RESUMO
AIMS/HYPOTHESIS: The Gq-coupled 5-hydroxytryptamine 2B (5-HT2B) receptor is known to regulate the proliferation of islet beta cells during pregnancy. However, the role of serotonin in the control of insulin release is still controversial. The aim of the present study was to explore the role of the 5-HT2B receptor in the regulation of insulin secretion in mouse and human islets, as well as in clonal INS-1(832/13) cells. METHODS: Expression of HTR2B mRNA and 5-HT2B protein was examined with quantitative real-time PCR, RNA sequencing and immunohistochemistry. α-Methyl serotonin maleate salt (AMS), a serotonin receptor agonist, was employed for robust 5-HT2B receptor activation. Htr2b was silenced with small interfering RNA in INS-1(832/13) cells. Insulin secretion, Ca(2+) response and oxygen consumption rate were determined. RESULTS: Immunohistochemistry revealed that 5-HT2B is expressed in human and mouse islet beta cells. Activation of 5-HT2B receptors by AMS enhanced glucose-stimulated insulin secretion (GSIS) in human and mouse islets as well as in INS-1(832/13) cells. Silencing Htr2b in INS-1(832/13) cells led to a 30% reduction in GSIS. 5-HT2B receptor activation produced robust, regular and sustained Ca(2+) oscillations in mouse islets with an increase in both peak distance (period) and time in the active phase as compared with control. Enhanced insulin secretion and Ca(2+) changes induced by AMS coincided with an increase in oxygen consumption in INS-1(832/13) cells. CONCLUSIONS/INTERPRETATION: Activation of 5-HT2B receptors stimulates GSIS in beta cells by triggering downstream changes in cellular Ca(2+) flux that enhance mitochondrial metabolism. Our findings suggest that serotonin and the 5-HT2B receptor stimulate insulin release.
Assuntos
Glucose/farmacologia , Ilhotas Pancreáticas/metabolismo , Receptor 5-HT2B de Serotonina/metabolismo , Animais , Células Cultivadas , Feminino , Humanos , Técnicas In Vitro , Ilhotas Pancreáticas/efeitos dos fármacos , Camundongos , Receptor 5-HT2B de Serotonina/genéticaRESUMO
Lipotoxicity is a presumed pathogenetic process whereby elevated circulating and stored lipids in type 2 diabetes cause pancreatic ß-cell failure. To resolve the underlying molecular mechanisms, we exposed clonal INS-1 832/13 ß-cells to palmitate for 48 h. We observed elevated basal insulin secretion but impaired glucose-stimulated insulin secretion in palmitate-exposed cells. Glucose utilization was unchanged, palmitate oxidation was increased, and oxygen consumption was impaired. Halting exposure of the clonal INS-1 832/13 ß-cells to palmitate largely recovered all of the lipid-induced functional changes. Metabolite profiling revealed profound but reversible increases in cellular lipids. Glucose-induced increases in tricarboxylic acid cycle intermediates were attenuated by exposure to palmitate. Analysis of gene expression by microarray showed increased expression of 982 genes and decreased expression of 1032 genes after exposure to palmitate. Increases were seen in pathways for steroid biosynthesis, cell cycle, fatty acid metabolism, DNA replication, and biosynthesis of unsaturated fatty acids; decreases occurred in the aminoacyl-tRNA synthesis pathway. The activity of histone-modifying enzymes and histone modifications of differentially expressed genes were reversibly altered upon exposure to palmitate. Thus, Insig1, Lss, Peci, Idi1, Hmgcs1, and Casr were subject to epigenetic regulation. Our analyses demonstrate that coordinate changes in histone modifications, mRNA levels, and metabolite profiles accompanied functional adaptations of clonal ß-cells to lipotoxicity. It is highly likely that these changes are pathogenetic, accounting for loss of glucose responsiveness and perturbed insulin secretion.
