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1.
J Biol Chem ; 299(8): 105065, 2023 08.
Artigo em Inglês | MEDLINE | ID: mdl-37468098

RESUMO

Pancreatic beta cells maintain glucose homeostasis by secreting pulses of insulin in response to a rise in plasma glucose. Pulsatile insulin secretion occurs as a result of glucose-induced oscillations in beta-cell cytosolic Ca2+. The endoplasmic reticulum (ER) helps regulate beta-cell cytosolic Ca2+, and ER stress can lead to ER Ca2+ reduction, beta-cell dysfunction, and an increased risk of type 2 diabetes. However, the mechanistic effects of ER stress on individual calcium channels are not well understood. To determine the effects of tunicamycin-induced ER stress on ER inositol 1,4,5-triphosphate receptors (IP3Rs) and ryanodine receptors (RyRs) and their involvement in subsequent Ca2+ dysregulation, we treated INS-1 832/13 cells and primary mouse islets with ER stress inducer tunicamycin (TM). We showed TM treatment increased RyR1 mRNA without affecting RyR2 mRNA and decreased both IP3R1 and IP3R3 mRNA. Furthermore, we found stress reduced ER Ca2+ levels, triggered oscillations in cytosolic Ca2+ under subthreshold glucose conditions, and increased apoptosis and that these changes were prevented by cotreatment with the RyR1 inhibitor dantrolene. In addition, we demonstrated silencing RyR1-suppressed TM-induced subthreshold cytosolic Ca2+ oscillations, but silencing RyR2 did not affect these oscillations. In contrast, inhibiting IP3Rs with xestospongin-C failed to suppress the TM-induced cytosolic Ca2+ oscillations and did not protect beta cells from TM-induced apoptosis although xestospongin-C inclusion did prevent ER Ca2+ reduction. Taken together, these results show changes in RyR1 play a critical role in ER stress-induced Ca2+ dysfunction and beta-cell apoptosis.


Assuntos
Sinalização do Cálcio , Estresse do Retículo Endoplasmático , Células Secretoras de Insulina , Canal de Liberação de Cálcio do Receptor de Rianodina , Animais , Camundongos , Apoptose , Diabetes Mellitus Tipo 2/metabolismo , Glucose/metabolismo , Homeostase , Células Secretoras de Insulina/metabolismo , Canal de Liberação de Cálcio do Receptor de Rianodina/genética , Canal de Liberação de Cálcio do Receptor de Rianodina/metabolismo , Tunicamicina , Ratos , Linhagem Celular
2.
J Cell Sci ; 135(5)2022 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-34821358

RESUMO

Endoplasmic reticulum (ER)-plasma membrane (PM) contacts are sites of lipid exchange and Ca2+ transport, and both lipid transport proteins and Ca2+ channels specifically accumulate at these locations. In pancreatic ß-cells, both lipid and Ca2+ signaling are essential for insulin secretion. The recently characterized lipid transfer protein TMEM24 (also known as C2CD2L) dynamically localizes to ER-PM contact sites and provides phosphatidylinositol, a precursor of phosphatidylinositol-4-phosphate [PI(4)P] and phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2], to the PM. ß-cells lacking TMEM24 exhibit markedly suppressed glucose-induced Ca2+ oscillations and insulin secretion, but the underlying mechanism is not known. We now show that TMEM24 only weakly interacts with the PM, and dissociates in response to both diacylglycerol and nanomolar elevations of cytosolic Ca2+. Loss of TMEM24 results in hyper-accumulation of Ca2+ in the ER and in excess Ca2+ entry into mitochondria, with resulting impairment in glucose-stimulated ATP production.


Assuntos
Cálcio , Proteínas de Membrana , Membrana Celular , Retículo Endoplasmático/genética , Homeostase , Proteínas de Membrana/genética
3.
Diabetologia ; 66(4): 709-723, 2023 04.
Artigo em Inglês | MEDLINE | ID: mdl-36459178

RESUMO

AIMS/HYPOTHESIS: The rapid remission of type 2 diabetes by a diet very low in energy correlates with a marked improvement in glucose-stimulated insulin secretion (GSIS), emphasising the role of beta cell dysfunction in the early stages of the disease. In search of novel mechanisms of beta cell dysfunction after long-term exposure to mild to severe glucotoxic conditions, we extensively characterised the alterations in insulin secretion and upstream coupling events in human islets cultured for 1-3 weeks at ~5, 8, 10 or 20 mmol/l glucose and subsequently stimulated by an acute stepwise increase in glucose concentration. METHODS: Human islets from 49 non-diabetic donors (ND-islets) and six type 2 diabetic donors (T2D-islets) were obtained from five isolation centres. After shipment, the islets were precultured for 3-7 days in RPMI medium containing ~5 mmol/l glucose and 10% (vol/vol) heat-inactivated FBS with selective islet picking at each medium renewal. Islets were then cultured for 1-3 weeks in RPMI containing ~5, 8, 10 or 20 mmol/l glucose before measurement of insulin secretion during culture, islet insulin and DNA content, beta cell apoptosis and cytosolic and mitochondrial glutathione redox state, and assessment of dynamic insulin secretion and upstream coupling events during acute stepwise stimulation with glucose [NAD(P)H autofluorescence, ATP/(ATP+ADP) ratio, electrical activity, cytosolic Ca2+ concentration ([Ca2+]c)]. RESULTS: Culture of ND-islets for 1-3 weeks at 8, 10 or 20 vs 5 mmol/l glucose did not significantly increase beta cell apoptosis or oxidative stress but decreased insulin content in a concentration-dependent manner and increased beta cell sensitivity to subsequent acute stimulation with glucose. Islet glucose responsiveness was higher after culture at 8 or 10 vs 5 mmol/l glucose and markedly reduced after culture at 20 vs 5 mmol/l glucose. In addition, the [Ca2+]c and insulin secretion responses to acute stepwise stimulation with glucose were no longer sigmoid but bell-shaped, with maximal stimulation at 5 or 10 mmol/l glucose and rapid sustained inhibition above that concentration. Such paradoxical inhibition was, however, no longer observed when islets were acutely depolarised by 30 mmol/l extracellular K+. The glucotoxic alterations of beta cell function were fully reversible after culture at 5 mmol/l glucose and were mimicked by pharmacological activation of glucokinase during culture at 5 mmol/l glucose. Similar results to those seen in ND-islets were obtained in T2D-islets, except that their rate of insulin secretion during culture at 8 and 20 mmol/l glucose was lower, their cytosolic glutathione oxidation increased after culture at 8 and 20 mmol/l glucose, and the alterations in GSIS and upstream coupling events were greater after culture at 8 mmol/l glucose. CONCLUSIONS/INTERPRETATION: Prolonged culture of human islets under moderate to severe glucotoxic conditions markedly increased their glucose sensitivity and revealed a bell-shaped acute glucose response curve for changes in [Ca2+]c and insulin secretion, with maximal stimulation at 5 or 10 mmol/l glucose and rapid inhibition above that concentration. This novel glucotoxic alteration may contribute to beta cell dysfunction in type 2 diabetes independently from a detectable increase in beta cell apoptosis.


