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1.
Diabetologia ; 63(12): 2628-2640, 2020 12.
Artículo en Inglés | MEDLINE | ID: mdl-32960311

RESUMEN

AIMS/HYPOTHESIS: In islets from individuals with type 2 diabetes and in islets exposed to chronic elevated glucose, mitochondrial energy metabolism is impaired. Here, we studied early metabolic changes and mitochondrial adaptations in human beta cells during chronic glucose stress. METHODS: Respiration and cytosolic ATP changes were measured in human islet cell clusters after culture for 4 days in 11.1 mmol/l glucose. Metabolomics was applied to analyse intracellular metabolite changes as a result of glucose stress conditions. Alterations in beta cell function were followed using insulin secretion assays or cytosolic calcium signalling after expression of the calcium probe YC3.6 specifically in beta cells of islet clusters. RESULTS: At early stages of glucose stress, mitochondrial energy metabolism was augmented in contrast to the previously described mitochondrial dysfunction in beta cells from islets of diabetic donors. Following chronic glucose stress, mitochondrial respiration increased (by 52.4%, p < 0.001) and, as a consequence, the cytosolic ATP/ADP ratio in resting human pancreatic islet cells was elevated (by 27.8%, p < 0.05). Because of mitochondrial overactivation in the resting state, nutrient-induced beta cell activation was reduced. In addition, chronic glucose stress caused metabolic adaptations that resulted in the accumulation of intermediates of the glycolytic pathway, the pentose phosphate pathway and the TCA cycle; the most strongly augmented metabolite was glycerol 3-phosphate. The changes in metabolites observed are likely to be due to the inability of mitochondria to cope with continuous nutrient oversupply. To protect beta cells from chronic glucose stress, we inhibited mitochondrial pyruvate transport. Metabolite concentrations were partially normalised and the mitochondrial respiratory response to nutrients was markedly improved. Furthermore, stimulus-secretion coupling as assessed by cytosolic calcium signalling, was restored. CONCLUSION/INTERPRETATION: We propose that metabolic changes and associated mitochondrial overactivation are early adaptations to glucose stress, and may reflect what happens as a result of poor blood glucose control. Inhibition of mitochondrial pyruvate transport reduces mitochondrial nutrient overload and allows beta cells to recover from chronic glucose stress. Graphical abstract.


Asunto(s)
Células Secretoras de Insulina/metabolismo , Mitocondrias/metabolismo , Adenosina Trifosfato/metabolismo , Calcio/metabolismo , Metabolismo Energético/fisiología , Glucosa/metabolismo , Humanos , Metabolómica/métodos
2.
Diabetologia ; 61(3): 641-657, 2018 03.
Artículo en Inglés | MEDLINE | ID: mdl-29185012

RESUMEN

AIMS/HYPOTHESIS: Pancreatic islet beta cell failure causes type 2 diabetes in humans. To identify transcriptomic changes in type 2 diabetic islets, the Innovative Medicines Initiative for Diabetes: Improving beta-cell function and identification of diagnostic biomarkers for treatment monitoring in Diabetes (IMIDIA) consortium ( www.imidia.org ) established a comprehensive, unique multicentre biobank of human islets and pancreas tissues from organ donors and metabolically phenotyped pancreatectomised patients (PPP). METHODS: Affymetrix microarrays were used to assess the islet transcriptome of islets isolated either by enzymatic digestion from 103 organ donors (OD), including 84 non-diabetic and 19 type 2 diabetic individuals, or by laser capture microdissection (LCM) from surgical specimens of 103 PPP, including 32 non-diabetic, 36 with type 2 diabetes, 15 with impaired glucose tolerance (IGT) and 20 with recent-onset diabetes (<1 year), conceivably secondary to the pancreatic disorder leading to surgery (type 3c diabetes). Bioinformatics tools were used to (1) compare the islet transcriptome of type 2 diabetic vs non-diabetic OD and PPP as well as vs IGT and type 3c diabetes within the PPP group; and (2) identify transcription factors driving gene co-expression modules correlated with insulin secretion ex vivo and glucose tolerance in vivo. Selected genes of interest were validated for their expression and function in beta cells. RESULTS: Comparative transcriptomic analysis identified 19 genes differentially expressed (false discovery rate ≤0.05, fold change ≥1.5) in type 2 diabetic vs non-diabetic islets from OD and PPP. Nine out of these 19 dysregulated genes were not previously reported to be dysregulated in type 2 diabetic islets. Signature genes included TMEM37, which inhibited Ca2+-influx and insulin secretion in beta cells, and ARG2 and PPP1R1A, which promoted insulin secretion. Systems biology approaches identified HNF1A, PDX1 and REST as drivers of gene co-expression modules correlated with impaired insulin secretion or glucose tolerance, and 14 out of 19 differentially expressed type 2 diabetic islet signature genes were enriched in these modules. None of these signature genes was significantly dysregulated in islets of PPP with impaired glucose tolerance or type 3c diabetes. CONCLUSIONS/INTERPRETATION: These studies enabled the stringent definition of a novel transcriptomic signature of type 2 diabetic islets, regardless of islet source and isolation procedure. Lack of this signature in islets from PPP with IGT or type 3c diabetes indicates differences possibly due to peculiarities of these hyperglycaemic conditions and/or a role for duration and severity of hyperglycaemia. Alternatively, these transcriptomic changes capture, but may not precede, beta cell failure.


