postendocytic trafficking profiles, and interactions with model and cellular membranes in HEK293 and HEK293T cells. Both ligands had similar cAMP potency, but exendin-4-C16 showed ∼2.5-fold bias toward G protein recruitment and a ∼60% reduction in ß-arrestin-2 recruitment efficacy compared with exendin-4, as well as reduced GLP-1R endocytosis and preferential targeting toward recycling pathways. These effects were associated with reduced movement of the GLP-1R extracellular domain measured using a conformational biosensor approach and a ∼70% increase in insulin secretion in INS-1 832/3 cells. Interactions with plasma membrane lipids were enhanced by the acyl chain. Exendin-4-C16 showed extensive albumin binding and was highly effective for lowering of blood glucose in mice over at least 72 hours. Our study highlights the importance of a broad approach to the evaluation of GLP-1RA pharmacology. SIGNIFICANCE STATEMENT: Acylation is a common strategy to enhance the pharmacokinetics of peptide-based drugs. This work shows how acylation can also affect various other pharmacological parameters, including biased agonism, receptor trafficking, and interactions with the plasma membrane, which may be therapeutically important.


Assuntos
Exenatida/metabolismo , Receptor do Peptídeo Semelhante ao Glucagon 1/metabolismo , Incretinas/metabolismo , Transdução de Sinais/fisiologia , Acilação/efeitos dos fármacos , Acilação/fisiologia , Animais , Exenatida/farmacologia , Células HEK293 , Humanos , Incretinas/farmacologia , Secreção de Insulina/efeitos dos fármacos , Secreção de Insulina/fisiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Transporte Proteico/efeitos dos fármacos , Transporte Proteico/fisiologia , Transdução de Sinais/efeitos dos fármacos
9.
FASEB J ; 34(5): 6284-6301, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-32201986

RESUMO

Mitophagy is a key process regulating mitochondrial quality control. Several mechanisms have been proposed to regulate mitophagy, but these have mostly been studied using stably expressed non-native proteins in immortalized cell lines. In skeletal muscle, mitophagy and its molecular mechanisms require more thorough investigation. To measure mitophagy directly, we generated a stable skeletal muscle C2C12 cell line, expressing a mitophagy reporter construct (mCherry-green fluorescence protein-mtFIS1101-152 ). Here, we report that both carbonyl cyanide m-chlorophenyl hydrazone (CCCP) treatment and adenosine monophosphate activated protein kinase (AMPK) activation by 991 promote mitochondrial fission via phosphorylation of MFF and induce mitophagy by ~20%. Upon CCCP treatment, but not 991, ubiquitin phosphorylation, a read-out of PTEN-induced kinase 1 (PINK1) activity, and Parkin E3 ligase activity toward CDGSH iron sulfur domain 1 (CISD1) were increased. Although the PINK1-Parkin signaling pathway is active in response to CCCP treatment, we observed no change in markers of mitochondrial protein content. Interestingly, our data shows that TANK-binding kinase 1 (TBK1) phosphorylation is increased after both CCCP and 991 treatments, suggesting TBK1 activation to be independent of both PINK1 and Parkin. Finally, we confirmed in non-muscle cell lines that TBK1 phosphorylation occurs in the absence of PINK1 and is regulated by AMPK-dependent signaling. Thus, AMPK activation promotes mitophagy by enhancing mitochondrial fission (via MFF phosphorylation) and autophagosomal engulfment (via TBK1 activation) in a PINK1-Parkin independent manner.


