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1.
Diabetes Obes Metab ; 24(11): 2090-2101, 2022 11.
Artigo em Inglês | MEDLINE | ID: mdl-35676825

RESUMO

AIMS: To describe the in vitro characteristics and antidiabetic in vivo efficacy of the novel glucagon-like peptide-1 receptor agonist (GLP-1RA) GL0034. MATERIALS AND METHODS: Glucagon-like peptide-1 receptor (GLP-1R) kinetic binding parameters, cyclic adenosine monophosphate (cAMP) signalling, endocytosis and recycling were measured using HEK293 and INS-1832/3 cells expressing human GLP-1R. Insulin secretion was measured in vitro using INS-1832/3 cells, mouse islets and human islets. Chronic administration studies to evaluate weight loss and glycaemic effects were performed in db/db and diet-induced obese mice. RESULTS: Compared to the leading GLP-1RA semaglutide, GL0034 showed increased binding affinity and potency-driven bias in favour of cAMP over GLP-1R endocytosis and ß-arrestin-2 recruitment. Insulin secretory responses were similar for both ligands. GL0034 (6 nmol/kg) led to at least as much weight loss and lowering of blood glucose as did semaglutide at a higher dose (14 nmol/kg). CONCLUSIONS: GL0034 is a G protein-biased agonist that shows powerful antidiabetic effects in mice, and may serve as a promising new GLP-1RA for obese patients with type 2 diabetes.


Assuntos
Diabetes Mellitus Tipo 2 , Insulinas , Monofosfato de Adenosina , Animais , Glicemia , AMP Cíclico/metabolismo , Diabetes Mellitus Tipo 2/tratamento farmacológico , Receptor do Peptídeo Semelhante ao Glucagon 1/agonistas , Células HEK293 , Humanos , Hipoglicemiantes/farmacologia , Hipoglicemiantes/uso terapêutico , Ligantes , Camundongos , Redução de Peso , beta-Arrestinas/metabolismo
3.
Cell Rep ; 34(5): 108703, 2021 02 02.
Artigo em Inglês | MEDLINE | ID: mdl-33535042

RESUMO

Using chromatin conformation capture, we show that an enhancer cluster in the STARD10 type 2 diabetes (T2D) locus forms a defined 3-dimensional (3D) chromatin domain. A 4.1-kb region within this locus, carrying 5 T2D-associated variants, physically interacts with CTCF-binding regions and with an enhancer possessing strong transcriptional activity. Analysis of human islet 3D chromatin interaction maps identifies the FCHSD2 gene as an additional target of the enhancer cluster. CRISPR-Cas9-mediated deletion of the variant region, or of the associated enhancer, from human pancreas-derived EndoC-ßH1 cells impairs glucose-stimulated insulin secretion. Expression of both STARD10 and FCHSD2 is reduced in cells harboring CRISPR deletions, and lower expression of STARD10 and FCHSD2 is associated, the latter nominally, with the possession of risk variant alleles in human islets. Finally, CRISPR-Cas9-mediated loss of STARD10 or FCHSD2, but not ARAP1, impairs regulated insulin secretion. Thus, multiple genes at the STARD10 locus influence ß cell function.


Assuntos
Proteínas de Transporte/metabolismo , Cromatina/metabolismo , Células Secretoras de Insulina/metabolismo , Proteínas de Membrana/metabolismo , Fosfoproteínas/metabolismo , Humanos
4.
Cell Rep ; 33(9): 108466, 2020 12 01.
Artigo em Inglês | MEDLINE | ID: mdl-33264613

RESUMO

Pancreatic ß cell failure is key to type 2 diabetes (T2D) onset and progression. Here, we assess whether human ß cell dysfunction induced by metabolic stress is reversible, evaluate the molecular pathways underlying persistent or transient damage, and explore the relationships with T2D islet traits. Twenty-six islet preparations are exposed to several lipotoxic/glucotoxic conditions, some of which impair insulin release, depending on stressor type, concentration, and combination. The reversal of dysfunction occurs after washout for some, although not all, of the lipoglucotoxic insults. Islet transcriptomes assessed by RNA sequencing and expression quantitative trait loci (eQTL) analysis identify specific pathways underlying ß cell failure and recovery. Comparison of a large number of human T2D islet transcriptomes with those of persistent or reversible ß cell lipoglucotoxicity show shared gene expression signatures. The identification of mechanisms associated with human ß cell dysfunction and recovery and their overlap with T2D islet traits provide insights into T2D pathogenesis, fostering the development of improved ß cell-targeted therapeutic strategies.