Asunto(s)
Bancos de Muestras Biológicas , Diabetes Mellitus Tipo 2/metabolismo , Biología de Sistemas/métodos , Donantes de Tejidos , Transcriptoma/genética , Anciano , Anciano de 80 o más Años , Biología Computacional , Femenino , Humanos , Masculino , Pancreatectomía
3.
J Biol Chem ; 291(12): 6146-57, 2016 Mar 18.
Artículo en Inglés | MEDLINE | ID: mdl-26792861

RESUMEN

The transcription factor, X-box-binding protein-1 (XBP1), controls the development and maintenance of the endoplasmic reticulum (ER) in multiple secretory cell lineages. We show here that Hepatocyte Nuclear Factor 4α (HNF4α) directly induces XBP1 expression. Mutations in HNF4α cause Mature-Onset Diabetes of the Young I (MODYI), a subset of diabetes characterized by diminished GSIS. In mouse models, cell lines, and ex vivo islets, using dominant negative and human- disease-allele point mutants or knock-out and knockdown models, we show that disruption of HNF4α caused decreased expression of XBP1 and reduced cellular ER networks. GSIS depends on ER Ca(2+) signaling; we show that diminished XBP1 and/or HNF4α in ß-cells led to impaired ER Ca(2+) homeostasis. Restoring XBP1 expression was sufficient to completely rescue GSIS in HNF4α-deficient ß-cells. Our findings uncover a transcriptional relationship between HNF4α and Xbp1 with potentially broader implications about MODYI and the importance of transcription factor signaling in the regulation of secretion.


Asunto(s)
Proteínas de Unión al ADN/genética , Factor Nuclear 4 del Hepatocito/fisiología , Células Secretoras de Insulina/fisiología , Factores de Transcripción/genética , Transcripción Genética , Animales , Calcio/metabolismo , Línea Celular , Proteínas de Unión al ADN/metabolismo , Diabetes Mellitus Tipo 2/genética , Retículo Endoplásmico/metabolismo , Regulación de la Expresión Génica , Glucosa/fisiología , Células HEK293 , Homeostasis , Humanos , Insulina/metabolismo , Secreción de Insulina , Ratones , Factores de Transcripción del Factor Regulador X , Factores de Transcripción/metabolismo , Proteína 1 de Unión a la X-Box
4.
Hum Mol Genet ; 24(7): 1945-55, 2015 Apr 01.
Artículo en Inglés | MEDLINE | ID: mdl-25489054

RESUMEN

Normal glucose homeostasis is characterized by appropriate insulin secretion and low HbA1c. Gene expression signatures associated with these two phenotypes could be essential for islet function and pathophysiology of type 2 diabetes (T2D). Herein, we employed a novel approach to identify candidate genes involved in T2D by correlating islet microarray gene expression data (78 donors) with insulin secretion and HbA1c level. The expression of 649 genes (P < 0.05) was correlated with insulin secretion and HbA1c. Of them, five genes (GLR1A, PPP1R1A, PLCDXD3, FAM105A and ENO2) correlated positively with insulin secretion/negatively with HbA1c and one gene (GNG5) correlated negatively with insulin secretion/positively with HbA1c were followed up. The five positively correlated genes have lower expression levels in diabetic islets, whereas GNG5 expression is higher. Exposure of human islets to high glucose for 24 h resulted in up-regulation of GNG5 and PPP1R1A expression, whereas the expression of ENO2 and GLRA1 was down-regulated. No effect was seen on the expression of FAM105A and PLCXD3. siRNA silencing in INS-1 832/13 cells showed reduction in insulin secretion for PPP1R1A, PLXCD3, ENO2, FAM105A and GNG5 but not GLRA1. Although no SNP in these gene loci passed the genome-wide significance for association with T2D in DIAGRAM+ database, four SNPs influenced gene expression in cis in human islets. In conclusion, we identified and confirmed PPP1R1A, FAM105A, ENO2, PLCDX3 and GNG5 as potential regulators of islet function. We provide a list of candidate genes as a resource for exploring their role in the pathogenesis of T2D.


Asunto(s)
Diabetes Mellitus Tipo 2/genética , Regulación de la Expresión Génica , Glucosa/metabolismo , Insulina/metabolismo , Islotes Pancreáticos/metabolismo , Adulto , Anciano , Diabetes Mellitus Tipo 2/metabolismo , Femenino , Hemoglobina Glucada/metabolismo , Humanos , Secreción de Insulina , Masculino , Persona de Mediana Edad
5.
FASEB J ; 30(12): 3979-3988, 2016 12.
Artículo en Inglés | MEDLINE | ID: mdl-27565711

RESUMEN

Elevated plasma levels of inorganic phosphate (Pi) are harmful, causing, among other complications, vascular calcification and defective insulin secretion. The underlying molecular mechanisms of these complications remain poorly understood. We demonstrated the role of Pi transport across the plasmalemma on Pi toxicity in INS-1E rat clonal ß cells and rat pancreatic islet cells. Type III sodium-phosphate cotransporters (NaPis) are the predominant Pi transporters expressed in insulin-secreting cells. Transcript and protein levels of sodium-dependent phosphate transporter 1 and 2 (PiT-1 and -2), isotypes of type III NaPi, were up-regulated by high-Pi incubation. In patch-clamp experiments, extracellular Pi elicited a Na+-dependent, inwardly rectifying current, which was markedly reduced under acidic extracellular conditions. Cellular uptake of Pi elicited cytosolic alkalinization; intriguingly, this pH change facilitated Pi transport into the mitochondrial matrix. Increased mitochondrial Pi uptake accelerated superoxide generation, mitochondrial permeability transition (mPT), and endoplasmic reticulum stress-mediated translational attenuation, leading to reduced insulin content and impaired glucose-stimulated insulin secretion. Silencing of PiT-1/2 prevented Pi-induced superoxide generation and mPT, and restored insulin secretion. We propose that Pi transport across the plasma membrane and consequent cytosolic alkalinization could be a therapeutic target for protection from Pi toxicity in insulin-secreting cells, as well as in other cell types.-Nguyen, T. T., Quan, X., Xu, S., Das, R., Cha, S.-K., Kong, I. D., Shong, M., Wollheim, C. B., Park, K.-S. Intracellular alkalinization by phosphate uptake via type III sodium-phosphate cotransporter participates in high-phosphate-induced mitochondrial oxidative stress and defective insulin secretion.