Assuntos
Diabetes Mellitus Tipo 2 , Ilhotas Pancreáticas , Humanos , Glucose/metabolismo , Secreção de Insulina , Cálcio/metabolismo , Diabetes Mellitus Tipo 2/metabolismo , Ilhotas Pancreáticas/metabolismo , Insulina/metabolismo , Glutationa/metabolismo , Trifosfato de Adenosina/metabolismo , Células Cultivadas
4.
Diabetologia ; 66(7): 1306-1321, 2023 07.
Artigo em Inglês | MEDLINE | ID: mdl-36995380

RESUMO

AIMS/HYPOTHESIS: Wolfram syndrome is a rare autosomal recessive disorder caused by pathogenic variants in the WFS1 gene. It is characterised by insulin-dependent diabetes mellitus, optic nerve atrophy, diabetes insipidus, hearing loss and neurodegeneration. Considering the unmet treatment need for this orphan disease, this study aimed to evaluate the therapeutic potential of glucagon-like peptide 1 receptor (GLP-1R) agonists under wolframin (WFS1) deficiency with a particular focus on human beta cells and neurons. METHODS: The effect of the GLP-1R agonists dulaglutide and exenatide was examined in Wfs1 knockout mice and in an array of human preclinical models of Wolfram syndrome, including WFS1-deficient human beta cells, human induced pluripotent stem cell (iPSC)-derived beta-like cells and neurons from control individuals and individuals affected by Wolfram syndrome, and humanised mice. RESULTS: Our study shows that the long-lasting GLP-1R agonist dulaglutide reverses impaired glucose tolerance in WFS1-deficient mice, and that exenatide and dulaglutide improve beta cell function and prevent apoptosis in different human WFS1-deficient models including iPSC-derived beta cells from people with Wolfram syndrome. Exenatide improved mitochondrial function, reduced oxidative stress and prevented apoptosis in Wolfram syndrome iPSC-derived neural precursors and cerebellar neurons. CONCLUSIONS/INTERPRETATION: Our study provides novel evidence for the beneficial effect of GLP-1R agonists on WFS1-deficient human pancreatic beta cells and neurons, suggesting that these drugs may be considered as a treatment for individuals with Wolfram syndrome.


Assuntos
Células-Tronco Pluripotentes Induzidas , Células Secretoras de Insulina , Atrofia Óptica , Síndrome de Wolfram , Humanos , Animais , Camundongos , Síndrome de Wolfram/tratamento farmacológico , Síndrome de Wolfram/genética , Exenatida/uso terapêutico , Atrofia Óptica/patologia , Células Secretoras de Insulina/patologia , Camundongos Knockout
5.
Int J Mol Sci ; 24(11)2023 May 23.
Artigo em Inglês | MEDLINE | ID: mdl-37298086

RESUMO

Loss of the ß cell population is a crucial feature of type 2 diabetes. Restoring the ß cell mass by stimulating ß cell proliferation and preventing its apoptosis was proposed as a therapeutic approach to treating diabetes. Therefore, researchers have been increasingly interested in identifying exogenous factors that can stimulate ß cell proliferation in situ and in vitro. Adipokine chemerin, which is secreted from adipose tissue and the liver, has been identified as a chemokine that plays a critical role in the regulation of metabolism. In this study, we demonstrate that chemerin as a circulating adipokine promotes ß cell proliferation in vivo and in vitro. Chemerin serum levels and the expression of the main receptors within islets are highly regulated under a variety of challenging conditions, including obesity and type 2 diabetes. As compared to their littermates, mice overexpressing chemerin had a larger islet area and increased ß cell mass with both a normal and high-fat diet. Moreover, in chemerin-overexpressed mice, we observed improved mitochondrial homeostasis and increased insulin synthesis. In summary, our findings confirm the potential role of chemerin as an inducer of ß cell proliferation, and they provide novel insights into the helpful strategy to expand ß cell population.


Assuntos
Diabetes Mellitus Tipo 2 , Camundongos , Animais , Diabetes Mellitus Tipo 2/genética , Receptores de Quimiocinas/genética , Receptores de Quimiocinas/metabolismo , Peptídeos e Proteínas de Sinalização Intercelular/genética , Quimiocinas/metabolismo , Adipocinas , Homeostase , Proliferação de Células
6.
J Biol Chem ; 295(16): 5419-5426, 2020 04 17.
Artigo em Inglês | MEDLINE | ID: mdl-32156704

RESUMO

Hypersecretion of glucagon from pancreatic α-cells strongly contributes to diabetic hyperglycemia. Moreover, failure of α-cells to increase glucagon secretion in response to falling blood glucose concentrations compromises the defense against hypoglycemia, a common complication in diabetes therapy. However, the mechanisms underlying glucose regulation of glucagon secretion are poorly understood and likely involve both α-cell-intrinsic and intraislet paracrine signaling. Among paracrine factors, glucose-stimulated release of the GABA metabolite γ-hydroxybutyric acid (GHB) from pancreatic ß-cells might mediate glucose suppression of glucagon release via GHB receptors on α-cells. However, the direct effects of GHB on α-cell signaling and glucagon release have not been investigated. Here, we found that GHB (4-10 µm) lacked effects on the cytoplasmic concentrations of the secretion-regulating messengers Ca2+ and cAMP in mouse α-cells. Glucagon secretion from perifused mouse islets was also unaffected by GHB at both 1 and 7 mm glucose. The GHB receptor agonist 3-chloropropanoic acid and the antagonist NCS-382 had no effects on glucagon secretion and did not affect stimulation of secretion induced by a drop in glucose from 7 to 1 mm Inhibition of endogenous GHB formation with the GABA transaminase inhibitor vigabatrin also failed to influence glucagon secretion at 1 mm glucose and did not prevent the suppressive effect of 7 mm glucose. In human islets, GHB tended to stimulate glucagon secretion at 1 mm glucose, an effect mimicked by 3-chloropropanoic acid. We conclude that GHB does not mediate the inhibitory effect of glucose on glucagon secretion.