Assuntos
Diabetes Mellitus Tipo 2/genética , Expressão Gênica/genética , Células Secretoras de Insulina/metabolismo , Estresse Fisiológico/genética , Diabetes Mellitus Tipo 2/patologia , Humanos
5.
Mol Metab ; 40: 101015, 2020 10.
Artigo em Inglês | MEDLINE | ID: mdl-32416313

RESUMO

OBJECTIVE: Risk alleles for type 2 diabetes at the STARD10 locus are associated with lowered STARD10 expression in the ß-cell, impaired glucose-induced insulin secretion, and decreased circulating proinsulin:insulin ratios. Although likely to serve as a mediator of intracellular lipid transfer, the identity of the transported lipids and thus the pathways through which STARD10 regulates ß-cell function are not understood. The aim of this study was to identify the lipids transported and affected by STARD10 in the ß-cell and the role of the protein in controlling proinsulin processing and insulin granule biogenesis and maturation. METHODS: We used isolated islets from mice deleted selectively in the ß-cell for Stard10 (ßStard10KO) and performed electron microscopy, pulse-chase, RNA sequencing, and lipidomic analyses. Proteomic analysis of STARD10 binding partners was executed in the INS1 (832/13) cell line. X-ray crystallography followed by molecular docking and lipid overlay assay was performed on purified STARD10 protein. RESULTS: ßStard10KO islets had a sharply altered dense core granule appearance, with a dramatic increase in the number of "rod-like" dense cores. Correspondingly, basal secretion of proinsulin was increased versus wild-type islets. The solution of the crystal structure of STARD10 to 2.3 Å resolution revealed a binding pocket capable of accommodating polyphosphoinositides, and STARD10 was shown to bind to inositides phosphorylated at the 3' position. Lipidomic analysis of ßStard10KO islets demonstrated changes in phosphatidylinositol levels, and the inositol lipid kinase PIP4K2C was identified as a STARD10 binding partner. Also consistent with roles for STARD10 in phosphoinositide signalling, the phosphoinositide-binding proteins Pirt and Synaptotagmin 1 were amongst the differentially expressed genes in ßStard10KO islets. CONCLUSION: Our data indicate that STARD10 binds to, and may transport, phosphatidylinositides, influencing membrane lipid composition, insulin granule biosynthesis, and insulin processing.


Assuntos
Diabetes Mellitus Tipo 2/genética , Fosfoproteínas/metabolismo , Alelos , Animais , Proteínas de Transporte/metabolismo , Diabetes Mellitus Tipo 2/metabolismo , Modelos Animais de Doenças , Feminino , Insulina/metabolismo , Secreção de Insulina/fisiologia , Células Secretoras de Insulina/metabolismo , Metabolismo dos Lipídeos/genética , Metabolismo dos Lipídeos/fisiologia , Lipídeos/fisiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Simulação de Acoplamento Molecular , Fosfatidilinositóis/metabolismo , Fosfoproteínas/genética , Ligação Proteica , Proteômica , Fatores de Risco , Vesículas Secretórias/metabolismo
6.
Nat Metab ; 1(6): 615-629, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-32694805