Assuntos
Inibidores Enzimáticos/efeitos adversos , Epigênese Genética/efeitos dos fármacos , Histonas/metabolismo , Células Secretoras de Insulina/metabolismo , Ácido Palmítico/efeitos adversos , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , RNA Mensageiro/metabolismo , Animais , Linhagem Celular Tumoral , Ciclo do Ácido Cítrico/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Histonas/genética , Humanos , Insulina/genética , Insulina/metabolismo , Secreção de Insulina , Consumo de Oxigênio/efeitos dos fármacos , Ácido Palmítico/farmacologia , RNA Mensageiro/genética , RatosRESUMO
Insulin secretion is coupled with changes in ß-cell metabolism. To define this process, 195 putative metabolites, mitochondrial respiration, NADP+, NADPH and insulin secretion were measured within 15 min of stimulation of clonal INS-1 832/13 ß-cells with glucose. Rapid responses in the major metabolic pathways of glucose occurred, involving several previously suggested metabolic coupling factors. The complexity of metabolite changes observed disagreed with the concept of one single metabolite controlling insulin secretion. The complex alterations in metabolite levels suggest that a coupling signal should reflect large parts of the ß-cell metabolic response. This was fulfilled by the NADPH/NADP+ ratio, which was elevated (8-fold; P<0.01) at 6 min after glucose stimulation. The NADPH/NADP+ ratio paralleled an increase in ribose 5-phosphate (>2.5-fold; P<0.001). Inhibition of the pentose phosphate pathway by trans-dehydroepiandrosterone (DHEA) suppressed ribose 5-phosphate levels and production of reduced glutathione, as well as insulin secretion in INS-1 832/13 ß-cells and rat islets without affecting ATP production. Metabolite profiling of rat islets confirmed the glucose-induced rise in ribose 5-phosphate, which was prevented by DHEA. These findings implicate the pentose phosphate pathway, and support a role for NADPH and glutathione, in ß-cell stimulus-secretion coupling.
Assuntos
Células Secretoras de Insulina/metabolismo , Insulina/metabolismo , Metabolômica/métodos , Via de Pentose Fosfato/fisiologia , Animais , Respiração Celular/fisiologia , Células Cultivadas , Glucose/farmacologia , Secreção de Insulina , Células Secretoras de Insulina/química , Ilhotas Pancreáticas/metabolismo , Masculino , Metaboloma , Mitocôndrias/metabolismo , Mitocôndrias/fisiologia , Via de Pentose Fosfato/efeitos dos fármacos , Ratos , Ratos Wistar , Via Secretória/efeitos dos fármacosRESUMO
Circadian rhythms are ubiquitous in biological systems and regulate metabolic processes throughout the body. Misalliance of these circadian rhythms and the systems they regulate has a profound impact on hormone levels and increases risk of developing metabolic diseases. Melatonin, a hormone secreted by the pineal gland, is one of the major signaling molecules used by the master circadian oscillator to entrain downstream circadian rhythms. Several recent genetic studies have pointed out that a common variant in the gene that encodes the melatonin receptor 2 (MTNR1B) is associated with impaired glucose homeostasis, reduced insulin secretion, and an increased risk of developing type 2 diabetes. Here, we try to review the role of this receptor and its signaling pathways in respect to glucose homeostasis and development of the disease.
Assuntos
Ritmo Circadiano/genética , Diabetes Mellitus Tipo 2/genética , Insulina/sangue , Melatonina/biossíntese , Receptor MT1 de Melatonina/genética , Receptor MT2 de Melatonina/genética , Diabetes Mellitus Tipo 2/sangue , Feminino , Variação Genética , Humanos , Insulina/metabolismo , Secreção de Insulina , Células Secretoras de Insulina , Masculino , Receptor MT1 de Melatonina/sangue , Receptor MT2 de Melatonina/sangue , Fatores de Risco , Transdução de SinaisRESUMO
Melatonin has multiple receptor-dependent and receptor-independent functions. At the cell membrane, melatonin interacts with its receptors MT1 and MT2, which are expressed in numerous tissues. Genome-wide association studies have recently shown that the MTNR1B/MT2 receptor may be involved in the pathogenesis of type 2 diabetes mellitus. In line with these findings, expression of melatonin receptors has been shown in mouse, rat, and human pancreatic islets. MT1 and MT2 are G-protein-coupled receptors and are proposed to exert inhibitory effects on insulin secretion. Here, we show by immunocytochemistry that these membrane melatonin receptors have distinct locations in the mouse islet. MT1 is expressed in α-cells while MT2 is located to the ß-cells. These findings help to unravel the complex machinery underlying melatonin's role in the regulation of islet function.