Assuntos
Células Secretoras de Glucagon/metabolismo , Glucagon/metabolismo , Glucose/metabolismo , Oxibato de Sódio/farmacologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Animais , Benzocicloeptenos/farmacologia , Células Cultivadas , Inibidores Enzimáticos/farmacologia , Feminino , GABAérgicos/farmacologia , Células Secretoras de Glucagon/efeitos dos fármacos , Glucose/farmacologia , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Pessoa de Meia-Idade , Propionatos/farmacologia , Vigabatrina/farmacologia
7.
Biochem Biophys Res Commun ; 568: 158-166, 2021 09 03.
Artigo em Inglês | MEDLINE | ID: mdl-34217973

RESUMO

The lactate dehydrogenase isoform A (LDHA) is a key metabolic enzyme that preferentially catalyzes the conversion of pyruvate to lactate. Whereas LDHA is highly expressed in many tissues, its expression is turned off in the differentiated adult ß-cell within the pancreatic islets. The repression of LDHA under normal physiological condition and its inappropriate upregulation under a diabetogenic environment is well-documented in rodent islets/ß-cells but little is known about LDHA expression in human islet cells and whether its abundance is altered under diabetic conditions. Analysis of public single-cell RNA-seq (sc-RNA seq) data as well as cell type-specific immunolabeling of human pancreatic islets showed that LDHA was mainly localized in human α-cells while it is expressed at a very low level in ß-cells. Furthermore, LDHA, both at mRNA and protein, as well as lactate production is upregulated in human pancreatic islets exposed to chronic high glucose treatment. Microscopic analysis of stressed human islets and autopsy pancreases from individuals with type 2 diabetes (T2D) showed LDHA upregulation mainly in human α-cells. Pharmacological inhibition of LDHA in isolated human islets enhanced insulin secretion under physiological conditions but did not significantly correct the deregulated secretion of insulin or glucagon under diabetic conditions.


Assuntos
Diabetes Mellitus Tipo 2/genética , Células Secretoras de Glucagon/metabolismo , L-Lactato Desidrogenase/genética , Células Cultivadas , Diabetes Mellitus Tipo 2/metabolismo , Células Secretoras de Glucagon/citologia , Glucose/metabolismo , Humanos , Secreção de Insulina , L-Lactato Desidrogenase/análise , L-Lactato Desidrogenase/metabolismo , RNA Mensageiro/análise , RNA Mensageiro/genética , Regulação para Cima
8.
Diabetologia ; 62(12): 2273-2286, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31624901

RESUMO

AIMS/HYPOTHESIS: The mechanisms responsible for beta cell compensation in obesity and for beta cell failure in type 2 diabetes are poorly defined. The mRNA levels of several metallothionein (MT) genes are upregulated in islets from individuals with type 2 diabetes, but their role in beta cells is not clear. Here we examined: (1) the temporal changes of islet Mt1 and Mt2 gene expression in mouse models of beta cell compensation and failure; and (2) the role of Mt1 and Mt2 in beta cell function and glucose homeostasis in mice. METHODS: Mt1 and Mt2 expression was assessed in islets from: (1) control lean (chow diet-fed) and diet-induced obese (high-fat diet-fed for 6 weeks) mice; (2) mouse models of diabetes (db/db mice) at 6 weeks old (prediabetes) and 16 weeks old (after diabetes onset) and age-matched db/+ (control) mice; and (3) obese non-diabetic ob/ob mice (16-week-old) and age-matched ob/+ (control) mice. MT1E, MT1X and MT2A expression was assessed in islets from humans with and without type 2 diabetes. Mt1-Mt2 double-knockout (KO) mice, transgenic mice overexpressing Mt1 under the control of its natural promoter (Tg-Mt1) and corresponding control mice were also studied. In MIN6 cells, MT1 and MT2 were inhibited by small interfering RNAs. mRNA levels were assessed by real-time RT-PCR, plasma insulin and islet MT levels by ELISA, glucose tolerance by i.p. glucose tolerance tests and overnight fasting-1 h refeeding tests, insulin tolerance by i.p. insulin tolerance tests, insulin secretion by RIA, cytosolic free Ca2+ concentration with Fura-2 leakage resistant (Fura-2 LR), cytosolic free Zn2+ concentration with Fluozin-3, and NAD(P)H by autofluorescence. RESULTS: Mt1 and Mt2 mRNA levels were reduced in islets of murine models of beta cell compensation, whereas they were increased in diabetic db/db mice. In humans, MT1X mRNA levels were significantly upregulated in islets from individuals with type 2 diabetes in comparison with non-diabetic donors, while MT1E and MT2A mRNA levels were unchanged. Ex vivo, islet Mt1 and Mt2 mRNA and MT1 and MT2 protein levels were downregulated after culture with glucose at 10-30 mmol/l vs 2-5 mmol/l, in association with increased insulin secretion. In human islets, mRNA levels of MT1E, MT1X and MT2A were downregulated by stimulation with physiological and supraphysiological levels of glucose. In comparison with wild-type (WT) mice, Mt1-Mt2 double-KO mice displayed improved glucose tolerance in association with increased insulin levels and enhanced insulin release from isolated islets. In contrast, isolated islets from Tg-Mt1 mice displayed impaired glucose-stimulated insulin secretion (GSIS). In both Mt1-Mt2 double-KO and Tg-Mt1 models, the changes in GSIS occurred despite similar islet insulin content, rises in cytosolic free Ca2+ concentration and NAD(P)H levels, or intracellular Zn2+ concentration vs WT mice. In MIN6 cells, knockdown of MT1 but not MT2 potentiated GSIS, suggesting that Mt1 rather than Mt2 affects beta cell function. CONCLUSIONS/INTERPRETATION: These findings implicate Mt1 as a negative regulator of insulin secretion. The downregulation of Mt1 is associated with beta cell compensation in obesity, whereas increased Mt1 accompanies beta cell failure and type 2 diabetes.