RESUMO

Pancreatic ß-cells form highly connected networks within isolated islets. Whether this behaviour pertains to the situation in vivo, after innervation and during continuous perfusion with blood, is unclear. In the present study, we used the recombinant Ca2+ sensor GCaMP6 to assess glucose-regulated connectivity in living zebrafish Danio rerio, and in murine or human islets transplanted into the anterior eye chamber. In each setting, Ca2+ waves emanated from temporally defined leader ß-cells, and three-dimensional connectivity across the islet increased with glucose stimulation. Photoablation of zebrafish leader cells disrupted pan-islet signalling, identifying these as likely pacemakers. Correspondingly, in engrafted mouse islets, connectivity was sustained during prolonged glucose exposure, and super-connected 'hub' cells were identified. Granger causality analysis revealed a controlling role for temporally defined leaders, and transcriptomic analyses revealed a discrete hub cell fingerprint. We thus define a population of regulatory ß-cells within coordinated islet networks in vivo. This population may drive Ca2+ dynamics and pulsatile insulin secretion.


Assuntos
Cálcio/metabolismo , Células Secretoras de Insulina/metabolismo , Animais , Glucose/metabolismo , Técnicas In Vitro , Insulina/metabolismo , Transdução de Sinais , Peixe-Zebra/metabolismo
7.
Proc Natl Acad Sci U S A ; 115(32): E7642-E7649, 2018 08 07.
Artigo em Inglês | MEDLINE | ID: mdl-30038024

RESUMO

SLC30A8 encodes a zinc transporter that is primarily expressed in the pancreatic islets of Langerhans. In ß-cells it transports zinc into insulin-containing secretory granules. Loss-of-function (LOF) mutations in SLC30A8 protect against type 2 diabetes in humans. In this study, we generated a knockin mouse model carrying one of the most common human LOF mutations for SLC30A8, R138X. The R138X mice had normal body weight, glucose tolerance, and pancreatic ß-cell mass. Interestingly, in hyperglycemic conditions induced by the insulin receptor antagonist S961, the R138X mice showed a 50% increase in insulin secretion. This effect was not associated with enhanced ß-cell proliferation or mass. Our data suggest that the SLC30A8 R138X LOF mutation may exert beneficial effects on glucose metabolism by increasing the capacity of ß-cells to secrete insulin under hyperglycemic conditions.


Assuntos
Diabetes Mellitus Tipo 2/genética , Glucose/metabolismo , Células Secretoras de Insulina/metabolismo , Insulina/metabolismo , Transportador 8 de Zinco/genética , Alelos , Animais , Glicemia , Diabetes Mellitus Tipo 2/metabolismo , Modelos Animais de Doenças , Técnicas de Introdução de Genes , Humanos , Hiperglicemia/sangue , Hiperglicemia/induzido quimicamente , Hiperglicemia/metabolismo , Secreção de Insulina , Mutação com Perda de Função , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Peptídeos/farmacologia , Receptor de Insulina/antagonistas & inibidores , Receptor de Insulina/metabolismo , Transportador 8 de Zinco/metabolismo
8.
Diabetes Obes Metab ; 20(12): 2800-2810, 2018 12.
Artigo em Inglês | MEDLINE | ID: mdl-29974637

RESUMO

AIMS: To investigate the effect of kisspeptin on glucose-stimulated insulin secretion and appetite in humans. MATERIALS AND METHODS: In 15 healthy men (age: 25.2 ± 1.1 years; BMI: 22.3 ± 0.5 kg m-2 ), we compared the effects of 1 nmol kg-1 h-1 kisspeptin versus vehicle administration on glucose-stimulated insulin secretion, metabolites, gut hormones, appetite and food intake. In addition, we assessed the effect of kisspeptin on glucose-stimulated insulin secretion in vitro in human pancreatic islets and a human ß-cell line (EndoC-ßH1 cells). RESULTS: Kisspeptin administration to healthy men enhanced insulin secretion following an intravenous glucose load, and modulated serum metabolites. In keeping with this, kisspeptin increased glucose-stimulated insulin secretion from human islets and a human pancreatic cell line in vitro. In addition, kisspeptin administration did not alter gut hormones, appetite or food intake in healthy men. CONCLUSIONS: Collectively, these data demonstrate for the first time a beneficial role for kisspeptin in insulin secretion in humans in vivo. This has important implications for our understanding of the links between reproduction and metabolism in humans, as well as for the ongoing translational development of kisspeptin-based therapies for reproductive and potentially metabolic conditions.


