RESUMEN
Mahvash disease is an exceedingly rare genetic disorder of glucagon signaling characterized by hyperglucagonemia, hyperaminoacidemia, and pancreatic α-cell hyperplasia. Although there is no known definitive treatment, octreotide has been used to decrease systemic glucagon levels. We describe a woman who presented to our medical center after three episodes of small-volume hematemesis. She was found to have hyperglucagonemia and pancreatic hypertrophy with genetically confirmed Mahvash disease and also had evidence of portal hypertension (recurrent portosystemic encephalopathy and variceal hemorrhage) in the absence of cirrhosis. These findings established a diagnosis of portosinusoidal vascular disease, a presinusoidal type of portal hypertension previously known as noncirrhotic portal hypertension. Liver transplantation was followed by normalization of serum glucagon and ammonia levels, reversal of pancreatic hypertrophy, and resolution of recurrent encephalopathy and bleeding varices.
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Enfermedades Genéticas Congénitas , Glucagón , Hipertensión Portal , Trasplante de Hígado , Femenino , Humanos , Várices Esofágicas y Gástricas/etiología , Várices Esofágicas y Gástricas/cirugía , Hemorragia Gastrointestinal/etiología , Hemorragia Gastrointestinal/cirugía , Glucagón/sangre , Glucagón/genética , Hipertensión Portal/sangre , Hipertensión Portal/etiología , Hipertensión Portal/genética , Hipertensión Portal/cirugía , Hipertrofia/genética , Cirrosis Hepática , Enfermedades Genéticas Congénitas/sangre , Enfermedades Genéticas Congénitas/diagnóstico , Enfermedades Genéticas Congénitas/genética , Enfermedades Genéticas Congénitas/cirugía , Enfermedades Pancreáticas/genética , Enfermedades Pancreáticas/patología , Enfermedades Pancreáticas/cirugía , Células Secretoras de Glucagón/patologíaAsunto(s)
Glucagón , Trasplante de Hígado , Errores Innatos del Metabolismo , Enfermedades Raras , Humanos , Hígado/metabolismo , Hígado/cirugía , Errores Innatos del Metabolismo/sangre , Errores Innatos del Metabolismo/genética , Errores Innatos del Metabolismo/cirugía , Síndromes Neoplásicos Hereditarios/genética , Síndromes Neoplásicos Hereditarios/cirugía , Enfermedades Raras/sangre , Enfermedades Raras/genética , Enfermedades Raras/cirugía , Glucagón/sangre , Glucagón/genética , Glucagón/metabolismoRESUMEN
BACKGROUND: High throughput sequencing has enabled the interrogation of the transcriptomic landscape of glucagon-secreting alpha cells, insulin-secreting beta cells, and somatostatin-secreting delta cells. These approaches have furthered our understanding of expression patterns that define healthy or diseased islet cell types and helped explicate some of the intricacies between major islet cell crosstalk and glucose regulation. All three endocrine cell types derive from a common pancreatic progenitor, yet alpha and beta cells have partially opposing functions, and delta cells modulate and control insulin and glucagon release. While gene expression signatures that define and maintain cellular identity have been widely explored, the underlying epigenetic components are incompletely characterized and understood. However, chromatin accessibility and remodeling is a dynamic attribute that plays a critical role to determine and maintain cellular identity. RESULTS: Here, we compare and contrast the chromatin landscape between mouse alpha, beta, and delta cells using ATAC-Seq to evaluate the significant differences in chromatin accessibility. The similarities and differences in chromatin accessibility between these related islet endocrine cells help define their fate in support of their distinct functional roles. We identify patterns that suggest that both alpha and delta cells are poised, but repressed, from becoming beta-like. We also identify patterns in differentially enriched chromatin that have transcription factor motifs preferentially associated with different regions of the genome. Finally, we not only confirm and visualize previously discovered common endocrine- and cell specific- enhancer regions across differentially enriched chromatin, but identify novel regions as well. We compiled our chromatin accessibility data in a freely accessible database of common endocrine- and cell specific-enhancer regions that can be navigated with minimal bioinformatics expertise. CONCLUSIONS: Both alpha and delta cells appear poised, but repressed, from becoming beta cells in murine pancreatic islets. These data broadly support earlier findings on the plasticity in identity of non-beta cells under certain circumstances. Furthermore, differential chromatin accessibility shows preferentially enriched distal-intergenic regions in beta cells, when compared to either alpha or delta cells.