Asunto(s)
Transporte Biológico/efectos de los fármacos , Transporte Iónico/genética , Mitocondrias/efectos de los fármacos , Estrés Oxidativo/efectos de los fármacos , Fosfatos/farmacología , Proteínas Cotransportadoras de Sodio-Fosfato de Tipo III/metabolismo , Animales , Membrana Celular/metabolismo , Células Cultivadas , Homeostasis/efectos de los fármacos , Insulina/metabolismo , Secreción de Insulina , Células Secretoras de Insulina/efectos de los fármacos , Células Secretoras de Insulina/metabolismo , Mitocondrias/metabolismo , Ratas
7.
PLoS Genet ; 10(3): e1004160, 2014 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-24603685

RESUMEN

Impaired insulin secretion is a hallmark of type 2 diabetes (T2D). Epigenetics may affect disease susceptibility. To describe the human methylome in pancreatic islets and determine the epigenetic basis of T2D, we analyzed DNA methylation of 479,927 CpG sites and the transcriptome in pancreatic islets from T2D and non-diabetic donors. We provide a detailed map of the global DNA methylation pattern in human islets, ß- and α-cells. Genomic regions close to the transcription start site showed low degrees of methylation and regions further away from the transcription start site such as the gene body, 3'UTR and intergenic regions showed a higher degree of methylation. While CpG islands were hypomethylated, the surrounding 2 kb shores showed an intermediate degree of methylation, whereas regions further away (shelves and open sea) were hypermethylated in human islets, ß- and α-cells. We identified 1,649 CpG sites and 853 genes, including TCF7L2, FTO and KCNQ1, with differential DNA methylation in T2D islets after correction for multiple testing. The majority of the differentially methylated CpG sites had an intermediate degree of methylation and were underrepresented in CpG islands (∼ 7%) and overrepresented in the open sea (∼ 60%). 102 of the differentially methylated genes, including CDKN1A, PDE7B, SEPT9 and EXOC3L2, were differentially expressed in T2D islets. Methylation of CDKN1A and PDE7B promoters in vitro suppressed their transcriptional activity. Functional analyses demonstrated that identified candidate genes affect pancreatic ß- and α-cells as Exoc3l silencing reduced exocytosis and overexpression of Cdkn1a, Pde7b and Sept9 perturbed insulin and glucagon secretion in clonal ß- and α-cells, respectively. Together, our data can serve as a reference methylome in human islets. We provide new target genes with altered DNA methylation and expression in human T2D islets that contribute to perturbed insulin and glucagon secretion. These results highlight the importance of epigenetics in the pathogenesis of T2D.


Asunto(s)
Metilación de ADN/genética , Diabetes Mellitus Tipo 2/genética , Epigénesis Genética , Insulina/genética , Islas de CpG/genética , Diabetes Mellitus Tipo 2/metabolismo , Diabetes Mellitus Tipo 2/patología , Susceptibilidad a Enfermedades , Exocitosis/genética , Genoma Humano , Humanos , Insulina/metabolismo , Secreción de Insulina , Células Secretoras de Insulina/metabolismo , Islotes Pancreáticos/metabolismo , Islotes Pancreáticos/patología , Regiones Promotoras Genéticas
8.
Proc Natl Acad Sci U S A ; 111(38): 13924-9, 2014 Sep 23.
Artículo en Inglés | MEDLINE | ID: mdl-25201977

RESUMEN

Genetic variation can modulate gene expression, and thereby phenotypic variation and susceptibility to complex diseases such as type 2 diabetes (T2D). Here we harnessed the potential of DNA and RNA sequencing in human pancreatic islets from 89 deceased donors to identify genes of potential importance in the pathogenesis of T2D. We present a catalog of genetic variants regulating gene expression (eQTL) and exon use (sQTL), including many long noncoding RNAs, which are enriched in known T2D-associated loci. Of 35 eQTL genes, whose expression differed between normoglycemic and hyperglycemic individuals, siRNA of tetraspanin 33 (TSPAN33), 5'-nucleotidase, ecto (NT5E), transmembrane emp24 protein transport domain containing 6 (TMED6), and p21 protein activated kinase 7 (PAK7) in INS1 cells resulted in reduced glucose-stimulated insulin secretion. In addition, we provide a genome-wide catalog of allelic expression imbalance, which is also enriched in known T2D-associated loci. Notably, allelic imbalance in paternally expressed gene 3 (PEG3) was associated with its promoter methylation and T2D status. Finally, RNA editing events were less common in islets than previously suggested in other tissues. Taken together, this study provides new insights into the complexity of gene regulation in human pancreatic islets and better understanding of how genetic variation can influence glucose metabolism.