Assuntos
Glicemia/metabolismo , Diabetes Mellitus Tipo 2/metabolismo , Glucose/farmacologia , Secreção de Insulina/fisiologia , Células Secretoras de Insulina/metabolismo , Metalotioneína/metabolismo , Acrilatos , Animais , Linhagem Celular , Diabetes Mellitus Tipo 2/genética , Dieta Hiperlipídica , Feminino , Expressão Gênica , Teste de Tolerância a Glucose , Humanos , Insulina/sangue , Secreção de Insulina/efeitos dos fármacos , Ilhotas Pancreáticas/efeitos dos fármacos , Ilhotas Pancreáticas/metabolismo , Metalotioneína/genética , Camundongos , Obesidade/genética , Obesidade/metabolismo , Éteres Fenílicos , Estado Pré-Diabético/genética , Estado Pré-Diabético/metabolismo
9.
Diabetologia ; 59(9): 1855-9, 2016 09.
Artigo em Inglês | MEDLINE | ID: mdl-27421727

RESUMO

Type 2 diabetes is characterised by chronic hyperglycaemia and its incidence is highly increased by exaggerated food consumption. It results from a lack of insulin action/production, but growing evidence suggests that it might also involve hyperglucagonaemia and impaired control of glucose homeostasis by the brain. In recent years, the cocaine and amphetamine-regulated transcript (CART) peptides have generated a lot of interest in the battle against obesity because, via the brain, they exert anorexic effects and they increase energy expenditure. They are also localised, outside the brain, in discrete regions of the body and play a hormonal role in controlling various functions. In this issue of Diabetologia, the Wierup group (doi: 10.1007/s00125-016-4020-6 ) shows that CART peptides are expressed heterogeneously in islet cells of various species, including humans, and that their expression is upregulated in diabetes. The authors also shine a spotlight on some interesting effects of CART peptides on islet function, including stimulation of insulin secretion and inhibition of glucagon release. CART peptides would thus be at the centre of a cooperation between the brain and the endocrine pancreas to control glucose homeostasis. Although the mechanisms of action of CART peptides remain enigmatic because no specific receptor for these peptides has so far been discovered, their potential therapeutic use is evident and represents a new challenge for future research.


Assuntos
Ilhotas Pancreáticas/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Animais , Encéfalo/metabolismo , Diabetes Mellitus Tipo 2/metabolismo , Glucagon/metabolismo , Homeostase/genética , Homeostase/fisiologia , Humanos , Hiperglicemia/metabolismo , Insulina/metabolismo
10.
J Biol Chem ; 289(47): 32798-810, 2014 Nov 21.
Artigo em Inglês | MEDLINE | ID: mdl-25271154

RESUMO

Although the pancreatic duodenal homeobox 1 (Pdx-1) transcription factor is known to play an indispensable role in ß cell development and secretory function, recent data also implicate Pdx-1 in the maintenance of endoplasmic reticulum (ER) health. The sarco-endoplasmic reticulum Ca(2+) ATPase 2b (SERCA2b) pump maintains a steep Ca(2+) gradient between the cytosol and ER lumen. In models of diabetes, our data demonstrated loss of ß cell Pdx-1 that occurs in parallel with altered SERCA2b expression, whereas in silico analysis of the SERCA2b promoter revealed multiple putative Pdx-1 binding sites. We hypothesized that Pdx-1 loss under inflammatory and diabetic conditions leads to decreased SERCA2b levels and activity with concomitant alterations in ER health. To test this, siRNA-mediated knockdown of Pdx-1 was performed in INS-1 cells. The results revealed reduced SERCA2b expression and decreased ER Ca(2+), which was measured using fluorescence lifetime imaging microscopy. Cotransfection of human Pdx-1 with a reporter fused to the human SERCA2 promoter increased luciferase activity 3- to 4-fold relative to an empty vector control, and direct binding of Pdx-1 to the proximal SERCA2 promoter was confirmed by chromatin immunoprecipitation. To determine whether restoration of SERCA2b could rescue ER stress induced by Pdx-1 loss, Pdx1(+/-) mice were fed a high-fat diet. Isolated islets demonstrated an increased spliced-to-total Xbp1 ratio, whereas SERCA2b overexpression reduced the Xbp1 ratio to that of wild-type controls. Together, these results identify SERCA2b as a novel transcriptional target of Pdx-1 and define a role for altered ER Ca(2+) regulation in Pdx-1-deficient states.


Assuntos
Cálcio/metabolismo , Retículo Endoplasmático/metabolismo , Proteínas de Homeodomínio/genética , Células Secretoras de Insulina/metabolismo , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/genética , Transativadores/genética , Animais , Linhagem Celular Tumoral , Diabetes Mellitus/genética , Diabetes Mellitus/metabolismo , Expressão Gênica , Regulação da Expressão Gênica , Células HEK293 , Proteínas de Homeodomínio/metabolismo , Humanos , Immunoblotting , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Mutantes , Microscopia de Fluorescência/métodos , Células NIH 3T3 , Interferência de RNA , Reação em Cadeia da Polimerase Via Transcriptase Reversa , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/metabolismo , Transativadores/metabolismo
11.
Am J Physiol Heart Circ Physiol ; 307(8): H1120-33, 2014 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-25128166