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Cromatina , Elementos de Facilitación Genéticos , Islotes Pancreáticos , Células Secretoras de Somatostatina , Animales , Ratones , Cromatina/genética , Cromatina/metabolismo , Glucagón/genética , Glucagón/metabolismo , Islotes Pancreáticos/metabolismo , Células Secretoras de Somatostatina/metabolismoRESUMEN
Hormone secretion from pancreatic islets is essential for glucose homeostasis, and loss or dysfunction of islet cells is a hallmark of type 2 diabetes. Maf transcription factors are crucial for establishing and maintaining adult endocrine cell function. However, during pancreas development, MafB is not only expressed in insulin- and glucagon-producing cells, but also in Neurog3+ endocrine progenitor cells, suggesting additional functions in cell differentiation and islet formation. Here, we report that MafB deficiency impairs ß cell clustering and islet formation, but also coincides with loss of neurotransmitter and axon guidance receptor gene expression. Moreover, the observed loss of nicotinic receptor gene expression in human and mouse ß cells implied that signaling through these receptors contributes to islet cell migration/formation. Inhibition of nicotinic receptor activity resulted in reduced ß cell migration towards autonomic nerves and impaired ß cell clustering. These findings highlight a novel function of MafB in controlling neuronal-directed signaling events required for islet formation.
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Diabetes Mellitus Tipo 2 , Células Secretoras de Insulina , Islotes Pancreáticos , Ratones , Adulto , Animales , Humanos , Glucagón/genética , Glucagón/metabolismo , Diabetes Mellitus Tipo 2/metabolismo , Islotes Pancreáticos/metabolismo , Insulina/metabolismo , Páncreas/metabolismo , Factor de Transcripción MafB/genética , Factor de Transcripción MafB/metabolismoRESUMEN
Glucagon receptor plays an important role in the regulation of glucose metabolism. Studies have revealed that glucagon receptor antagonism is a potential effective treatment for diabetes. However, the functions of GCGR have not been fully illustrated. Although two Gcgr truncation knockout mice models have been widely used for GCGR function studies, truncated gene may remain neomorphic and/or dominant-negative function. In this study, we took the advantages of Crispr-Cas9 technique and generated a novel allele of GCGR in the mouse that yields complete loss of GCGR protein. Our studies reveal that complete deletion of Gcgr results in hyperglucagonemia, α-cell hyperplasia, improvement of glucose tolerance. These results are similar to the Gcgr-truncated mutation in mice. Hence, we provide a novel strain of GCGR knockout mice for the GCGR function studies.
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Sistemas CRISPR-Cas , Receptores de Glucagón , Animales , Ratones , Receptores de Glucagón/genética , Hiperplasia/genética , Glucagón/genética , Glucagón/metabolismo , Ratones NoqueadosRESUMEN
Hypoglycemia, as a complication of type 2 diabetes (T2D), causes increased morbidity and mortality but the physiological response underlying hypoglycemia has not been fully elucidated. Small noncoding microRNA (miRNA) have multiple downstream biological effects. This pilot exploratory study was undertaken to determine if induced miRNA changes would persist and contribute to effects seen 24 h post-hypoglycemia. A parallel, prospective study design was employed, involving T2D (n = 23) and control (n = 23) subjects. The subjects underwent insulin-induced hypoglycemia (2 mmol/L; 36 mg/dL); blood samples were drawn at baseline, upon the induction of hypoglycemia, and 4 h and 24 h post-hypoglycemia, with a quantitative polymerase chain reaction analysis of miRNA undertaken. The baseline miRNAs did not differ. In the controls, 15 miRNAs were downregulated and one was upregulated (FDR < 0.05) from the induction of hypoglycemia to 4 h later while, in T2D, only four miRNAs were altered (downregulated), and these were common to both cohorts (miR-191-5p; miR-143-3p; let-7b-5p; let-7g-5p), correlated with elevated glucagon levels, and all were associated with energy balance. From the induction of hypoglycemia to 24 h, 14 miRNAs were downregulated and 5 were upregulated (FDR < 0.05) in the controls; 7 miRNAs were downregulated and 7 upregulated (FDR < 0.05) in T2D; a total of 6 miRNAs were common between cohorts, 5 were downregulated (miR-93-5p, let-7b-5p, miR-191-5p, miR-185-5p, and miR-652-3p), and 1 was upregulated (miR-369-3p). An ingenuity pathway analysis indicated that many of the altered miRNAs were associated with metabolic and coagulation pathways; however, of the inflammatory proteins expressed, only miR-143-3p at 24 h correlated positively with tumor necrosis factor-α (TNFa; p < 0.05 and r = 0.46) and negatively with toll-like receptor-4 (TLR4; p < 0.05 and r = 0.43). The MiRNA levels altered by hypoglycemia reflected changes in counter-regulatory glucagon and differed between cohorts, and their expression at 24 h suggests miRNAs may potentiate and prolong the physiological response. Trial registration: ClinicalTrials.gov NCT03102801.