Asunto(s)
Genómica , Glucosa , Transcriptoma/fisiología , 5'-Nucleotidasa/biosíntesis , 5'-Nucleotidasa/genética , Línea Celular , Diabetes Mellitus Tipo 2/genética , Diabetes Mellitus Tipo 2/metabolismo , Femenino , Proteínas Ligadas a GPI/biosíntesis , Proteínas Ligadas a GPI/genética , Glucosa/genética , Glucosa/metabolismo , Humanos , Islotes Pancreáticos , Masculino , Edición de ARN/fisiología , ARN Largo no Codificante/biosíntesis , ARN Largo no Codificante/genética , Tetraspaninas/biosíntesis , Tetraspaninas/genética , Proteínas de Transporte Vesicular/biosíntesis , Proteínas de Transporte Vesicular/genética , Quinasas p21 Activadas/biosíntesis , Quinasas p21 Activadas/genética
9.
Diabetologia ; 59(8): 1702-13, 2016 08.
Artículo en Inglés | MEDLINE | ID: mdl-27155871

RESUMEN

AIMS/HYPOTHESIS: Genome-wide association studies (GWAS) have identified more than 65 genetic loci associated with risk of type 2 diabetes. However, the contribution of distorted parental transmission of alleles to risk of type 2 diabetes has been mostly unexplored. Our goal was therefore to search for parent-of-origin effects (POE) among type 2 diabetes loci in families. METHODS: Families from the Botnia study (n = 4,211, 1,083 families) were genotyped for 72 single-nucleotide polymorphisms (SNPs) associated with type 2 diabetes and assessed for POE on type 2 diabetes. The family-based Hungarian Transdanubian Biobank (HTB) (n = 1,463, >135 families) was used to replicate SNPs showing POE. Association of type 2 diabetes loci within families was also tested. RESULTS: Three loci showed nominal POE, including the previously reported variants in KCNQ1, for type 2 diabetes in families from Botnia (rs2237895: p POE = 0.037), which can be considered positive controls. The strongest POE was seen for rs7578597 SNP in the THADA gene, showing excess transmission of the maternal risk allele T to diabetic offspring (Botnia: p POE = 0.01; HTB p POE = 0.045). These data are consistent with previous evidence of allelic imbalance for expression in islets, suggesting that the THADA gene can be imprinted in a POE-specific fashion. Five CpG sites, including those flanking rs7578597, showed differential methylation between diabetic and non-diabetic donor islets. CONCLUSIONS/INTERPRETATION: Taken together, the data emphasise the need for genetic studies to consider from which parent an offspring has inherited a susceptibility allele.


Asunto(s)
Diabetes Mellitus Tipo 2/genética , Proteínas de Neoplasias/genética , Adulto , Alelos , Femenino , Predisposición Genética a la Enfermedad/genética , Estudio de Asociación del Genoma Completo , Genotipo , Humanos , Canal de Potasio KCNQ1/genética , Herencia Materna/genética , Persona de Mediana Edad , Polimorfismo de Nucleótido Simple/genética
10.
Diabetologia ; 59(9): 1928-37, 2016 09.
Artículo en Inglés | MEDLINE | ID: mdl-27338624

RESUMEN

AIMS/HYPOTHESIS: Insufficient insulin release and hyperglucagonaemia are culprits in type 2 diabetes. Cocaine- and amphetamine-regulated transcript (CART, encoded by Cartpt) affects islet hormone secretion and beta cell survival in vitro in rats, and Cart (-/-) mice have diminished insulin secretion. We aimed to test if CART is differentially regulated in human type 2 diabetic islets and if CART affects insulin and glucagon secretion in vitro in humans and in vivo in mice. METHODS: CART expression was assessed in human type 2 diabetic and non-diabetic control pancreases and rodent models of diabetes. Insulin and glucagon secretion was examined in isolated islets and in vivo in mice. Ca(2+) oscillation patterns and exocytosis were studied in mouse islets. RESULTS: We report an important role of CART in human islet function and glucose homeostasis in mice. CART was found to be expressed in human alpha and beta cells and in a subpopulation of mouse beta cells. Notably, CART expression was several fold higher in islets of type 2 diabetic humans and rodents. CART increased insulin secretion in vivo in mice and in human and mouse islets. Furthermore, CART increased beta cell exocytosis, altered the glucose-induced Ca(2+) signalling pattern in mouse islets from fast to slow oscillations and improved synchronisation of the oscillations between different islet regions. Finally, CART reduced glucagon secretion in human and mouse islets, as well as in vivo in mice via diminished alpha cell exocytosis. CONCLUSIONS/INTERPRETATION: We conclude that CART is a regulator of glucose homeostasis and could play an important role in the pathophysiology of type 2 diabetes. Based on the ability of CART to increase insulin secretion and reduce glucagon secretion, CART-based agents could be a therapeutic modality in type 2 diabetes.