Assuntos
Proteínas Quinases Ativadas por AMP/metabolismo , Dinâmica Mitocondrial , Mitofagia , Músculo Esquelético/patologia , Proteínas Quinases/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Proteínas Quinases Ativadas por AMP/genética , Animais , Carbonil Cianeto m-Clorofenil Hidrazona/farmacologia , Ativação Enzimática , Células HeLa , Humanos , Camundongos , Músculo Esquelético/efeitos dos fármacos , Músculo Esquelético/metabolismo , Proteínas Quinases/genética , Proteínas Serina-Treonina Quinases/genética , Ionóforos de Próton/farmacologia , Transdução de Sinais , Ubiquitina/metabolismo , Ubiquitina-Proteína Ligases/genética , Ubiquitinação
10.
Diabetologia ; 63(9): 1822-1835, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32472193

RESUMO

AIMS/HYPOTHESIS: During pregnancy, maternal metabolic disease and hormonal imbalance may alter fetal beta cell development and/or proliferation, thus leading to an increased risk for developing type 2 diabetes in adulthood. Although thyroid hormones play an important role in fetal endocrine pancreas development, the impact of maternal hypothyroidism on glucose homeostasis in adult offspring remains poorly understood. METHODS: We investigated this using a mouse model of hypothyroidism, induced by administration of an iodine-deficient diet supplemented with propylthiouracil during gestation. RESULTS: Here, we show that, when fed normal chow, adult mice born to hypothyroid mothers were more glucose-tolerant due to beta cell hyperproliferation (two- to threefold increase in Ki67-positive beta cells) and increased insulin sensitivity. However, following 8 weeks of high-fat feeding, these offspring gained 20% more body weight, became profoundly hyperinsulinaemic (with a 50% increase in fasting insulin concentration), insulin-resistant and glucose-intolerant compared with controls from euthyroid mothers. Furthermore, altered glucose metabolism was maintained in a second generation of animals. CONCLUSIONS/INTERPRETATION: Therefore, gestational hypothyroidism induces long-term alterations in endocrine pancreas function, which may have implications for type 2 diabetes prevention in affected individuals.


Assuntos
Glicemia/metabolismo , Intolerância à Glucose/metabolismo , Hipotireoidismo/metabolismo , Células Secretoras de Insulina/metabolismo , Ilhotas Pancreáticas/embriologia , Complicações na Gravidez/metabolismo , Efeitos Tardios da Exposição Pré-Natal/metabolismo , Animais , Antitireóideos/toxicidade , Proliferação de Células , Dieta Hiperlipídica , Modelos Animais de Doenças , Feminino , Hiperinsulinismo/metabolismo , Resistência à Insulina , Iodo/deficiência , Ilhotas Pancreáticas/metabolismo , Camundongos , Gravidez , Propiltiouracila/toxicidade , Estresse Fisiológico
11.
Diabetologia ; 63(2): 313-323, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-31732790