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Diabetes Mellitus Tipo 2 , MicroARNs , Humanos , Diabetes Mellitus Tipo 2/complicaciones , Diabetes Mellitus Tipo 2/genética , Perfilación de la Expresión Génica , Glucagón/genética , MicroARNs/genética , MicroARNs/metabolismo , Estudios ProspectivosRESUMEN
Characterization of gene expression in pancreatic islets and its alteration in type 2 diabetes (T2D) are vital in understanding islet function and T2D pathogenesis. We leveraged RNA sequencing and genome-wide genotyping in islets from 188 donors to create the Islet Gene View (IGW) platform to make this information easily accessible to the scientific community. Expression data were related to islet phenotypes, diabetes status, other islet-expressed genes, islet hormone-encoding genes and for expression in insulin target tissues. The IGW web application produces output graphs for a particular gene of interest. In IGW, 284 differentially expressed genes (DEGs) were identified in T2D donor islets compared with controls. Forty percent of DEGs showed cell-type enrichment and a large proportion significantly co-expressed with islet hormone-encoding genes; glucagon (<i>GCG</i>, 56%), amylin (<i>IAPP</i>, 52%), insulin (<i>INS</i>, 44%), and somatostatin (<i>SST</i>, 24%). Inhibition of two DEGs, <i>UNC5D</i> and <i>SERPINE2</i>, impaired glucose-stimulated insulin secretion and impacted cell survival in a human ß-cell model. The exploratory use of IGW could help designing more comprehensive functional follow-up studies and serve to identify therapeutic targets in T2D.
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Diabetes Mellitus Tipo 2 , Islotes Pancreáticos , Diabetes Mellitus Tipo 2/genética , Glucagón/genética , Glucagón/metabolismo , Humanos , Insulina/genética , Insulina/metabolismo , Islotes Pancreáticos/metabolismo , Serpina E2/metabolismoRESUMEN
A number of G protein-coupled receptors (GPCRs) are now thought to use endocytosis to promote cellular cAMP signaling that drives downstream transcription of cAMP-dependent genes. We tested if this is true for the glucagon receptor (GCGR), which mediates physiological regulation of hepatic glucose metabolism via cAMP signaling. We show that epitope-tagged GCGRs undergo clathrin- and dynamin-dependent endocytosis in HEK293 and Huh-7-Lunet cells after activation by glucagon within 5 min and transit via EEA1-marked endosomes shown previously to be sites of GPCR/Gs-stimulated production of cAMP. We further show that endocytosis potentiates cytoplasmic cAMP elevation produced by GCGR activation and promotes expression of phosphoenolpyruvate carboxykinase 1 (PCK1), the enzyme catalyzing the rate-limiting step in gluconeogenesis. We verify endocytosis-dependent induction of PCK1 expression by endogenous GCGRs in primary hepatocytes and show similar control of two other gluconeogenic genes (PGC1α and G6PC). Together, these results implicate the endosomal signaling paradigm in metabolic regulation by glucagon.