RESUMO

Exposure of cardiomyocytes to high glucose concentrations (HG) stimulates reactive oxygen species (ROS) production by NADPH oxidase (NOX2). NOX2 activation is triggered by enhanced glucose transport through a sodium-glucose cotransporter (SGLT) but not by a stimulation of glucose metabolism. The aim of this work was to identify potential therapeutic approaches to counteract this glucotoxicity. In cultured adult rat cardiomyocytes incubated with 21 mM glucose (HG), AMP-activated protein kinase (AMPK) activation by A769662 or phenformin nearly suppressed ROS production. Interestingly, glucagon-like peptide 1 (GLP-1), a new antidiabetic drug, concomitantly induced AMPK activation and prevented the HG-mediated ROS production (maximal effect at 100 nM). α2-AMPK, the major isoform expressed in cardiomyocytes (but not α1-AMPK), was activated in response to GLP-1. Anti-ROS properties of AMPK activators were not related to changes in glucose uptake or glycolysis. Using in situ proximity ligation assay, we demonstrated that AMPK activation prevented the HG-induced p47phox translocation to caveolae, whatever the AMPK activators used. NOX2 activation by either α-methyl-d-glucopyranoside, a glucose analog transported through SGLT, or angiotensin II was also counteracted by GLP-1. The crucial role of AMPK in limiting HG-mediated NOX2 activation was demonstrated by overexpressing a constitutively active form of α2-AMPK using adenoviral infection. This overexpression prevented NOX2 activation in response to HG, whereas GLP-1 lost its protective action in α2-AMPK-deficient mouse cardiomyocytes. Under HG, the GLP-1/AMPK pathway inhibited PKC-ß2 phosphorylation, a key element mediating p47phox translocation. In conclusion, GLP-1 induces α2-AMPK activation and blocks HG-induced p47phox translocation to the plasma membrane, thereby preventing glucotoxicity.


Assuntos
Proteínas Quinases Ativadas por AMP/metabolismo , Peptídeo 1 Semelhante ao Glucagon/farmacologia , Glucose/farmacologia , Hipoglicemiantes/farmacologia , Miócitos Cardíacos/metabolismo , NADPH Oxidases/metabolismo , Proteínas Quinases Ativadas por AMP/genética , Animais , Compostos de Bifenilo , Células Cultivadas , Masculino , Glicoproteínas de Membrana/metabolismo , Metilglucosídeos/farmacologia , Miócitos Cardíacos/efeitos dos fármacos , NADPH Oxidase 2 , NADPH Oxidases/genética , Fenformin/farmacologia , Proteína Quinase C/metabolismo , Transporte Proteico , Pironas/farmacologia , Ratos , Ratos Wistar , Espécies Reativas de Oxigênio/metabolismo , Tiofenos/farmacologia
12.
Genome Res ; 21(1): 95-105, 2011 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-21088282

RESUMO

We report on a hitherto poorly characterized class of genes that are expressed in all tissues, except in one. Often, these genes have been classified as housekeeping genes, based on their nearly ubiquitous expression. However, the specific repression in one tissue defines a special class of "disallowed genes." In this paper, we used the intersection-union test to screen for such genes in a multi-tissue panel of genome-wide mRNA expression data. We propose that disallowed genes need to be repressed in the specific target tissue to ensure correct tissue function. We provide mechanistic data of repression with two metabolic examples, exercise-induced inappropriate insulin release and interference with ketogenesis in liver. Developmentally, this repression is established during tissue maturation in the early postnatal period involving epigenetic changes in histone methylation. In addition, tissue-specific expression of microRNAs can further diminish these repressed mRNAs. Together, we provide a systematic analysis of tissue-specific repression of housekeeping genes, a phenomenon that has not been studied so far on a genome-wide basis and, when perturbed, can lead to human disease.


Assuntos
Diferenciação Celular , Regulação da Expressão Gênica no Desenvolvimento , Fígado/metabolismo , Pâncreas/metabolismo , Animais , Epigenômica , Feminino , Ilhotas Pancreáticas/citologia , Ilhotas Pancreáticas/metabolismo , Lactato Desidrogenases/genética , Lactato Desidrogenases/metabolismo , Fígado/citologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , MicroRNAs/genética , MicroRNAs/metabolismo , Transportadores de Ácidos Monocarboxílicos/genética , Transportadores de Ácidos Monocarboxílicos/metabolismo , Análise de Sequência com Séries de Oligonucleotídeos , Especificidade de Órgãos , Pâncreas/citologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos , Simportadores/genética , Simportadores/metabolismo
13.
Islets ; 16(1): 2298518, 2024 Dec 31.
Artigo em Inglês | MEDLINE | ID: mdl-38267218

RESUMO

Pancreatic islet transplantation is a promising treatment for type 1 diabetes, but the survival and function of transplanted islets are hindered by the loss of extracellular matrix (ECM) during islet isolation and by low oxygenation upon implantation. This study aimed to evaluate the impact of hypoxia on ECM using a cutting-edge imaging approach based on tissue clearing and 3D microscopy. Human and rat islets were cultured under normoxic (O2 21%) or hypoxic (O2 1%) conditions. Immunofluorescence staining targeting insulin, glucagon, CA9 (a hypoxia marker), ECM proteins (collagen 4, fibronectin, laminin), and E-cadherin (intercellular adhesion protein) was performed on fixed whole islets. The cleared islets were imaged using Light Sheet Fluorescence Microscopy (LSFM) and digitally analyzed. The volumetric analysis of target proteins did not show significant differences in abundance between the experimental groups. However, 3D projections revealed distinct morphological features that differentiated normoxic and hypoxic islets. Under normoxic conditions, ECM could be found throughout the islets. Hypoxic islets exhibited areas of scattered nuclei and central clusters of ECM proteins, indicating central necrosis. E-cadherin was absent in these areas. Our results, demonstrating a diminution of islets' functional mass in hypoxia, align with the functional decline observed in transplanted islets experiencing low oxygenation after grafting. This study provides a methodology combining tissue clearing, multiplex immunofluorescence, Light Sheet Fluorescence Microscopy, and digital image analysis to investigate pancreatic islet morphology. This 3D approach allowed us to highlight ECM organizational changes during hypoxia from a morphological perspective.