Asunto(s)
Diabetes Mellitus Tipo 2/metabolismo , Diabetes Mellitus Tipo 2/fisiopatología , Glucagón/metabolismo , Insulina/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Animales , Western Blotting , Señalización del Calcio/fisiología , Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Tipo 2/sangre , Electrofisiología , Exocitosis/genética , Exocitosis/fisiología , Femenino , Células Secretoras de Glucagón/metabolismo , Glucosa/metabolismo , Homeostasis , Humanos , Inmunohistoquímica , Hibridación in Situ , Secreción de Insulina , Células Secretoras de Insulina/metabolismo , Islotes Pancreáticos/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Persona de Mediana Edad , Proteínas del Tejido Nervioso/genética , Reacción en Cadena en Tiempo Real de la Polimerasa
11.
J Biol Chem ; 290(7): 4086-96, 2015 Feb 13.
Artículo en Inglés | MEDLINE | ID: mdl-25548283

RESUMEN

In pancreatic ß-cells, ATP acts as a signaling molecule initiating plasma membrane electrical activity linked to Ca(2+) influx, which triggers insulin exocytosis. The mitochondrial Ca(2+) uniporter (MCU) mediates Ca(2+) uptake into the organelle, where energy metabolism is further stimulated for sustained second phase insulin secretion. Here, we have studied the contribution of the MCU to the regulation of oxidative phosphorylation and metabolism-secretion coupling in intact and permeabilized clonal ß-cells as well as rat pancreatic islets. Knockdown of MCU with siRNA transfection blunted matrix Ca(2+) rises, decreased nutrient-stimulated ATP production as well as insulin secretion. Furthermore, MCU knockdown lowered the expression of respiratory chain complexes, mitochondrial metabolic activity, and oxygen consumption. The pH gradient formed across the inner mitochondrial membrane following nutrient stimulation was markedly lowered in MCU-silenced cells. In contrast, nutrient-induced hyperpolarization of the electrical gradient was not altered. In permeabilized cells, knockdown of MCU ablated matrix acidification in response to extramitochondrial Ca(2+). Suppression of the putative Ca(2+)/H(+) antiporter leucine zipper-EF hand-containing transmembrane protein 1 (LETM1) also abolished Ca(2+)-induced matrix acidification. These results demonstrate that MCU-mediated Ca(2+) uptake is essential to establish a nutrient-induced mitochondrial pH gradient which is critical for sustained ATP synthesis and metabolism-secretion coupling in insulin-releasing cells.


Asunto(s)
Canales de Calcio/metabolismo , Proteínas de Unión al Calcio/metabolismo , Calcio/metabolismo , Proteínas de Transporte de Catión/metabolismo , Células Secretoras de Insulina/metabolismo , Insulina/metabolismo , Insulinoma/metabolismo , Mitocondrias/metabolismo , Adenosina Trifosfato/metabolismo , Animales , Western Blotting , Canales de Calcio/química , Canales de Calcio/genética , Proteínas de Unión al Calcio/antagonistas & inhibidores , Proteínas de Unión al Calcio/genética , Proteínas de Transporte de Catión/antagonistas & inhibidores , Proteínas de Transporte de Catión/genética , Proliferación Celular , Células Cultivadas , Metabolismo Energético , Glucosa/metabolismo , Concentración de Iones de Hidrógeno , Técnicas para Inmunoenzimas , Secreción de Insulina , Células Secretoras de Insulina/citología , Insulinoma/genética , Insulinoma/patología , Masculino , Potencial de la Membrana Mitocondrial , Fosforilación Oxidativa , ARN Mensajero/genética , ARN Interferente Pequeño/genética , Ratas , Ratas Sprague-Dawley , Reacción en Cadena en Tiempo Real de la Polimerasa , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa
12.
Hum Mol Genet ; 23(24): 6419-31, 2014 Dec 15.
Artículo en Inglés | MEDLINE | ID: mdl-25015099

RESUMEN

Genome-wide association studies have revealed >60 loci associated with type 2 diabetes (T2D), but the underlying causal variants and functional mechanisms remain largely elusive. Although variants in TCF7L2 confer the strongest risk of T2D among common variants by presumed effects on islet function, the molecular mechanisms are not yet well understood. Using RNA-sequencing, we have identified a TCF7L2-regulated transcriptional network responsible for its effect on insulin secretion in rodent and human pancreatic islets. ISL1 is a primary target of TCF7L2 and regulates proinsulin production and processing via MAFA, PDX1, NKX6.1, PCSK1, PCSK2 and SLC30A8, thereby providing evidence for a coordinated regulation of insulin production and processing. The risk T-allele of rs7903146 was associated with increased TCF7L2 expression, and decreased insulin content and secretion. Using gene expression profiles of 66 human pancreatic islets donors', we also show that the identified TCF7L2-ISL1 transcriptional network is regulated in a genotype-dependent manner. Taken together, these results demonstrate that not only synthesis of proinsulin is regulated by TCF7L2 but also processing and possibly clearance of proinsulin and insulin. These multiple targets in key pathways may explain why TCF7L2 has emerged as the gene showing one of the strongest associations with T2D.


Asunto(s)
Diabetes Mellitus Tipo 2/genética , Predisposición Genética a la Enfermedad , Insulina/genética , Proteínas con Homeodominio LIM/genética , Proinsulina/genética , Proteína 2 Similar al Factor de Transcripción 7/genética , Factores de Transcripción/genética , Alelos , Animales , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Diabetes Mellitus Tipo 2/metabolismo , Diabetes Mellitus Tipo 2/patología , Regulación de la Expresión Génica , Sitios Genéticos , Estudio de Asociación del Genoma Completo , Secuenciación de Nucleótidos de Alto Rendimiento , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/metabolismo , Humanos , Insulina/metabolismo , Islotes Pancreáticos/metabolismo , Islotes Pancreáticos/patología , Proteínas con Homeodominio LIM/metabolismo , Factores de Transcripción Maf de Gran Tamaño/genética , Factores de Transcripción Maf de Gran Tamaño/metabolismo , Ratones , Ratones Transgénicos , Polimorfismo de Nucleótido Simple , Proinsulina/metabolismo , Transducción de Señal , Transactivadores/genética , Transactivadores/metabolismo , Proteína 2 Similar al Factor de Transcripción 7/metabolismo , Factores de Transcripción/metabolismo , Transcripción Genética
13.
Biochem J ; 468(1): 49-63, 2015 May 15.
Artículo en Inglés | MEDLINE | ID: mdl-25731850