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Gluconeogénesis , Receptores de Glucagón , Endocitosis , Regulación de la Expresión Génica , Glucagón/genética , Glucagón/metabolismo , Glucagón/farmacología , Gluconeogénesis/genética , Células HEK293 , Hepatocitos/metabolismo , Humanos , Hígado/metabolismo , Fosfoenolpiruvato Carboxiquinasa (GTP)/genética , Fosfoenolpiruvato Carboxiquinasa (GTP)/metabolismo , Receptores de Glucagón/genética , Receptores de Glucagón/metabolismo , Transcripción GenéticaRESUMEN
Background: Colorectal cancer is highly prevalent and causes high global mortality, and glucagon axis has been implicated in colon cancer. The present study is aimed at investigating the regulating mechanisms of glucagon involvement in colorectal cancer. Methods: Publicly available data from the TCGA database was utilized to explore the expression pattern and regulating role of glucagon (GCG) in colorectal cancer (COADREAD) including colon adenocarcinomas (COAD) and rectum adenocarcinomas (READ). Statistical analyses were performed using the R software packages and public web servers. The expression pattern and prognostic significance of GCG gene in pan-cancer and TCGA-COADREAD data were investigated by performing unpaired and paired sample analyses. The association of GCG expression with clinical characteristics was investigated using logistic regression analysis. Univariate cox regression analysis was performed to test the prognostic value of GCG expression for overall survival in COADREAD patients. GCG-significantly correlated genes were obtained. Biological functions and signaling pathways were identified by performing functional enrichment analysis and Gene Set Enrichment Analysis (GSEA). Additionally, the potential involvement of GCG in tumor immunity was researched by investigating the correlation between GCG expression and 24 tumor infiltrating immune cells. Results: GCG was found to be significantly downregulated in COADREAD tumor samples compared with healthy control samples. GCG gene was shown to be associated with the prognostic outcomes of COADREAD, whereby its upregulation predicted improved survival outcomes. Functional enrichment analysis showed that the top 100 positively and top 100 negatively GCG-correlated genes were mainly enriched in three signaling pathways including ribosome, nitrogen metabolism, and proximal tubule bicarbonate reclamation. The GSEA showed that GCG-significantly correlated genes were mainly enriched in cell cycle-related pathways (reactome cell cycle, reactome cell cycle mitotic, reactome cell cycle checkpoints, reactome M phase, Reactome G2 M DNA damage checkpoint, and Reactome G2 M checkpoints), neuropeptide ligand receptor interaction, RHO GTPases signaling, WNT signaling, RUNX1 signaling, NOTCH signaling, ESR signaling, HCMV infection, and oxidative stress-related signaling. GCG was positively correlated with Th17 cells, pDC, macrophages, TFH cells, iDC, Tem, B cells, dendritic cells, neutrophils, mast cells, and eosinophils and was negatively associated with NK cells. Conclusions: GCG dysregulation with high prognostic value in COADREAD was noted. Several tumor progression-related pathways and tumor immune-modulatory cells were linked to GCG expression in COADREAD. Therefore, GCG may be regarded as a potential therapeutic target for treating colorectal cancer.
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Neoplasias del Colon , Glucagón , Biomarcadores de Tumor/metabolismo , Neoplasias del Colon/genética , Biología Computacional , Regulación Neoplásica de la Expresión Génica , Glucagón/genética , Glucagón/metabolismo , HumanosRESUMEN
Leptin, insulin, and glucagon are involved in regulating glycaemia in vertebrates and play a role in the progression of obesity and type 2 diabetes. While mammals possess an 'adipoinsular axis' whereby insulin stimulates leptin release from adipocytes and leptin in turn feeds back on the pancreas to inhibit further insulin secretion, evidence of such an axis in non-mammalian vertebrates is unknown. We investigated the interactions between these glycaemic hormones and provide evidence for a leptin-insulin axis in a teleost fish, the tilapia. In the first study, we exposed hepatocytes to various concentrations of either insulin or glucagon to determine effects on leptin a (lepa) and then examined this in vivo with i.p. injections of both hormones. We also exposed isolated Brockmann bodies (pancreatic islets) to recombinant tilapia leptin A (rtLepA) and again followed this up with an i.p. injection to examine changes in insulin a and glucagon b. We found that glucagon increases lepa in vitroand in vivo, with the latter being 18-fold higher than saline-injected controls; however, the effects of rtLepA on glub were more variable. Insulin increased lepa by 2.5-fold in vitro and 70-fold in vivo, while rtLepA decreased insa at basal and increased it at high glucose concentrations. These data indicate that a leptin-insulin axis may be conserved among vertebrates and is thus essential for regulating nutrient balance but that the relationship is likely much more dynamic in teleosts as glycaemia is not as tightly regulated as it is in mammals.
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Proteínas de Peces/genética , Insulina/genética , Leptina/genética , Transducción de Señal/genética , Tilapia/genética , Animales , Glucemia/metabolismo , Células Cultivadas , Proteínas de Peces/metabolismo , Regulación de la Expresión Génica , Glucagón/genética , Glucagón/metabolismo , Glucosa/metabolismo , Hepatocitos/metabolismo , Insulina/metabolismo , Secreción de Insulina , Leptina/metabolismo , Masculino , ARN Mensajero/genética , ARN Mensajero/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Tilapia/metabolismo , Factores de TiempoRESUMEN
In contrast to mammals, glucagon is reported as a much more potent blood glucose modulator in birds. Interestingly, we have found p.Thr16Ser mutation, a variation in the highly conserved glucagon hormone, in Galliformes as well as Strigiformes. To check the effect of this mutation on the receptor binding of glucagon, we predicted the ancestral glucagon receptor sequence of all available Galliformes and Strigiformes species. Subsequently, we analysed their binding to the mutated and wild type glucagon (ancestral) by molecular dynamics simulation. At first, we made a model of ancestral glucagon receptor and ancestral mutated, and wild type glucagon in the order Galliformes and Strigiformes. Then we performed molecular dynamics for each Galliformes and Strigiformes receptor as well as each glucagon peptide, respectively. The final structures were used for docking simulation of glucagon to their receptors. The results of the docking simulations showed a stronger binding affinity of mutated glucagon to glucagon receptors. Afterward, we obtained blood glucose concentrations of all available Galliformes members, as well as all available members of its only taxonomic neighbour (order Anseriformes) in superorder Galloanserae. Interestingly the p.Thr16Ser mutation could finely cluster these two orders into two groups: higher blood glucose concentration (order Galliformes, 17.64 ± 1.66 mMol/L) and lower blood glucose concentration (order Anseriformes, 11.34 ± 1.11 mMol/L). Strigiformes which carry the mutated glucagon peptide show also high blood glucose concentrations (17.40 ± 1.51 mMol/L). Therefore, the results suggest this mutation, which leads to stronger binding affinity of mutated glucagon to its receptor, may be a driving force for higher blood glucose homeostasis in the related birds.