Assuntos
Apetite/efeitos dos fármacos , Secreção de Insulina/efeitos dos fármacos , Células Secretoras de Insulina/efeitos dos fármacos , Kisspeptinas/farmacologia , Adolescente , Adulto , Linhagem Celular , Glucose/metabolismo , Voluntários Saudáveis , Humanos , Insulina/sangue , Masculino , Adulto Jovem
9.
Diabetes Obes Metab ; 20(9): 2246-2254, 2018 09.
Artigo em Inglês | MEDLINE | ID: mdl-29748994

RESUMO

AIMS: To investigate the role of arcuate glucokinase (GK) in the regulation of glucose homeostasis. MATERIALS AND METHODS: A recombinant adeno-associated virus expressing either GK or an antisense GK construct was used to alter GK activity specifically in the hypothalamic arcuate nucleus (arc). GK activity in this nucleus was also increased by stereotactic injection of the GK activator, compound A. The effect of altered arc GK activity on glucose homeostasis was subsequently investigated using glucose and insulin tolerance tests. RESULTS: Increased GK activity specifically within the arc increased insulin secretion and improved glucose tolerance in rats during oral glucose tolerance tests. Decreased GK activity in this nucleus reduced insulin secretion and increased glucose levels during the same tests. Insulin sensitivity was not affected in either case. The effect of arc GK was maintained in a model of type 2 diabetes. CONCLUSIONS: These results demonstrate a role for arc GK in systemic glucose homeostasis.


Assuntos
Núcleo Arqueado do Hipotálamo/enzimologia , Diabetes Mellitus Experimental/enzimologia , Diabetes Mellitus Tipo 2/enzimologia , Glucoquinase/metabolismo , Glucose/metabolismo , Secreção de Insulina/fisiologia , Animais , Diabetes Mellitus Experimental/sangue , Diabetes Mellitus Tipo 2/sangue , Teste de Tolerância a Glucose , Homeostase/fisiologia , Masculino , Ratos , Ratos Wistar , Ratos Zucker
10.
Diabetes ; 66(5): 1346-1358, 2017 05.
Artigo em Inglês | MEDLINE | ID: mdl-28232492

RESUMO

Changes in cellular free Zn2+ concentration, including those in the sarco(endo)plasmic reticulum [S(E)R], are primarily coordinated by Zn2+ transporters (ZnTs) whose identity and role in the heart are not well established. We hypothesized that ZIP7 and ZnT7 transport Zn2+ in opposing directions across the S(E)R membrane in cardiomyocytes and that changes in their activity play an important role in the development of ER stress during hyperglycemia. The subcellular S(E)R localization of ZIP7 and ZnT7 was determined in cardiomyocytes and in isolated S(E)R preparations. Markedly increased mRNA and protein levels of ZIP7 were observed in ventricular cardiomyocytes from diabetic rats or high-glucose-treated H9c2 cells while ZnT7 expression was low. In addition, we observed increased ZIP7 phosphorylation in response to high glucose in vivo and in vitro. By using recombinant-targeted Förster resonance energy transfer sensors, we show that hyperglycemia induces a marked redistribution of cellular free Zn2+, increasing cytosolic free Zn2+ and lowering free Zn2+ in the S(E)R. These changes involve alterations in ZIP7 phosphorylation and were suppressed by small interfering RNA-mediated silencing of CK2α. Opposing changes in the expression of ZIP7 and ZnT7 were also observed in hyperglycemia. We conclude that subcellular free Zn2+ redistribution in the hyperglycemic heart, resulting from altered ZIP7 and ZnT7 activity, contributes to cardiac dysfunction in diabetes.