Assuntos
Ilhotas Pancreáticas , Humanos , Animais , Ratos , Microscopia de Fluorescência , Matriz Extracelular , Hipóxia , Proteínas da Matriz Extracelular , Caderinas
14.
Pflugers Arch ; 465(4): 543-54, 2013 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-23149488

RESUMO

Pancreatic ß cells respond to increases in glucose concentration with enhanced metabolism, the closure of ATP-sensitive K(+) channels and electrical spiking. The latter results in oscillatory Ca(2+) influx through voltage-gated Ca(2+) channels and the activation of insulin release. The relationship between changes in cytosolic and mitochondrial free calcium concentration ([Ca(2+)]cyt and [Ca(2+)]mit, respectively) during these cycles is poorly understood. Importantly, the activation of Ca(2+)-sensitive intramitochondrial dehydrogenases, occurring alongside the stimulation of ATP consumption required for Ca(2+) pumping and other processes, may exert complex effects on cytosolic ATP/ADP ratios and hence insulin secretion. To explore the relationship between these parameters in single primary ß cells, we have deployed cytosolic (Fura red, Indo1) or green fluorescent protein-based recombinant-targeted (Pericam, 2mt8RP for mitochondria; D4ER for the ER) probes for Ca(2+) and cytosolic ATP/ADP (Perceval) alongside patch-clamp electrophysiology. We demonstrate that: (1) blockade of mitochondrial Ca(2+) uptake by shRNA-mediated silencing of the uniporter MCU attenuates glucose- and essentially blocks tolbutamide-stimulated, insulin secretion; (2) during electrical stimulation, mitochondria decode cytosolic Ca(2+) oscillation frequency as stable increases in [Ca(2+)]mit and cytosolic ATP/ADP; (3) mitochondrial Ca(2+) uptake rates remained constant between individual spikes, arguing against activity-dependent regulation ("plasticity") and (4) the relationship between [Ca(2+)]cyt and [Ca(2+)]mit is essentially unaffected by changes in endoplasmic reticulum Ca(2+) ([Ca(2+)]ER). Our findings thus highlight new aspects of Ca(2+) signalling in ß cells of relevance to the actions of both glucose and sulphonylureas.


Assuntos
Trifosfato de Adenosina/biossíntese , Sinalização do Cálcio , Cálcio/metabolismo , Células Secretoras de Insulina/metabolismo , Mitocôndrias/metabolismo , Potenciais de Ação , Difosfato de Adenosina/metabolismo , Animais , Canais de Cálcio/metabolismo , Células Cultivadas , Citosol/metabolismo , Retículo Endoplasmático/metabolismo , Feminino , Glucose/metabolismo , Insulina/metabolismo , Células Secretoras de Insulina/fisiologia , Camundongos
15.
Biochem J ; 441(3): 971-8, 2012 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-22050124

RESUMO

Using the ROS (reactive oxygen species)-sensitive fluorescent dyes dichlorodihydrofluorescein and dihydroethidine, previous studies yielded opposite results about the glucose regulation of oxidative stress in insulin-secreting pancreatic ß-cells. In the present paper, we used the ratiometric fluorescent proteins HyPer and roGFP1 (redox-sensitive green fluorescent protein 1) targeted to mitochondria [mt-HyPer (mitochondrial HyPer)/mt-roGFP1 (mitochondrial roGFP1)] to monitor glucose-induced changes in mitochondrial hydrogen peroxide concentration and glutathione redox state in adenovirus-infected rat islet cell clusters. Because of the reported pH sensitivity of HyPer, the results were compared with those obtained with the mitochondrial pH sensors mt-AlpHi and mt-SypHer. The fluorescence ratio of the mitochondrial probes slowly decreased (mt-HyPer) or increased (mt-roGFP1) in the presence of 10 mmol/l glucose. Besides its expected sensitivity to H2O2, mt-HyPer was also highly pH sensitive. In agreement, changes in mitochondrial metabolism similarly affected mt-HyPer, mt-AlpHi and mt-SypHer fluorescence signals. In contrast, the mt-roGFP1 fluorescence ratio was only slightly affected by pH and reversibly increased when glucose was lowered from 10 to 2 mmol/l. This increase was abrogated by the catalytic antioxidant Mn(III) tetrakis (4-benzoic acid) porphyrin but not by N-acetyl-L-cysteine. In conclusion, due to its pH sensitivity, mt-HyPer is not a reliable indicator of mitochondrial H2O2 in ß-cells. In contrast, the mt-roGFP1 fluorescence ratio monitors changes in ß-cell mitochondrial glutathione redox state with little interference from pH changes. Our results also show that glucose acutely decreases rather than increases mitochondrial thiol oxidation in rat ß-cells.


Assuntos
Glutationa/análise , Proteínas de Fluorescência Verde/análise , Peróxido de Hidrogênio/análise , Células Secretoras de Insulina/química , Medições Luminescentes/métodos , Mitocôndrias/química , Animais , Glutationa/metabolismo , Proteínas de Fluorescência Verde/metabolismo , Células HEK293 , Humanos , Peróxido de Hidrogênio/metabolismo , Concentração de Íons de Hidrogênio , Células Secretoras de Insulina/metabolismo , Células Secretoras de Insulina/ultraestrutura , Cinética , Masculino , Mitocôndrias/metabolismo , Concentração Osmolar , Oxirredução , Ratos , Ratos Wistar , Espécies Reativas de Oxigênio/metabolismo , Sensibilidade e Especificidade
16.
Proc Natl Acad Sci U S A ; 107(11): 5208-13, 2010 Mar 16.
Artigo em Inglês | MEDLINE | ID: mdl-20194741

RESUMO

Glucose homeostasis is critically dependent on insulin release from pancreatic beta-cells, which is strictly regulated by glucose-induced oscillations in membrane potential (V(m)) and the cytosolic calcium level ([Ca(2+)](cyt)). We propose that TRPM5, a Ca(2+)-activated monovalent cation channel, is a positive regulator of glucose-induced insulin release. Immunofluorescence revealed expression of TRPM5 in pancreatic islets. A Ca(2+)-activated nonselective cation current with TRPM5-like properties is significantly reduced in Trpm5(-/-) cells. Ca(2+)-imaging and electrophysiological analysis show that glucose-induced oscillations of V(m) and [Ca(2+)](cyt) have on average a reduced frequency in Trpm5(-/-) islets, specifically due to a lack of fast oscillations. As a consequence, glucose-induced insulin release from Trpm5(-/-) pancreatic islets is significantly reduced, resulting in an impaired glucose tolerance in Trpm5(-/-) mice.