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Galliformes , Glucagón , Estrigiformes , Animales , Glucemia , Simulación por Computador , Glucagón/genética , Homeostasis , InsulinaRESUMEN
The mammalian proglucagon gene (Gcg) encodes three glucagon like sequences, glucagon, glucagon-like peptide-1 (GLP-1), and glucagon-like peptide-2 that are of similar length and share sequence similarity, with these hormones having cell surface receptors, glucagon receptor (Gcgr), GLP-1 receptor (Glp1r), and GLP-2 receptor (Glp2r), respectively. Gcgr, Glp1r, and Glp2r are all class B1 G protein-coupled receptors (GPCRs). Despite their sequence and structural similarity, analyses of sequences from rodents have found differences in patterns of sequence conservation and evolution. To determine whether these were rodent-specific traits or general features of these genes in mammals I analyzed coding and protein sequences for proglucagon and the receptors for proglucagon-derived peptides from the genomes of 168 mammalian species. Single copy genes for each gene were found in almost all genomes. In addition to glucagon sequences within Hystricognath rodents (e.g., guinea pig), glucagon sequences from a few other groups (e.g., pangolins and some bats) as well as changes in the proteolytic processing of GLP-1 in some bats are suggested to have functional effects. GLP-2 sequences display increased variability but accepted few substitutions that are predicted to have functional consequences. In parallel, Glp2r sequences display the most rapid protein sequence evolution, and show greater variability in amino acids at sites involved in ligand interaction, however most were not predicted to have a functional consequence. These observations suggest that a greater diversity in biological functions for proglucagon-derived peptides might exist in mammals.
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Evolución Molecular , Variación Genética , Receptor del Péptido 1 Similar al Glucagón/genética , Receptor del Péptido 2 Similar al Glucagón/genética , Glucagón/genética , Proglucagón/genética , Receptores de Glucagón/genética , Secuencia de Aminoácidos , Animales , Genoma , Péptido 1 Similar al Glucagón , Péptido 2 Similar al Glucagón , Mamíferos , FilogeniaRESUMEN
Strong efforts have been placed on understanding the physiological roles and therapeutic potential of the proglucagon peptide hormones including glucagon, GLP-1 and GLP-2. However, little is known about the extent and magnitude of variability in the amino acid composition of the proglucagon precursor and its mature peptides. Here, we identified 184 unique missense variants in the human proglucagon gene GCG obtained from exome and whole-genome sequencing of more than 450,000 individuals across diverse sub-populations. This provides an unprecedented source of population-wide genetic variation data on missense mutations and insights into the evolutionary constraint spectrum of proglucagon-derived peptides. We show that the stereotypical peptides glucagon, GLP-1 and GLP-2 display fewer evolutionary alterations and are more likely to be functionally affected by genetic variation compared to the rest of the gene products. Elucidating the spectrum of genetic variations and estimating the impact of how a peptide variant may influence human physiology and pathophysiology through changes in ligand binding and/or receptor signalling, are vital and serve as the first important step in understanding variability in glucose homeostasis, amino acid metabolism, intestinal epithelial growth, bone strength, appetite regulation, and other key physiological parameters controlled by these hormones.