Assuntos
Proteínas de Transporte de Cátions/genética , Diabetes Mellitus Experimental/genética , Diabetes Mellitus Tipo 1/genética , Estresse do Retículo Endoplasmático/genética , Retículo Endoplasmático/metabolismo , Miócitos Cardíacos/metabolismo , Retículo Sarcoplasmático/metabolismo , Animais , Western Blotting , Caseína Quinase II/genética , Proteínas de Transporte de Cátions/metabolismo , Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Tipo 1/metabolismo , Ventrículos do Coração/citologia , Hiperglicemia/genética , Hiperglicemia/metabolismo , Imunoprecipitação , Fosforilação , RNA Mensageiro/metabolismo , Ratos , Ratos Wistar , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Zinco/metabolismo
11.
Am J Hum Genet ; 100(2): 238-256, 2017 02 02.
Artigo em Inglês | MEDLINE | ID: mdl-28132686

RESUMO

Genetic variants near ARAP1 (CENTD2) and STARD10 influence type 2 diabetes (T2D) risk. The risk alleles impair glucose-induced insulin secretion and, paradoxically but characteristically, are associated with decreased proinsulin:insulin ratios, indicating improved proinsulin conversion. Neither the identity of the causal variants nor the gene(s) through which risk is conferred have been firmly established. Whereas ARAP1 encodes a GTPase activating protein, STARD10 is a member of the steroidogenic acute regulatory protein (StAR)-related lipid transfer protein family. By integrating genetic fine-mapping and epigenomic annotation data and performing promoter-reporter and chromatin conformational capture (3C) studies in ß cell lines, we localize the causal variant(s) at this locus to a 5 kb region that overlaps a stretch-enhancer active in islets. This region contains several highly correlated T2D-risk variants, including the rs140130268 indel. Expression QTL analysis of islet transcriptomes from three independent subject groups demonstrated that T2D-risk allele carriers displayed reduced levels of STARD10 mRNA, with no concomitant change in ARAP1 mRNA levels. Correspondingly, ß-cell-selective deletion of StarD10 in mice led to impaired glucose-stimulated Ca2+ dynamics and insulin secretion and recapitulated the pattern of improved proinsulin processing observed at the human GWAS signal. Conversely, overexpression of StarD10 in the adult ß cell improved glucose tolerance in high fat-fed animals. In contrast, manipulation of Arap1 in ß cells had no impact on insulin secretion or proinsulin conversion in mice. This convergence of human and murine data provides compelling evidence that the T2D risk associated with variation at this locus is mediated through reduction in STARD10 expression in the ß cell.


Assuntos
Diabetes Mellitus Tipo 2/genética , Insulina/metabolismo , Fosfoproteínas/genética , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Alelos , Animais , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Clonagem Molecular , Diabetes Mellitus Tipo 2/sangue , Proteínas Ativadoras de GTPase/genética , Proteínas Ativadoras de GTPase/metabolismo , Regulação da Expressão Gênica , Variação Genética , Homeostase , Humanos , Insulina/sangue , Secreção de Insulina , Células Secretoras de Insulina/metabolismo , Fígado/metabolismo , Camundongos , Proinsulina/sangue , Proinsulina/metabolismo , Locos de Características Quantitativas , Transcriptoma
12.
Mol Pharmacol ; 78(5): 818-26, 2010 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-20699324