Assuntos
Sinalização do Cálcio/efeitos dos fármacos , Glucose/farmacologia , Ilhotas Pancreáticas/efeitos dos fármacos , Ilhotas Pancreáticas/metabolismo , Canais de Cátion TRPM/deficiência , Animais , Cátions , Regulação da Expressão Gênica/efeitos dos fármacos , Teste de Tolerância a Glucose , Insulina/metabolismo , Secreção de Insulina , Espaço Intracelular/efeitos dos fármacos , Espaço Intracelular/metabolismo , Ativação do Canal Iônico/efeitos dos fármacos , Ilhotas Pancreáticas/citologia , Potenciais da Membrana/efeitos dos fármacos , Camundongos , Fenótipo , Canais de Cátion TRPM/genética , Canais de Cátion TRPM/metabolismo
17.
Cell Rep ; 42(11): 113326, 2023 11 28.
Artigo em Inglês | MEDLINE | ID: mdl-37897727

RESUMO

Glucagon-like peptide 1 (GLP-1R) and glucose-dependent insulinotropic polypeptide (GIPR) receptors are G-protein-coupled receptors involved in glucose homeostasis. Diabetogenic conditions decrease ß-arrestin 2 (ARRB2) levels in human islets. In mouse ß cells, ARRB2 dampens insulin secretion by partially uncoupling cyclic AMP (cAMP)/protein kinase A (PKA) signaling at physiological doses of GLP-1, whereas at pharmacological doses, the activation of extracellular signal-related kinase (ERK)/cAMP-responsive element-binding protein (CREB) requires ARRB2. In contrast, GIP-potentiated insulin secretion needs ARRB2 in mouse and human islets. The GIPR-ARRB2 axis is not involved in cAMP/PKA or ERK signaling but does mediate GIP-induced F-actin depolymerization. Finally, the dual GLP-1/GIP agonist tirzepatide does not require ARRB2 for the potentiation of insulin secretion. Thus, ARRB2 plays distinct roles in regulating GLP-1R and GIPR signaling, and we highlight (1) its role in the physiological context and the possible functional consequences of its decreased expression in pathological situations such as diabetes and (2) the importance of assessing the signaling pathways engaged by the agonists (biased/dual) for therapeutic purposes.


Assuntos
Células Secretoras de Insulina , Camundongos , Humanos , Animais , Células Secretoras de Insulina/metabolismo , Peptídeo 1 Semelhante ao Glucagon/metabolismo , Insulina/metabolismo , beta-Arrestina 2/metabolismo , beta-Arrestina 1/metabolismo , Glucose/metabolismo
18.
Mol Metab ; 61: 101495, 2022 07.
Artigo em Inglês | MEDLINE | ID: mdl-35421610

RESUMO

OBJECTIVE: The mechanisms by which glucose stimulates insulin secretion from ß-cells are well established and involve inhibition of ATP-sensitive K+ (KATP) channels, followed by a rise in [Ca2+]c that triggers exocytosis. However, the mechanisms by which glucose controls glucagon release from α-cells are much less known. In particular, it is debated whether the sugar controls glucagon secretion by changing α-cell [Ca2+]c, and whether KATP channels or paracrine factors are involved. The present study addresses these issues. METHODS: We tested the effect of a decrease or an increase of glucose concentration (Gx, with x = concentration in mM) on α-cell [Ca2+]c and glucagon secretion. α-cell [Ca2+]c was monitored using GluCreGCaMP6f mice expressing the Ca2+-sensitive fluorescent protein, GCaMP6f, specifically in α-cells. [Ca2+]c was compared between dispersed α-cells and α-cells within islets to evaluate the potential contribution of an indirect effect of glucose. The same protocols were used for experiments of glucagon secretion from whole islets and [Ca2+]c measurements to test if changes in glucagon release mirror those in α-cell [Ca2+]c. RESULTS: Blockade of KATP channels by sulfonylureas (tolbutamide 100 µM or gliclazide 25 µM) strongly increased [Ca2+]c in both dispersed α-cells and α-cells within islets. By contrast, glucose had no effect on [Ca2+]c in dispersed α-cells, whereas it affected it in α-cells within islets. The effect of glucose was however different in islets expressing (Sst+/+) or not somatostatin (SST) (Sst-/-). Decreasing glucose concentration from G7 to G1 modestly increased α-cell [Ca2+]c, but to a slightly larger extent in Sst+/+ islets than in Sst-/- islets. This G1-induced [Ca2+]c rise was also observed in the continuous presence of sulfonylureas in both Sst+/+ and Sst-/- islets. Increasing glucose concentration from G7 to G20 did not affect α-cell [Ca2+]c in Sst+/+ islets which remained low, whereas it strongly increased it in Sst-/- islets. The observations that this increase was seen only in α-cells within islets but never in dispersed α-cells and that it was abrogated by the gap junction inhibitor, carbenoxolone, point to an indirect effect of G20 and suggest that, in Sst-/- islets, G20-stimulated ß-cells entrain α-cells whereas, in Sst+/+ islets, the concomitant release of SST keeps α-cell [Ca2+]c at low levels. The [Ca2+]c lowering effect of endogenous SST is also supported by the observation that SST receptor antagonists (SSTR2/3) increased [Ca2+]c in α-cells from Sst+/+ islets. All these [Ca2+]c changes induced parallel changes in glucagon release. To test if glucose also controls glucagon release independently of [Ca2+]c changes, additional experiments were performed in the continuous presence of 30 mM K+ and the KATP channel opener diazoxide (250 µM). In these conditions, α-cell [Ca2+]c within islets was elevated and its steady-state level was unaffected by glucose. However, decreasing the glucose concentration from G7 to G1 stimulated glucagon release whereas increasing it from G1 to G15 inhibited it. These effects were also evident in Sst-/- islets, and opposite to those on insulin secretion. CONCLUSIONS: We propose a model according to which glucose controls α-cell [Ca2+]c and glucagon secretion through multiple mechanisms. Increasing the glucose concentration modestly decreases [Ca2+]c in α-cells independently of their KATP channels and partly via SST. The involvement of SST increases with the glucose concentration, and one major effect of SST is to keep α-cell [Ca2+]c at low levels by counteracting the effect of an entrainment of α-cells by ß-cells when ß-cells become stimulated by glucose. All these [Ca2+]c changes induce parallel changes in glucagon release. Glucose also decreases the efficacy of Ca2+ on exocytosis by an attenuating pathway that is opposite to the well-established amplifying pathway controlling insulin release in ß-cells.