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Péptidos Similares al Glucagón/genética , Proglucagón/genética , Secuencia de Aminoácidos , Análisis Mutacional de ADN , Conjuntos de Datos como Asunto , Frecuencia de los Genes , Glucagón/química , Glucagón/genética , Péptido 1 Similar al Glucagón/química , Péptido 1 Similar al Glucagón/genética , Péptido 2 Similar al Glucagón/química , Péptido 2 Similar al Glucagón/genética , Péptidos Similares al Glucagón/química , Humanos , Modelos Moleculares , Mutación Missense , Pruebas de Farmacogenómica , Proglucagón/química , Precursores de Proteínas/química , Precursores de Proteínas/genética , Estructura Secundaria de Proteína/genéticaRESUMEN
Accumulating evidence has demonstrated that gene alterations play a crucial role in LUAD development, progression, and prognosis. The present study aimed to identify the hub genes associated with LUAD. In the present study, we used TCGA database to screen the hub genes. Then, we validated the results by GEO datasets. Finally, we used cBioPortal, UALCAN, qRT-PCR, HPA database, TCGA database, and Kaplan-Meier plotter database to estimate the gene mutation, gene transcription, protein expression, clinical features of hub genes in patients with LUAD. A total of 5930 DEGs were screened out in TCGA database. Enrichment analysis revealed that DEGs were involved in the transcriptional misregulation in cancer, viral carcinogenesis, cAMP signaling pathway, calcium signaling pathway, and ECM-receptor interaction. The combining results of MCODE and CytoHubba showed that ADCY8, ADRB2, CALCA, GCG, GNGT1, and NPSR1 were hub genes. Then, we verified the above results by GSE118370, GSE136043, and GSE140797 datasets. Compared with normal lung tissues, the expression levels of ADCY8 and ADRB2 were lower in LUAD tissues, but the expression levels of CALCA, GCG, GNGT1, and NPSR1 were higher. In the prognosis analyses, the low expression of ADCY8 and ADRB2 and the high expression of CALCA, GCG, GNGT1, and NPSR1 were correlated with poor OS and poor PFS. The significant differences in the relationship of the expression of 6 hub genes and clinical features were observed. In conclusion, 6 hub genes will not only contribute to elucidating the pathogenesis of LUAD and may be potential therapeutic targets for LUAD.
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Adenocarcinoma del Pulmón/genética , Biomarcadores de Tumor/genética , Perfilación de la Expresión Génica , Neoplasias Pulmonares/genética , Transcriptoma , Adenocarcinoma del Pulmón/patología , Adenilil Ciclasas/genética , Anciano , Péptido Relacionado con Gen de Calcitonina/genética , Bases de Datos Genéticas , Femenino , Subunidades gamma de la Proteína de Unión al GTP/genética , Regulación Neoplásica de la Expresión Génica , Redes Reguladoras de Genes , Glucagón/genética , Humanos , Neoplasias Pulmonares/patología , Masculino , Persona de Mediana Edad , Valor Predictivo de las Pruebas , Pronóstico , Mapas de Interacción de Proteínas , Receptores Adrenérgicos beta 2/genética , Receptores Acoplados a Proteínas G/genéticaRESUMEN
One polysaccharide, designated as WPEP-A, was isolated from Passiflora edulis Sims peel and its hypoglycemic effects on diabetic db/db mice were evaluated. Physicochemical characterization showed that WPEP-A was composed of galactose, glucose, xylose, rhamnose, galacturonic acid and glucuronic acid with a molecular weight of 9.51 × 104 Da. We observed an inhibition in weight gain and blood glucose levels. Glucose tolerance and insulin tolerance improved after the administration of WPEP-A. In addition, our data showed increased antioxidant enzyme activities. Furthermore, the levels of serum insulin and triglyceride decreased with the recovery of liver damage. Meanwhile, positive changes in short chain fatty acid content were observed, and the mRNA levels of glucagon-like peptide 1 receptor, glucagon and prohormone convertase 3 were up-regulated in the intestinal tract. In summary, our results showed that WPEP-A had hypoglycemic activity and improved intestinal function in diabetic mice, which may contribute to the attenuation of the hypoglycemia effects.