RESUMO

AIMS/HYPOTHESIS: Progressive decline in functional beta cell mass is central to the development of type 2 diabetes. Elevated serum levels of extracellular nicotinamide phosphoribosyltransferase (eNAMPT) are associated with beta cell failure in type 2 diabetes and eNAMPT immuno-neutralisation improves glucose tolerance in mouse models of diabetes. Despite this, the effects of eNAMPT on functional beta cell mass are poorly elucidated, with some studies having separately reported beta cell-protective effects of eNAMPT. eNAMPT exists in structurally and functionally distinct monomeric and dimeric forms. Dimerisation is essential for the NAD-biosynthetic capacity of NAMPT. Monomeric eNAMPT does not possess NAD-biosynthetic capacity and may exert distinct NAD-independent effects. This study aimed to fully characterise the structure-functional effects of eNAMPT on pancreatic beta cell functional mass and to relate these to beta cell failure in type 2 diabetes. METHODS: CD-1 mice and serum from obese humans who were without diabetes, with impaired fasting glucose (IFG) or with type 2 diabetes (from the Body Fat, Surgery and Hormone [BodyFatS&H] study) or with or at risk of developing type 2 diabetes (from the VaSera trial) were used in this study. We generated recombinant wild-type and monomeric eNAMPT to explore the effects of eNAMPT on functional beta cell mass in isolated mouse and human islets. Beta cell function was determined by static and dynamic insulin secretion and intracellular calcium microfluorimetry. NAD-biosynthetic capacity of eNAMPT was assessed by colorimetric and fluorescent assays and by native mass spectrometry. Islet cell number was determined by immunohistochemical staining for insulin, glucagon and somatostatin, with islet apoptosis determined by caspase 3/7 activity. Markers of inflammation and beta cell identity were determined by quantitative reverse transcription PCR. Total, monomeric and dimeric eNAMPT and nicotinamide mononucleotide (NMN) were evaluated by ELISA, western blot and fluorometric assay using serum from non-diabetic, glucose intolerant and type 2 diabetic individuals. RESULTS: eNAMPT exerts bimodal and concentration- and structure-functional-dependent effects on beta cell functional mass. At low physiological concentrations (~1 ng/ml), as seen in serum from humans without diabetes, eNAMPT enhances beta cell function through NAD-dependent mechanisms, consistent with eNAMPT being present as a dimer. However, as eNAMPT concentrations rise to ~5 ng/ml, as in type 2 diabetes, eNAMPT begins to adopt a monomeric form and mediates beta cell dysfunction, reduced beta cell identity and number, increased alpha cell number and increased apoptosis, through NAD-independent proinflammatory mechanisms. CONCLUSIONS/INTERPRETATION: We have characterised a novel mechanism of beta cell dysfunction in type 2 diabetes. At low physiological levels, eNAMPT exists in dimer form and maintains beta cell function and identity through NAD-dependent mechanisms. However, as eNAMPT levels rise, as in type 2 diabetes, structure-functional changes occur resulting in marked elevation of monomeric eNAMPT, which induces a diabetic phenotype in pancreatic islets. Strategies to selectively target monomeric eNAMPT could represent promising therapeutic strategies for the treatment of type 2 diabetes.


Assuntos
Citocinas/sangue , Citocinas/metabolismo , Diabetes Mellitus Tipo 2/sangue , Diabetes Mellitus Tipo 2/fisiopatologia , Nicotinamida Fosforribosiltransferase/sangue , Nicotinamida Fosforribosiltransferase/metabolismo , Animais , Células Cultivadas , Diabetes Mellitus Tipo 2/metabolismo , Glucagon/sangue , Glucagon/metabolismo , Humanos , Immunoblotting , Secreção de Insulina/fisiologia , Células Secretoras de Insulina/metabolismo , Masculino , Espectrometria de Massas , Camundongos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Somatostatina/sangue , Somatostatina/metabolismo , Relação Estrutura-Atividade
12.
Nat Chem Biol ; 14(8): 764-767, 2018 08.
Artigo em Inglês | MEDLINE | ID: mdl-30013061

RESUMO

L-type Ca2+ channels (LTCCs) play a crucial role in excitation-contraction coupling and release of hormones from secretory cells. They are targets of antihypertensive and antiarrhythmic drugs such as diltiazem. Here, we present a photoswitchable diltiazem, FHU-779, which can be used to reversibly block endogenous LTCCs by light. FHU-779 is as potent as diltiazem and can be used to place pancreatic ß-cell function and cardiac activity under optical control.


Assuntos
Canais de Cálcio Tipo L/metabolismo , Diltiazem/farmacologia , Corantes Fluorescentes/farmacologia , Coração/efeitos dos fármacos , Células Secretoras de Insulina/efeitos dos fármacos , Imagem Óptica , Canais de Cálcio Tipo L/química , Diltiazem/química , Corantes Fluorescentes/química , Humanos , Células Secretoras de Insulina/metabolismo , Luz , Processos Fotoquímicos
13.
Proc Natl Acad Sci U S A ; 114(9): 2379-2382, 2017 02 28.
Artigo em Inglês | MEDLINE | ID: mdl-28193889