Assuntos
Receptores de Melatonina/metabolismo , Animais , Feminino , Imuno-Histoquímica , Ilhotas Pancreáticas/metabolismo , Masculino , Camundongos , Receptor MT1 de Melatonina/genética , Receptor MT1 de Melatonina/metabolismo , Receptor MT2 de Melatonina/genética , Receptor MT2 de Melatonina/metabolismo , Receptores de Melatonina/genética , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Type 2 diabetes (T2D) is a global pandemic. Genome-wide association studies (GWASs) have identified >100 genetic variants associated with the disease, including a common variant in the melatonin receptor 1 b gene (MTNR1B). Here, we demonstrate increased MTNR1B expression in human islets from risk G-allele carriers, which likely leads to a reduction in insulin release, increasing T2D risk. Accordingly, in insulin-secreting cells, melatonin reduced cAMP levels, and MTNR1B overexpression exaggerated the inhibition of insulin release exerted by melatonin. Conversely, mice with a disruption of the receptor secreted more insulin. Melatonin treatment in a human recall-by-genotype study reduced insulin secretion and raised glucose levels more extensively in risk G-allele carriers. Thus, our data support a model where enhanced melatonin signaling in islets reduces insulin secretion, leading to hyperglycemia and greater future risk of T2D. The findings also imply that melatonin physiologically serves to inhibit nocturnal insulin release.
Assuntos
Diabetes Mellitus Tipo 2/metabolismo , Melatonina/metabolismo , Transdução de Sinais , Animais , AMP Cíclico/metabolismo , Predisposição Genética para Doença , Glucose/metabolismo , Heterozigoto , Humanos , Insulina/metabolismo , Secreção de Insulina , Células Secretoras de Insulina/efeitos dos fármacos , Células Secretoras de Insulina/metabolismo , Melatonina/farmacologia , Camundongos Knockout , Polimorfismo de Nucleotídeo Único/genética , Locos de Características Quantitativas/genética , Receptores de Melatonina/genética , Fatores de Risco , Transdução de Sinais/efeitos dos fármacosRESUMO
Nearly all mammalian cells express a set of genes known as clock genes. These regulate the circadian rhythm of cellular processes by means of negative and positive autoregulatory feedback loops of transcription and translation. Recent genomewide association studies have demonstrated an association between a polymorphism near the circadian clock gene CRY2 and elevated fasting glucose. To determine whether clock genes could play a pathogenetic role in the disease, we examined messenger RNA (mRNA) expression of core clock genes in human islets from donors with or without type 2 diabetes mellitus. Microarray and quantitative real-time polymerase chain reaction analyses were used to assess expression of the core clock genes CLOCK, BMAL-1, PER1 to 3, and CRY1 and 2 in human islets. Insulin secretion and insulin content in human islets were measured by radioimmunoassay. The mRNA levels of PER2, PER3, and CRY2 were significantly lower in islets from donors with type 2 diabetes mellitus. To investigate the functional relevance of these clock genes, we correlated their expression to insulin content and glycated hemoglobin levels: mRNA levels of PER2 (ρ = 0.33, P = .012), PER3 (ρ = 0.30, P = .023), and CRY2 (ρ = 0.37, P = .0047) correlated positively with insulin content. Of these genes, expression of PER3 and CRY2 correlated negatively with glycated hemoglobin levels (ρ = -0.44, P = .0012; ρ = -0.28, P = .042). Furthermore, in an in vitro model mimicking pathogenetic conditions, the PER3 mRNA level was reduced in human islets exposed to 16.7 mmol/L glucose per 1 mmol/L palmitate for 48 hours (P = .003). Core clock genes are regulated in human islets. The data suggest that perturbations of circadian clock components may contribute to islet pathophysiology in human type 2 diabetes mellitus.