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Diabetes Mellitus Tipo 2/metabolismo , Hipoglucemiantes/química , Hipoglucemiantes/farmacología , Passiflora/química , Polisacáridos/química , Polisacáridos/farmacología , Animales , Antioxidantes/química , Antioxidantes/farmacología , Glucemia/análisis , Carbohidratos de la Dieta/farmacología , Ácidos Grasos Volátiles/metabolismo , Glucagón/genética , Receptor del Péptido 1 Similar al Glucagón/genética , Insulina/sangre , Mucosa Intestinal/metabolismo , Ratones , Polisacáridos/aislamiento & purificación , Proproteína Convertasa 1/genética , Triglicéridos/sangre , Regulación hacia Arriba , Aumento de Peso/efectos de los fármacosRESUMEN
Glucagon-like peptide-1 (GLP-1) is an incretin secreted from enteroendocrine preproglucagon (PPG)-expressing cells (traditionally known as L cells) in response to luminal nutrients that potentiates insulin secretion. Augmentation of endogenous GLP-1 secretion might well represent a novel therapeutic target for diabetes treatment in addition to the incretin-associated drugs currently in use. In this study, we found that PPG cells substantially express carbonic anhydrase 8 (CAR8), which has been reported to inhibit inositol 1,4,5-trisphosphate (IP3) binding to the IP3 receptor and subsequent Ca2+ efflux from the endoplasmic reticulum in neuronal cells. In vitro experiments using STC-1 cells demonstrated that Car8 knockdown increases long-chain fatty acid (LCFA)-stimulated GLP-1 secretion. This effect was reduced in the presence of phospholipase C (PLC) inhibitor; in addition, Car8 knockdown increased the intracellular Ca2+ elevation caused by α-linolenic acid, indicating that CAR8 exerts its effect on GLP-1 secretion via the PLC/IP3/Ca2+ pathway. Car8wdl null mutant mice showed significant increase in GLP-1 response to oral corn oil administration compared with that in wild-type littermates, with no significant change in intestinal GLP-1 content. These results demonstrate that CAR8 negatively regulates GLP-1 secretion from PPG cells in response to LCFAs, suggesting the possibility of augmentation of postprandial GLP-1 secretion by CAR8 inhibition.NEW & NOTEWORTHY This study focused on the physiological significance of carbonic anhydrase 8 (CAR8) in GLP-1 secretion from enteroendocrine preproglucagon (PPG)-expressing cells. We found an inhibitory role of CAR8 in LCFA-induced GLP-1 secretion in vitro and in vivo, suggesting a novel therapeutic approach to diabetes and obesity through augmentation of postprandial GLP-1 secretion by CAR8 inhibition.
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Biomarcadores de Tumor/metabolismo , Aceite de Maíz/farmacología , Células Enteroendocrinas/efectos de los fármacos , Ácidos Grasos/farmacología , Péptido 1 Similar al Glucagón/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Animales , Biomarcadores de Tumor/genética , Señalización del Calcio , Línea Celular , Células Enteroendocrinas/enzimología , Glucagón/genética , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Inositol 1,4,5-Trifosfato/metabolismo , Masculino , Ratones Endogámicos C57BL , Ratones Transgénicos , Mutación , Proteínas del Tejido Nervioso/genética , Vías Secretoras , Fosfolipasas de Tipo C/metabolismoRESUMEN
Mitochondrial gene expression is pivotal to cell metabolism. Nevertheless, it is unknown whether it diverges within a given cell type. Here, we analysed single-cell RNA-seq experiments from human pancreatic alpha (N = 3471) and beta cells (N = 1989), as well as mouse beta cells (N = 1094). Cluster analysis revealed two distinct human beta cells populations, which diverged by mitochondrial (mtDNA) and nuclear DNA (nDNA)-encoded oxidative phosphorylation (OXPHOS) gene expression in healthy and diabetic individuals, and in newborn but not in adult mice. Insulin gene expression was elevated in beta cells with higher mtDNA gene expression in humans and in young mice. Such human beta cell populations also diverged in mitochondrial RNA mutational repertoire, and in their selective signature, thus implying the existence of two previously overlooked distinct and conserved beta cell populations. While applying our approach to human alpha cells, two sub-populations of cells were identified which diverged in mtDNA gene expression, yet these cellular populations did not consistently diverge in nDNA OXPHOS genes expression, nor did they correlate with the expression of glucagon, the hallmark of alpha cells. Thus, pancreatic beta cells within an individual are divided into distinct groups with unique metabolic-mitochondrial signature.