RESUMO

Small assemblies of hypothalamic "parvocellular" neurons release their neuroendocrine signals at the median eminence (ME) to control long-lasting pituitary hormone rhythms essential for homeostasis. How such rapid hypothalamic neurotransmission leads to slowly evolving hormonal signals remains unknown. Here, we show that the temporal organization of dopamine (DA) release events in freely behaving animals relies on a set of characteristic features that are adapted to the dynamic dopaminergic control of pituitary prolactin secretion, a key reproductive hormone. First, locally generated DA release signals are organized over more than four orders of magnitude (0.001 Hz-10 Hz). Second, these DA events are finely tuned within and between frequency domains as building blocks that recur over days to weeks. Third, an integration time window is detected across the ME and consists of high-frequency DA discharges that are coordinated within the minutes range. Thus, a hierarchical combination of time-scaled neuroendocrine signals displays local-global integration to connect brain-pituitary rhythms and pace hormone secretion.


Assuntos
Hipotálamo/fisiologia , Eminência Mediana/fisiologia , Hipófise/fisiologia , Sistema Hipófise-Suprarrenal/fisiologia , Prolactina/metabolismo , Ritmo Ultradiano/fisiologia , Potenciais de Ação/fisiologia , Animais , Relógios Biológicos/fisiologia , Técnicas Eletroquímicas , Feminino , Camundongos , Camundongos Endogâmicos C57BL , Microeletrodos
14.
Int J Mol Sci ; 21(21)2020 Nov 09.
Artigo em Inglês | MEDLINE | ID: mdl-33182425

RESUMO

The glucagon-like peptide-1 receptor (GLP-1R) is an important regulator of blood glucose homeostasis. Ligand-specific differences in membrane trafficking of the GLP-1R influence its signalling properties and therapeutic potential in type 2 diabetes. Here, we have evaluated how different factors combine to control the post-endocytic trafficking of GLP-1R to recycling versus degradative pathways. Experiments were performed in primary islet cells, INS-1 832/3 clonal beta cells and HEK293 cells, using biorthogonal labelling of GLP-1R to determine its localisation and degradation after treatment with GLP-1, exendin-4 and several further GLP-1R agonist peptides. We also characterised the effect of a rare GLP1R coding variant, T149M, and the role of endosomal peptidase endothelin-converting enzyme-1 (ECE-1), in GLP1R trafficking. Our data reveal how treatment with GLP-1 versus exendin-4 is associated with preferential GLP-1R targeting towards a recycling pathway. GLP-1, but not exendin-4, is a substrate for ECE-1, and the resultant propensity to intra-endosomal degradation, in conjunction with differences in binding affinity, contributes to alterations in GLP-1R trafficking behaviours and degradation. The T149M GLP-1R variant shows reduced signalling and internalisation responses, which is likely to be due to disruption of the cytoplasmic region that couples to intracellular effectors. These observations provide insights into how ligand- and genotype-specific factors can influence GLP-1R trafficking.


Assuntos
Endocitose/fisiologia , Receptor do Peptídeo Semelhante ao Glucagon 1/metabolismo , Células Secretoras de Insulina/metabolismo , Células Secretoras de Insulina/fisiologia , Transporte Proteico/fisiologia , Animais , Linhagem Celular , Citoplasma/metabolismo , Endossomos/metabolismo , Endossomos/fisiologia , Enzimas Conversoras de Endotelina/metabolismo , Células HEK293 , Humanos , Ligantes , Camundongos
15.
Cell Mol Life Sci ; 75(7): 1303-1305, 2018 04.
Artigo em Inglês | MEDLINE | ID: mdl-29450573

RESUMO

The original version of this article unfortunately contained a mistake. Legends of Figures 1 and 2 were interchanged. The correct versions are given below.