RESUMEN

Altered secretion of insulin as well as glucagon has been implicated in the pathogenesis of Type 2 diabetes (T2D), but the mechanisms controlling glucagon secretion from α-cells largely remain unresolved. Therefore, we studied the regulation of glucagon secretion from αTC1-6 (αTC1 clone 6) cells and compared it with insulin release from INS-1 832/13 cells. We found that INS-1 832/13 and αTC1-6 cells respectively secreted insulin and glucagon concentration-dependently in response to glucose. In contrast, tight coupling of glycolytic and mitochondrial metabolism was observed only in INS-1 832/13 cells. Although glycolytic metabolism was similar in the two cell lines, TCA (tricarboxylic acid) cycle metabolism, respiration and ATP levels were less glucose-responsive in αTC1-6 cells. Inhibition of the malate-aspartate shuttle, using phenyl succinate (PhS), abolished glucose-provoked ATP production and hormone secretion from αTC1-6 but not INS-1 832/13 cells. Blocking the malate-aspartate shuttle increased levels of glycerol 3-phosphate only in INS-1 832/13 cells. Accordingly, relative expression of constituents in the glycerol phosphate shuttle compared with malate-aspartate shuttle was lower in αTC1-6 cells. Our data suggest that the glycerol phosphate shuttle augments the malate-aspartate shuttle in INS-1 832/13 but not αTC1-6 cells. These results were confirmed in mouse islets, where PhS abrogated secretion of glucagon but not insulin. Furthermore, expression of the rate-limiting enzyme of the glycerol phosphate shuttle was higher in sorted primary ß- than in α-cells. Thus, suppressed glycerol phosphate shuttle activity in the α-cell may prevent a high rate of glycolysis and consequently glucagon secretion in response to glucose. Accordingly, pyruvate- and lactate-elicited glucagon secretion remains unaffected since their signalling is independent of mitochondrial shuttles.


Asunto(s)
Glucagón/metabolismo , Insulina/metabolismo , Islotes Pancreáticos/metabolismo , Animales , Ácido Aspártico/metabolismo , Línea Celular , Diabetes Mellitus Tipo 2/metabolismo , Diabetes Mellitus Tipo 2/fisiopatología , Células Secretoras de Glucagón/efectos de los fármacos , Células Secretoras de Glucagón/metabolismo , Glucosa/metabolismo , Glucosa/farmacología , Glicerofosfatos/metabolismo , Glucólisis , Técnicas In Vitro , Secreción de Insulina , Células Secretoras de Insulina/efectos de los fármacos , Células Secretoras de Insulina/metabolismo , Islotes Pancreáticos/efectos de los fármacos , Cinética , Malatos/metabolismo , Masculino , Proteínas de Transporte de Membrana/metabolismo , Metaboloma , Ratones , Ratones Endogámicos C3H , Ratones Transgénicos , Mitocondrias/metabolismo
14.
Am J Physiol Endocrinol Metab ; 308(11): E933-41, 2015 Jun 01.
Artículo en Inglés | MEDLINE | ID: mdl-25852001

RESUMEN

Inorganic phosphate (Pi) plays an important role in cell signaling and energy metabolism. In insulin-releasing cells, Pi transport into mitochondria is essential for the generation of ATP, a signaling factor in metabolism-secretion coupling. Elevated Pi concentrations, however, can have toxic effects in various cell types. The underlying molecular mechanisms are poorly understood. Here, we have investigated the effect of Pi on secretory function and apoptosis in INS-1E clonal ß-cells and rat pancreatic islets. Elevated extracellular Pi (1~5 mM) increased the mitochondrial membrane potential (ΔΨm), superoxide generation, caspase activation, and cell death. Depolarization of the ΔΨm abolished Pi-induced superoxide generation. Butylmalonate, a nonselective blocker of mitochondrial phosphate transporters, prevented ΔΨm hyperpolarization, superoxide generation, and cytotoxicity caused by Pi. High Pi also promoted the opening of the mitochondrial permeability transition (PT) pore, leading to apoptosis, which was also prevented by butylmalonate. The mitochondrial antioxidants mitoTEMPO or MnTBAP prevented Pi-triggered PT pore opening and cytotoxicity. Elevated extracellular Pi diminished ATP synthesis, cytosolic Ca(2+) oscillations, and insulin content and secretion in INS-1E cells as well as in dispersed islet cells. These parameters were restored following preincubation with mitochondrial antioxidants. This treatment also prevented high-Pi-induced phosphorylation of ER stress proteins. We propose that elevated extracellular Pi causes mitochondrial oxidative stress linked to mitochondrial hyperpolarization. Such stress results in reduced insulin content and defective insulin secretion and cytotoxicity. Our data explain the decreased insulin content and secretion observed under hyperphosphatemic states.