Asunto(s)
Expresión Génica , Células Secretoras de Insulina/metabolismo , Células Secretoras de Insulina/patología , Insulina/genética , Insulina/metabolismo , Mitocondrias/genética , Transducción de Señal/genética , Transducción de Señal/fisiología , Animales , Recuento de Células , ADN Mitocondrial/genética , ADN Mitocondrial/metabolismo , Glucagón/genética , Glucagón/metabolismo , Humanos , Ratones , Mitocondrias/metabolismo , Mutación , Fosforilación Oxidativa , Páncreas/citologíaRESUMEN
Increased glucagon is a hallmark of diabetes and leads to worsening of the hyperglycemia, but the molecular mechanisms causing it are still unknown. We therefore investigated the possibility that microRNAs might be involved in the regulation of glucagon. Indeed, analysis of the glucagon 3' untranslated region (UTR) revealed potential binding sites for miR-320a, and using luciferase reporter assays we found that miR-320a directly targets the 3' UTRs of human and rodent glucagon. In addition, endogenous glucagon mRNA and protein expression as well as glucagon secretion were reduced in response to miR-320a overexpression, whereas inhibition of miR-320a upregulated glucagon expression. Interestingly, miR-320a expression was decreased by high glucose, and this was associated with an increase in glucagon expression in human islets and mouse αTC1-6 cells. Moreover, miR-320a overexpression completely blunted these effects. Importantly, miR-320a was also significantly downregulated in human islets of subjects with type 2 diabetes and this was accompanied by increased glucagon expression. Thus, our data suggest that glucose-induced downregulation of miR-320a may contribute to the paradoxical increase in glucagon observed in type 2 diabetes and reveal for the first time that glucagon expression is under the control by a microRNA providing novel insight into the abnormal regulation of glucagon in diabetes.
Asunto(s)
Glucagón/genética , MicroARNs/fisiología , Regiones no Traducidas 3'/efectos de los fármacos , Regiones no Traducidas 3'/genética , Adolescente , Adulto , Anciano , Animales , Células Cultivadas , Femenino , Regulación de la Expresión Génica/efectos de los fármacos , Glucagón/metabolismo , Células Secretoras de Glucagón/efectos de los fármacos , Células Secretoras de Glucagón/metabolismo , Glucosa/farmacología , Células HEK293 , Humanos , Islotes Pancreáticos/efectos de los fármacos , Islotes Pancreáticos/metabolismo , Masculino , Ratones , Persona de Mediana EdadRESUMEN
The secretion of glucagon by pancreatic alpha cells is regulated by a number of external and intrinsic factors. While the electrophysiological processes linking a lowering of glucose concentrations to an increased glucagon release are well characterized, the evidence for the identity and function of the glucose sensor is still incomplete. In the present study we aimed to address two unsolved problems: (1) do individual alpha cells have the intrinsic capability to regulate glucagon secretion by glucose, and (2) is glucokinase the alpha cell glucose sensor in this scenario. Single cell RT-PCR was used to confirm that glucokinase is the main glucose-phosphorylating enzyme expressed in rat pancreatic alpha cells. Modulation of glucokinase activity by pharmacological activators and inhibitors led to a lowering or an increase of the glucose threshold of glucagon release from single alpha cells, measured by TIRF microscopy, respectively. Knockdown of glucokinase expression resulted in a loss of glucose control of glucagon secretion. Taken together this study provides evidence for a crucial role of glucokinase in intrinsic glucose regulation of glucagon release in rat alpha cells.
Asunto(s)
Células Secretoras de Glucagón/metabolismo , Glucagón/metabolismo , Glucoquinasa/metabolismo , Glucosa/metabolismo , Animales , Técnicas Biosensibles , Técnica del Anticuerpo Fluorescente , Regulación Enzimológica de la Expresión Génica , Glucagón/genética , Células Secretoras de Glucagón/efectos de los fármacos , Glucoquinasa/genética , Glucosa/farmacología , Isoenzimas/metabolismo , Manoheptulosa/farmacología , Microscopía Fluorescente , Ratas Wistar , Análisis de la Célula Individual/métodos , Sulfonas/farmacología , Tiazoles/farmacologíaRESUMEN
Previous morphological studies of mammalian pancreatic islets have been performed mainly in domestic and laboratory animals. Therefore, the present immunohistochemical investigation was conducted in a wild species, the European bison, using antibodies against glucagon-like peptide-1 (GLP1), glucagon, insulin and somatostatin. Morphological analyses revealed that the mean area of the endocrine pancreas constituted 2.1±0.1% of the whole area of the pancreas, while the mean area of a single pancreatic islet was 13301.5±686.5 µm2. Glucagon-immunoreac- tive cells accounted for 22.4±1.1% and occupied 19.4±0.4% of the average islet area. As many as 14.3±1.4% of pancreatic islet cells were shown to express GLP1, which constituted 12.6±0.8% of the mean area of the islet. Insulin expression was confirmed in 67.6±0.7% of pancreatic islet cells, which represented 62.3±4.9% of the mean total area of the pancreatic islet. As many as 8.5±1.3% of cells stained for somatostatin. The somatostatin-immunoreactive cell area was 4.9±0.3% of the mean pancreatic islet area. In summary, we have determined in detail for the first time the morphometry and islet composition of the European bison pancreas. The distri- bution patterns of immunoreactivities to the substances studied in the European bison show many similarities to those described in other ruminant species.