16.
J Biol Chem ; 292(21): 8892-8906, 2017 05 26.
Artigo em Inglês | MEDLINE | ID: mdl-28377501

RESUMO

Heterozygous mutations in the human paired box gene PAX6 lead to impaired glucose tolerance. Although embryonic deletion of the Pax6 gene in mice leads to loss of most pancreatic islet cell types, the functional consequences of Pax6 loss in adults are poorly defined. Here we developed a mouse line in which Pax6 was selectively inactivated in ß cells by crossing animals with floxed Pax6 alleles to mice expressing the inducible Pdx1CreERT transgene. Pax6 deficiency, achieved by tamoxifen injection, caused progressive hyperglycemia. Although ß cell mass was preserved 8 days post-injection, total insulin content and insulin:chromogranin A immunoreactivity were reduced by ∼60%, and glucose-stimulated insulin secretion was eliminated. RNA sequencing and quantitative real-time PCR analyses revealed that, although the expression of key ß cell genes, including Ins2, Slc30a8, MafA, Slc2a2, G6pc2, and Glp1r, was reduced after Pax6 deletion, that of several genes that are usually selectively repressed ("disallowed") in ß cells, including Slc16a1, was increased. Assessed in intact islets, glucose-induced ATP:ADP increases were significantly reduced (p < 0.05) in ßPax6KO versus control ß cells, and the former displayed attenuated increases in cytosolic Ca2+ Unexpectedly, glucose-induced increases in intercellular connectivity were enhanced after Pax6 deletion, consistent with increases in the expression of the glucose sensor glucokinase, but decreases in that of two transcription factors usually expressed in fully differentiated ß-cells, Pdx1 and Nkx6.1, were observed in islet "hub" cells. These results indicate that Pax6 is required for the functional identity of adult ß cells. Furthermore, deficiencies in ß cell glucose sensing are likely to contribute to defective insulin secretion in human carriers of PAX6 mutations.


Assuntos
Trifosfato de Adenosina/metabolismo , Sinalização do Cálcio , Cálcio/metabolismo , Regulação da Expressão Gênica , Glucose/metabolismo , Células Secretoras de Insulina/metabolismo , Fator de Transcrição PAX6/biossíntese , Trifosfato de Adenosina/genética , Animais , Humanos , Camundongos , Camundongos Knockout , Fator de Transcrição PAX6/genética
17.
Nat Chem Biol ; 12(9): 755-62, 2016 09.
Artigo em Inglês | MEDLINE | ID: mdl-27454932

RESUMO

Increased levels of the second messenger lipid diacylglycerol (DAG) induce downstream signaling events including the translocation of C1-domain-containing proteins toward the plasma membrane. Here, we introduce three light-sensitive DAGs, termed PhoDAGs, which feature a photoswitchable acyl chain. The PhoDAGs are inactive in the dark and promote the translocation of proteins that feature C1 domains toward the plasma membrane upon a flash of UV-A light. This effect is quickly reversed after the termination of photostimulation or by irradiation with blue light, permitting the generation of oscillation patterns. Both protein kinase C and Munc13 can thus be put under optical control. PhoDAGs control vesicle release in excitable cells, such as mouse pancreatic islets and hippocampal neurons, and modulate synaptic transmission in Caenorhabditis elegans. As such, the PhoDAGs afford an unprecedented degree of spatiotemporal control and are broadly applicable tools to study DAG signaling.


Assuntos
Diglicerídeos/metabolismo , Diglicerídeos/efeitos da radiação , Processos Fotoquímicos/efeitos da radiação , Proteína Quinase C/metabolismo , Proteína Quinase C/efeitos da radiação , Raios Ultravioleta , Animais , Caenorhabditis elegans/enzimologia , Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/efeitos da radiação , Diglicerídeos/química , Camundongos , Fenômenos Ópticos , Proteína Quinase C/química , Transdução de Sinais/efeitos da radiação
19.
Curr Diab Rep ; 18(11): 112, 2018 09 24.
Artigo em Inglês | MEDLINE | ID: mdl-30251179