Assuntos
Proteínas CLOCK/genética , Regulação da Expressão Gênica , Ilhotas Pancreáticas/metabolismo , Adulto , Idoso , Índice de Massa Corporal , Criptocromos/análise , Diabetes Mellitus Tipo 2/genética , Diabetes Mellitus Tipo 2/metabolismo , Diabetes Mellitus Tipo 2/fisiopatologia , Feminino , Hemoglobinas Glicadas/análise , Humanos , Insulina/análise , Insulina/metabolismo , Secreção de Insulina , Ilhotas Pancreáticas/fisiopatologia , Masculino , Pessoa de Meia-Idade , Proteínas Circadianas Period/análiseRESUMO
Genome-wide association studies have shown that variation in MTNR1B (melatonin receptor 1B) is associated with insulin and glucose concentrations. Here we show that the risk genotype of this SNP predicts future type 2 diabetes (T2D) in two large prospective studies. Specifically, the risk genotype was associated with impairment of early insulin response to both oral and intravenous glucose and with faster deterioration of insulin secretion over time. We also show that the MTNR1B mRNA is expressed in human islets, and immunocytochemistry confirms that it is primarily localized in beta cells in islets. Nondiabetic individuals carrying the risk allele and individuals with T2D showed increased expression of the receptor in islets. Insulin release from clonal beta cells in response to glucose was inhibited in the presence of melatonin. These data suggest that the circulating hormone melatonin, which is predominantly released from the pineal gland in the brain, is involved in the pathogenesis of T2D. Given the increased expression of MTNR1B in individuals at risk of T2D, the pathogenic effects are likely exerted via a direct inhibitory effect on beta cells. In view of these results, blocking the melatonin ligand-receptor system could be a therapeutic avenue in T2D.
Assuntos
Diabetes Mellitus Tipo 2/genética , Predisposição Genética para Doença , Insulina/metabolismo , Polimorfismo de Nucleotídeo Único/genética , Receptor MT2 de Melatonina/genética , Receptores de Melatonina/genética , Idoso , Animais , Estudos de Coortes , Feminino , Regulação da Expressão Gênica , Humanos , Secreção de Insulina , Ilhotas Pancreáticas/metabolismo , Ilhotas Pancreáticas/patologia , Masculino , Camundongos , Pessoa de Meia-Idade , Transporte Proteico , Ratos , Receptor MT2 de Melatonina/metabolismo , Receptores de Melatonina/metabolismoRESUMO
In clonal beta-cell lines and islets from different species, a variety of calcium channels are coupled to glucose-stimulated insulin secretion. The aim of this study was to identify the voltage-gated calcium channels that control insulin secretion in insulinoma (INS)-1 832/13 cells. The mRNA level of Ca(V)1.2 exceeded that of Ca(V)1.3 and Ca(V)2.3 two-fold. Insulin secretion, which rose tenfold in response to 16.7 mM glucose, was completely abolished by 5 microM isradipine that blocks Ca(V)1.2 and Ca(V)1.3. Similarly, the increase in intracellular calcium in response to 15 mM glucose was decreased in the presence of 5 microM isradipine, and the frequency of calcium spikes was decreased to the level seen at 2.8 mM glucose. By contrast, inhibition of Ca(V)2.3 with 100 nM SNX-482 did not significantly affect insulin secretion or intracellular calcium. Using RNA interference, Ca(V)1.2 mRNA and protein levels were knocked down by approximately 65% and approximately 34% respectively, which reduced insulin secretion in response to 16.7 mM glucose by 50%. Similar reductions in calcium currents and cell capacitance were seen in standard whole-cell patch-clamp experiments. The remaining secretion of insulin could be reduced to the basal level by 5 microM isradipine. Calcium influx underlying this residual insulin secretion could result from persisting Ca(V)1.2 expression in transfected cells since knock-down of Ca(V)1.3 did not affect glucose-stimulated insulin secretion. In summary, our results suggest that Ca(V)1.2 is critical for insulin secretion in INS-1 832/13 cells.