RESUMO

Serotonin (5-HT)(2C) receptor is a G(q)-coupled receptor exhibiting a high degree of constitutive activity toward phospholipase C effector pathway, a process regulated by receptor mRNA editing. In addition to G protein-dependent signaling, 5-HT(2C) receptors also activate the extracellular signal-regulated kinase (ERK) 1/2 pathway independently of receptor coupling to G proteins. Constitutive activity at ERK signaling has not yet been explored. Transient expression of unedited 5-HT(2C-INI) receptors in human embryonic kidney (HEK) 293 cells resulted in a marked increase in ERK1/2 phosphorylation compared with nontransfected cells. No increase in ERK1/2 phosphorylation was measured in cells expressing fully edited (5-HT(2C-VGV)) receptors. Basal ERK1/2 phosphorylation in 5-HT(2C-INI) receptor-expressing cells was abolished by 5-methyl-1-(3-pyridylcarbamoyl)-1,2,3,5-tetrahydropyrrolo[2,3-f]indole (SB206,553), a 5-HT(2C) inverse agonist toward phospholipase C. This effect was prevented by the neutral antagonist 6-chloro-5-methyl-1-[6-(2-methylpyridin-3-yloxy)pyridin-3-ylcarbamoyl]indoline (SB242,084), which alone did not alter basal activity. Similar observations were made in vivo in mouse choroid plexus, a structure rich in constitutively active 5-HT(2C) receptors. Reminiscent of agonist-induced ERK1/2 phosphorylation, basal activity in HEK 293 cells was unaffected by cellular depletion of Gα(q/11) and Gα(13) proteins but strongly reduced in cells expressing a dominant-negative ß-arrestin or depleted of ß-arrestin by RNA interference and in cells expressing a dominant-negative calmodulin or a 5-HT(2C-INI) receptor mutant not capable of interacting with calmodulin. The tetracyclic antidepressants mirtazapine and mianserin likewise reduced basal ERK activation. On the other hand, the m-chlorophenylpiperazine derivative trazodone and the selective serotonin reuptake inhibitor fluoxetine were inactive alone but blocked 5-HT-induced ERK1/2 phosphorylation. Together, these data provide the first evidence of constitutive activity of a G protein-coupled receptor toward G-independent, ß-arrestin-dependent, receptor signaling.


Assuntos
Antidepressivos/farmacologia , Proteínas de Ligação ao GTP/fisiologia , Edição de RNA/efeitos dos fármacos , Receptor 5-HT2C de Serotonina/fisiologia , Animais , Arrestinas/metabolismo , Calmodulina/genética , Calmodulina/metabolismo , Linhagem Celular , Plexo Corióideo/efeitos dos fármacos , Plexo Corióideo/metabolismo , Agonismo Inverso de Drogas , Ativação Enzimática , Humanos , Técnicas In Vitro , Masculino , Camundongos , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Mutação , Fosforilação , Multimerização Proteica , Ratos , Ratos Sprague-Dawley , Receptor 5-HT2C de Serotonina/genética , Agonistas do Receptor 5-HT2 de Serotonina/farmacologia , Antagonistas do Receptor 5-HT2 de Serotonina/farmacologia , Transdução de Sinais , beta-Arrestinas
13.
EMBO J ; 28(18): 2706-18, 2009 Sep 16.
Artigo em Inglês | MEDLINE | ID: mdl-19661922

RESUMO

G protein-coupled receptors (GPCRs) have been found to trigger G protein-independent signalling. However, the regulation of G protein-independent pathways, especially their desensitization, is poorly characterized. Here, we show that the G protein-independent 5-HT(4) receptor (5-HT(4)R)-operated Src/ERK (extracellular signal-regulated kinase) pathway, but not the G(s) pathway, is inhibited by GPCR kinase 5 (GRK5), physically associated with the proximal region of receptor' C-terminus in both human embryonic kidney (HEK)-293 cells and colliculi neurons. This inhibition required two sequences of events: the association of beta-arrestin1 to a phosphorylated serine/threonine cluster located within the receptor C-t domain and the phosphorylation, by GRK5, of beta-arrestin1 (at Ser(412)) bound to the receptor. Phosphorylated beta-arrestin1 in turn prevented activation of Src constitutively bound to 5-HT(4)Rs, a necessary step in receptor-stimulated ERK signalling. This is the first demonstration that beta-arrestin1 phosphorylation by GRK5 regulates G protein-independent signalling.


Assuntos
Arrestinas/biossíntese , Quinase 5 de Receptor Acoplado a Proteína G/metabolismo , Receptores 5-HT4 de Serotonina/metabolismo , Linhagem Celular , Proteínas de Ligação ao GTP/metabolismo , Humanos , Mutação , Neurônios/metabolismo , Peptídeos/química , Fosforilação , Mapeamento de Interação de Proteínas , Estrutura Terciária de Proteína , RNA Interferente Pequeno/metabolismo , Serina/química , beta-Arrestinas , Quinases da Família src/metabolismo
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