RESUMO

PURPOSE OF REVIEW: To discuss advances in our understanding of beta-cell heterogeneity and the ramifications of this for type 1 diabetes (T1D) and its therapy. RECENT FINDINGS: A number of studies have challenged the long-standing dogma that the majority of beta cells are eliminated in T1D. As many as 80% are present in some T1D subjects. Why don't these cells function properly to release insulin in response to high glucose? Other findings deploying single-cell "omics" to study both healthy and diseased cells-from patients with both T1D and type 2 diabetes (T2D)-have revealed cell subpopulations and heterogeneity at the transcriptomic/protein level between individual cells. Finally, our own and others' findings have demonstrated the importance of functional beta-cell subpopulations for insulin secretion. Heterogeneity may endow beta cells with molecular features that predispose them to failure/death during T1D.


Assuntos
Diabetes Mellitus Tipo 1/etiologia , Células Secretoras de Insulina/patologia , Diabetes Mellitus Tipo 1/terapia , Diabetes Mellitus Tipo 2/patologia , Humanos , Modelos Biológicos
20.
Hum Mol Genet ; 24(5): 1390-9, 2015 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-25355422

RESUMO

Type 2 diabetes (T2D) is characterized by ß cell dysfunction and loss. Single nucleotide polymorphisms in the T-cell factor 7-like 2 (TCF7L2) gene, associated with T2D by genome-wide association studies, lead to impaired ß cell function. While deletion of the homologous murine Tcf7l2 gene throughout the developing pancreas leads to impaired glucose tolerance, deletion in the ß cell in adult mice reportedly has more modest effects. To inactivate Tcf7l2 highly selectively in ß cells from the earliest expression of the Ins1 gene (∼E11.5) we have therefore used a Cre recombinase introduced at the Ins1 locus. Tcfl2(fl/fl)::Ins1Cre mice display impaired oral and intraperitoneal glucose tolerance by 8 and 16 weeks, respectively, and defective responses to the GLP-1 analogue liraglutide at 8 weeks. Tcfl2(fl/fl)::Ins1Cre islets displayed defective glucose- and GLP-1-stimulated insulin secretion and the expression of both the Ins2 (∼20%) and Glp1r (∼40%) genes were significantly reduced. Glucose- and GLP-1-induced intracellular free Ca(2+) increases, and connectivity between individual ß cells, were both lowered by Tcf7l2 deletion in islets from mice maintained on a high (60%) fat diet. Finally, analysis by optical projection tomography revealed ∼30% decrease in ß cell mass in pancreata from Tcfl2(fl/fl)::Ins1Cre mice. These data demonstrate that Tcf7l2 plays a cell autonomous role in the control of ß cell function and mass, serving as an important regulator of gene expression and islet cell coordination. The possible relevance of these findings for the action of TCF7L2 polymorphisms associated with Type 2 diabetes in man is discussed.


Assuntos
Células Secretoras de Insulina/metabolismo , Insulina/metabolismo , Pâncreas/fisiopatologia , Proteína 2 Semelhante ao Fator 7 de Transcrição/metabolismo , Animais , Glicemia/metabolismo , Diabetes Mellitus Tipo 2/genética , Dieta Hiperlipídica/veterinária , Modelos Animais de Doenças , Deleção de Genes , Loci Gênicos , Estudo de Associação Genômica Ampla , Glucagon/sangue , Peptídeo 1 Semelhante ao Glucagon/genética , Peptídeo 1 Semelhante ao Glucagon/metabolismo , Receptor do Peptídeo Semelhante ao Glucagon 1 , Insulina/sangue , Insulina/genética , Secreção de Insulina , Células Secretoras de Insulina/patologia , Integrases/genética , Integrases/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Peso Molecular , Pâncreas/metabolismo , Polimorfismo de Nucleotídeo Único , Receptores de Glucagon/genética , Receptores de Glucagon/metabolismo , Proteína 2 Semelhante ao Fator 7 de Transcrição/genética , Via de Sinalização Wnt
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