Assuntos
Cálcio , Exocitose , Células Secretoras de Glucagon , Glucagon , Glucose , Somatostatina , Trifosfato de Adenosina , Animais , Cálcio/metabolismo , Cátions Bivalentes/metabolismo , Exocitose/fisiologia , Glucagon/biossíntese , Glucagon/metabolismo , Células Secretoras de Glucagon/metabolismo , Glucose/análise , Glucose/metabolismo , Canais KATP/metabolismo , Camundongos , Somatostatina/metabolismo
19.
Front Cell Dev Biol ; 10: 967765, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-36060810

RESUMO

In vitro differentiation of human induced pluripotent stem cells (iPSCs) into beta cells represents an important cell source for diabetes research. Here, we fully characterized iPSC-derived beta cell function in vitro and in vivo in humanized mice. Using a 7-stage protocol, human iPSCs were differentiated into islet-like aggregates with a yield of insulin-positive beta cells comparable to that of human islets. The last three stages of differentiation were conducted with two different 3D culture systems, rotating suspension or static microwells. In the latter, homogeneously small-sized islet-like aggregates were obtained, while in rotating suspension size was heterogeneous and aggregates often clumped. In vitro function was assessed by glucose-stimulated insulin secretion, NAD(P)H and calcium fluctuations. Stage 7 aggregates slightly increased insulin release in response to glucose in vitro. Aggregates were transplanted under the kidney capsule of NOD-SCID mice to allow for further in vivo beta cell maturation. In transplanted mice, grafts showed glucose-responsiveness and maintained normoglycemia after streptozotocin injection. In situ kidney perfusion assays showed modulation of human insulin secretion in response to different secretagogues. In conclusion, iPSCs differentiated with equal efficiency into beta cells in microwells compared to rotating suspension, but the former had a higher experimental success rate. In vitro differentiation generated aggregates lacking fully mature beta cell function. In vivo, beta cells acquired the functional characteristics typical of human islets. With this technology an unlimited supply of islet-like organoids can be generated from human iPSCs that will be instrumental to study beta cell biology and dysfunction in diabetes.

20.
Mol Metab ; 53: 101268, 2021 11.
Artigo em Inglês | MEDLINE | ID: mdl-34118477

RESUMO

OBJECTIVE: Glucagon is secreted by pancreatic α-cells in response to hypoglycemia and its hyperglycemic effect helps to restore normal blood glucose. Insulin and somatostatin (SST) secretions from ß- and δ-cells, respectively, are stimulated by glucose by mechanisms involving an inhibition of their ATP-sensitive K+ (KATP) channels, leading to an increase in [Ca2+]c that triggers exocytosis. Drugs that close KATP channels, such as sulfonylureas, are used to stimulate insulin release in type 2 diabetic patients. α-cells also express KATP channels. However, the mechanisms by which sulfonylureas control glucagon secretion are still largely debated and were addressed in the present study. In particular, we studied the effects of KATP channel blockers on α-cell [Ca2+]c and glucagon secretion in the presence of a low (1 mM) or a high (15 mM) glucose concentration and evaluated the role of SST in these effects. METHODS: Using a transgenic mouse model expressing the Ca2+-sensitive fluorescent protein, GCaMP6f, specifically in α-cells, we measured [Ca2+]c in α-cells either dispersed or within whole islets (by confocal microscopy). By measuring [Ca2+]c in α-cells within islets and glucagon secretion using the same perifusion protocols, we tested whether glucagon secretion correlated with changes in [Ca2+]c in response to sulfonylureas. We studied the role of SST in the effects of sulfonylureas using multiple approaches including genetic ablation of SST, or application of SST-14 and SST receptor antagonists. RESULTS: Application of the sulfonylureas, tolbutamide, or gliclazide, to a medium containing 1 mM or 15 mM glucose increased [Ca2+]c in α-cells by a direct effect as in ß-cells. At low glucose, sulfonylureas inhibited glucagon secretion of islets despite the rise in α-cell [Ca2+]c that they triggered. This glucagonostatic effect was indirect and attributed to SST because, in the islets of SST-knockout mice, sulfonylureas induced a stimulation of glucagon secretion which correlated with an increase in α-cell [Ca2+]c. Experiments with exogenous SST-14 and SST receptor antagonists indicated that the glucagonostatic effect of sulfonylureas mainly resulted from an inhibition of the efficacy of cytosolic Ca2+ on exocytosis. Although SST-14 was also able to inhibit glucagon secretion by decreasing α-cell [Ca2+]c, no decrease in [Ca2+]c occurred during sulfonylurea application because it was largely counterbalanced by the direct stimulatory effect of these drugs on α-cell [Ca2+]c. At high glucose, i.e., in conditions where glucagon release was already low, sulfonylureas stimulated glucagon secretion because their direct stimulatory effect on α-cells exceeded the indirect effect by SST. Our results also indicated that, unexpectedly, SST-14 poorly decreased the efficacy of Ca2+ on exocytosis in ß-cells. CONCLUSIONS: Sulfonylureas exert two opposite actions on α-cells: a direct stimulation as in ß-cells and an indirect inhibition by SST. This suggests that any alteration of SST paracrine influence, as described in diabetes, will modify the effect of sulfonylureas on glucagon release. In addition, we suggest that δ-cells inhibit α-cells more efficiently than ß-cells.


Assuntos
Cálcio/metabolismo , Gliclazida/farmacologia , Células Secretoras de Glucagon/efeitos dos fármacos , Glucagon/metabolismo , Canais KATP/antagonistas & inibidores , Bloqueadores dos Canais de Potássio/farmacologia , Somatostatina/farmacologia , Tolbutamida/farmacologia , Animais , Gliclazida/química , Células Secretoras de Glucagon/metabolismo , Canais KATP/metabolismo , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Bloqueadores dos Canais de Potássio/química , Somatostatina/química , Tolbutamida/química
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