Asunto(s)
Apoptosis/efectos de los fármacos , Células Secretoras de Insulina/efectos de los fármacos , Insulina/metabolismo , Mitocondrias/metabolismo , Estrés Oxidativo/fisiología , Fosfatos/farmacología , Animales , Células Cultivadas , Secreción de Insulina , Células Secretoras de Insulina/fisiología , Masculino , Proteínas de Transporte de Membrana Mitocondrial/efectos de los fármacos , Proteínas de Transporte de Membrana Mitocondrial/metabolismo , Poro de Transición de la Permeabilidad Mitocondrial , Fosfatos/metabolismo , Ratas , Ratas Sprague-Dawley , Especies Reactivas de Oxígeno/metabolismo , Especies Reactivas de Oxígeno/farmacología , Superóxidos/metabolismo
16.
Mol Cell Proteomics ; 11(8): 244-54, 2012 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-22427704

RESUMEN

Insulin secretory granules are ß-cell vesicles dedicated to insulin processing, storage, and release. The secretion of insulin secretory granule content in response to an acute increase of glucose concentration is a highly regulated process allowing normal glycemic homeostasis. Type 2 diabetes is a metabolic disease characterized by chronic hyperglycemia. The consequent prolonged glucose exposure is known to exert deleterious effects on the function of various organs, notably impairment of insulin secretion by pancreatic ß-cells and induction of apoptosis. It has also been described as modifying gene and protein expression in ß-cells. Therefore, we hypothesized that a modulation of insulin secretory granule protein expression induced by chronic hyperglycemia may partially explain ß-cell dysfunction. To identify the potential early molecular mechanisms underlying ß-cell dysfunction during chronic hyperglycemia, we performed SILAC and mass spectrometry experiments to monitor changes in the insulin secretory granule proteome from INS-1E rat insulinoma ß-cells cultivated either with 11 or 30 mm of glucose for 24 h. Fourteen proteins were found to be differentially expressed between these two conditions, and several of these proteins were not described before to be present in ß-cells. Among them, neuronal pentraxin 1 was only described in neurons so far. Here we investigated its expression and intracellular localization in INS-1E cells. Furthermore, its overexpression in glucotoxic conditions was confirmed at the mRNA and protein levels. According to its role in hypoxia-ischemia-induced apoptosis described in neurons, this suggests that neuronal pentraxin 1 might be a new ß-cell mediator in the AKT/GSK3 apoptotic pathway. In conclusion, the modification of specific ß-cell pathways such as apoptosis and oxidative stress may partially explain the impairment of insulin secretion and ß-cell failure, observed after prolonged exposure to high glucose concentrations.


Asunto(s)
Proteína C-Reactiva/metabolismo , Células Secretoras de Insulina/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Proteoma/metabolismo , Vesículas Secretoras/metabolismo , Animales , Western Blotting , Proteína C-Reactiva/genética , Línea Celular Tumoral , Expresión Génica/efectos de los fármacos , Perfilación de la Expresión Génica/métodos , Glucosa/farmacología , Glucógeno Sintasa Quinasa 3/antagonistas & inhibidores , Glucógeno Sintasa Quinasa 3/metabolismo , Hiperglucemia/genética , Hiperglucemia/metabolismo , Inmunohistoquímica , Insulina/metabolismo , Secreción de Insulina , Células Secretoras de Insulina/efectos de los fármacos , Células Secretoras de Insulina/patología , Islotes Pancreáticos/efectos de los fármacos , Islotes Pancreáticos/metabolismo , Espectrometría de Masas , Proteínas del Tejido Nervioso/genética , Proteoma/genética , Proteómica/métodos , Proteínas Proto-Oncogénicas c-akt/metabolismo , Piridinas/farmacología , Pirimidinas/farmacología , Ratas , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Vesículas Secretoras/efectos de los fármacos , Transducción de Señal/efectos de los fármacos , Factores de Tiempo
17.
Biochem J ; 450(3): 595-605, 2013 Mar 15.
Artículo en Inglés | MEDLINE | ID: mdl-23282133

RESUMEN

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.


Asunto(s)
Células Secretoras de Insulina/metabolismo , Insulina/metabolismo , Metabolómica/métodos , Vía de Pentosa Fosfato/fisiología , Animales , Respiración de la Célula/fisiología , Células Cultivadas , Glucosa/farmacología , Secreción de Insulina , Células Secretoras de Insulina/química , Islotes Pancreáticos/metabolismo , Masculino , Metaboloma , Mitocondrias/metabolismo , Mitocondrias/fisiología , Vía de Pentosa Fosfato/efectos de los fármacos , Ratas , Ratas Wistar , Vías Secretoras/efectos de los fármacos
18.
Exp Mol Med ; 56(2): 273-288, 2024 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-38297165

RESUMEN

Autophagy is an essential quality control mechanism for maintaining organellar functions in eukaryotic cells. Defective autophagy in pancreatic beta cells has been shown to be involved in the progression of diabetes through impaired insulin secretion under glucolipotoxic stress. The underlying mechanism reveals the pathologic role of the hyperactivation of mechanistic target of rapamycin (mTOR), which inhibits lysosomal biogenesis and autophagic processes. Moreover, accumulating evidence suggests that oxidative stress induces Ca2+ depletion in the endoplasmic reticulum (ER) and cytosolic Ca2+ overload, which may contribute to mTOR activation in perilysosomal microdomains, leading to autophagic defects and ß-cell failure due to lipotoxicity. This review delineates the antagonistic regulation of autophagic flux by mTOR and AMP-dependent protein kinase (AMPK) at the lysosomal membrane, and both of these molecules could be activated by perilysosomal calcium signaling. However, aberrant and persistent Ca2+ elevation upon lipotoxic stress increases mTOR activity and suppresses autophagy. Therefore, normalization of autophagy is an attractive therapeutic strategy for patients with ß-cell failure and diabetes.


Asunto(s)
Diabetes Mellitus , Células Secretoras de Insulina , Humanos , Calcio , Adenilato Quinasa , Autofagia , Serina-Treonina Quinasas TOR
19.
Biochim Biophys Acta ; 1823(10): 1815-24, 2012 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-22809973

RESUMEN

Glucose-induced insulin secretion from pancreatic ß-cells depends on mitochondrial activation. In the organelle, glucose-derived pyruvate is metabolised along the oxidative and anaplerotic pathway to generate downstream signals leading to insulin granule exocytosis. Entry into the oxidative pathway is catalysed by pyruvate dehydrogenase (PDH) and controlled in part by phosphorylation of the PDH E1α subunit blocking enzyme activity. We find that glucose but not other nutrient secretagogues induce PDH E1α phosphorylation in INS-1E cells and rat islets. INS-1E cells and primary ß-cells express pyruvate dehydrogenase kinase (PDK) 1, 2 and 3, which mediate the observed phosphorylation. In INS-1E cells, suppression of the two main isoforms, PDK1 and PDK3, almost completely prevented PDH E1α phosphorylation. Under basal glucose conditions, phosphorylation was barely detectable and therefore the enzyme almost fully active (90% of maximal). During glucose stimulation, PDH is only partially inhibited (to 78% of maximal). Preventing PDH phosphorylation in situ after suppression of PDK1, 2 and 3 neither enhanced pyruvate oxidation nor insulin secretion. In conclusion, although glucose stimulates E1α phosphorylation and therefore inhibits PDH activity, this control mechanism by itself does not alter metabolism-secretion coupling in INS-1E clonal ß-cells.


Asunto(s)
Glucosa/farmacología , Células Secretoras de Insulina/metabolismo , Piruvato Deshidrogenasa (Lipoamida)/metabolismo , Animales , Calcio/farmacología , Supervivencia Celular/efectos de los fármacos , Células Clonales , Glucosa/toxicidad , Insulina/metabolismo , Secreción de Insulina , Células Secretoras de Insulina/efectos de los fármacos , Células Secretoras de Insulina/enzimología , Isoenzimas/metabolismo , Fosforilación/efectos de los fármacos , Proteínas Serina-Treonina Quinasas/metabolismo , Piruvato Deshidrogenasa Quinasa Acetil-Transferidora , ARN Interferente Pequeño/metabolismo , Ratas
20.
Hum Mol Genet ; 20(14): 2823-33, 2011 Jul 15.
Artículo en Inglés | MEDLINE | ID: mdl-21536586

RESUMEN

Liver receptor homolog (LRH-1) is an orphan nuclear receptor (NR5A2) that regulates cholesterol homeostasis and cell plasticity in endodermal-derived tissues. Estrogen increases LRH-1 expression conveying cell protection and proliferation. Independently, estrogen also protects isolated human islets against cytokine-induced apoptosis. Herein, we demonstrate that LRH-1 is expressed in islets, including ß-cells, and that transcript levels are modulated by 17ß-estradiol through the estrogen receptor (ER)α but not ERß signaling pathway. Repression of LRH-1 by siRNA abrogated the protective effect conveyed by estrogen on rat islets against cytokines. Adenoviral-mediated overexpression of LRH-1 in human islets did not alter proliferation but conferred protection against cytokines and streptozotocin-induced apoptosis. Expression levels of the cell cycle genes cyclin D1 and cyclin E1 as well as the antiapoptotic gene bcl-xl were unaltered in LRH-1 expressing islets. In contrast, the steroidogenic enzymes CYP11A1 and CYP11B1 involved in glucocorticoid biosynthesis were both stimulated in transduced islets. In parallel, graded overexpression of LRH-1 dose-dependently impaired glucose-induced insulin secretion. Our results demonstrate the crucial role of the estrogen target gene nr5a2 in protecting human islets against-stressed-induced apoptosis. We postulate that this effect is mediated through increased glucocorticoid production that blunts the pro-inflammatory response of islets.


Asunto(s)
Apoptosis , Regulación de la Expresión Génica , Células Secretoras de Insulina/metabolismo , Receptores Citoplasmáticos y Nucleares/biosíntesis , Estrés Fisiológico , Adenoviridae , Animales , Línea Celular Tumoral , Colesterol/biosíntesis , Colesterol/genética , Enzima de Desdoblamiento de la Cadena Lateral del Colesterol/genética , Enzima de Desdoblamiento de la Cadena Lateral del Colesterol/metabolismo , Ciclina D1/genética , Ciclina D1/metabolismo , Ciclina E/genética , Ciclina E/metabolismo , Citocinas/genética , Citocinas/metabolismo , Diabetes Mellitus Experimental/genética , Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Experimental/patología , Receptor alfa de Estrógeno/genética , Receptor alfa de Estrógeno/metabolismo , Estrógenos/genética , Estrógenos/metabolismo , Humanos , Insulina/genética , Insulina/metabolismo , Secreción de Insulina , Ratones , Ratones Noqueados , Proteínas Oncogénicas/genética , Proteínas Oncogénicas/metabolismo , Ratas , Ratas Wistar , Receptores Citoplasmáticos y Nucleares/genética , Esteroide 11-beta-Hidroxilasa/genética , Esteroide 11-beta-Hidroxilasa/metabolismo , Proteína bcl-X/genética , Proteína bcl-X/metabolismo
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