RESUMEN
The identification of cannabinoid ligands Cannabidiol and O-1918 as inverse agonists of the orphan receptor GPR52 is reported. Detailed characterisation of GPR52 pharmacology and modelling of the proposed receptor interaction is described. The identification of a novel and further CNS pharmacology for the polypharmacological agent and marketed drug Cannabidiol is noteworthy.
RESUMEN
Even though metformin is widely used to treat type2 diabetes, reducing glycaemia and body weight, the mechanisms of action are still elusive. Recent studies have identified the gastrointestinal tract as an important site of action. Here we used intestinal organoids to explore the effects of metformin on intestinal cell physiology. Bulk RNA-sequencing analysis identified changes in hexose metabolism pathways, particularly glycolytic genes. Metformin increased expression of Slc2a1 (GLUT1), decreased expression of Slc2a2 (GLUT2) and Slc5a1 (SGLT1) whilst increasing GLUT-dependent glucose uptake and glycolytic rate as observed by live cell imaging of genetically encoded metabolite sensors and measurement of oxygen consumption and extracellular acidification rates. Metformin caused mitochondrial dysfunction and metformin's effects on 2D-cultures were phenocopied by treatment with rotenone and antimycin-A, including upregulation of GDF15 expression, previously linked to metformin dependent weight loss. Gene expression changes elicited by metformin were replicated in 3D apical-out organoids and distal small intestines of metformin treated mice. We conclude that metformin affects glucose uptake, glycolysis and GDF-15 secretion, likely downstream of the observed mitochondrial dysfunction. This may explain the effects of metformin on intestinal glucose utilisation and food balance.
Asunto(s)
Glucosa/metabolismo , Factor 15 de Diferenciación de Crecimiento/biosíntesis , Metformina/farmacología , Mitocondrias/efectos de los fármacos , Mitocondrias/metabolismo , Animales , Transporte Biológico , Respiración de la Célula/efectos de los fármacos , Células Cultivadas , Biología Computacional/métodos , Perfilación de la Expresión Génica , Proteínas Facilitadoras del Transporte de la Glucosa/genética , Proteínas Facilitadoras del Transporte de la Glucosa/metabolismo , Glucólisis/efectos de los fármacos , Factor 15 de Diferenciación de Crecimiento/genética , Mucosa Intestinal/efectos de los fármacos , Mucosa Intestinal/metabolismo , Masculino , Ratones , Mitocondrias/genética , Fosforilación Oxidativa/efectos de los fármacos , TranscriptomaRESUMEN
The gut is the largest endocrine organ of the body, with hormone-secreting enteroendocrine cells located along the length of the gastrointestinal epithelium. Despite their physiological importance, enteroendocrine cells represent only a small fraction of the epithelial cell population and in the past, their characterization has presented a considerable challenge resulting in a reliance on cell line models. Here, we provide a detailed protocol for the isolation and culture of mixed murine small intestinal cells. These primary cultures have been used to identify the signaling pathways underlying the stimulation and inhibition of gut peptide secretion in response to a number of nutrients and neuropeptides as well as pharmacological agents. Furthermore, in combination with the use of transgenic fluorescent reporter mice, we have demonstrated that these primary cultures become a powerful tool for the examination of fluorescently-tagged enteroendocrine cells at the intracellular level, using methods such as patch clamping and single-cell calcium and cAMP-FRET imaging.
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Técnicas de Cultivo de Célula/métodos , Células Enteroendocrinas/metabolismo , Hormonas Gastrointestinales/metabolismo , Intestino Delgado/citología , Animales , Bombesina/farmacología , Técnicas de Cultivo de Célula/instrumentación , Células Enteroendocrinas/citología , Células Enteroendocrinas/efectos de los fármacos , Péptido 1 Similar al Glucagón/metabolismo , Ratones Endogámicos C57BL , Ratones Transgénicos , Cloruro de Potasio/farmacologíaRESUMEN
UNLABELLED: Glucagon-like peptide-1 (GLP-1) acts as a satiety signal and enhances insulin release. This study examined how GLP-1 production from intestinal L-cells is modified by dietary changes. METHODS: Transgenic mouse models were utilized in which L-cells could be purified by cell specific expression of a yellow fluorescent protein, Venus. Mice were fed on chow or 60% high fat diet (HFD) for 2 or 16 weeks. L-cells were purified by flow cytometry and analysed by microarray and quantitative RT-PCR. Enteroendocrine cell populations were examined by FACS analysis, and GLP-1 secretion was assessed in primary intestinal cultures. RESULTS: Two weeks HFD reduced the numbers of GLP-1 positive cells in the colon, and of GIP positive cells in the small intestine. Purified small intestinal L-cells showed major shifts in their gene expression profiles. In mice on HFD for 16 weeks, significant reductions were observed in the expression of L-cell specific genes, including those encoding gut hormones (Gip, Cck, Sct, Nts), prohormone processing enzymes (Pcsk1, Cpe), granins (Chgb, Scg2), nutrient sensing machinery (Slc5a1, Slc15a1, Abcc8, Gpr120) and enteroendocrine-specific transcription factors (Etv1, Isl1, Mlxipl, Nkx2.2 and Rfx6). A corresponding reduction in the GLP-1 secretory responsiveness to nutrient stimuli was observed in primary small intestinal cultures. CONCLUSION: Mice fed on HFD exhibited reduced expression in L-cells of many L-cell specific genes, suggesting an impairment of enteroendocrine cell function. Our results suggest that a western style diet may detrimentally affect the secretion of gut hormones and normal post-prandial signaling, which could impact on insulin secretion and satiety.
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Dieta Alta en Grasa/efectos adversos , Células Enteroendocrinas/fisiología , Péptido 1 Similar al Glucagón/metabolismo , Animales , Células Cultivadas , Expresión Génica , Péptido 1 Similar al Glucagón/genética , Proteína Homeobox Nkx-2.2 , Masculino , Ratones Endogámicos C57BL , Cultivo Primario de CélulasRESUMEN
Bile acids are well-recognized stimuli of glucagon-like peptide-1 (GLP-1) secretion. This action has been attributed to activation of the G protein-coupled bile acid receptor GPBAR1 (TGR5), although other potential bile acid sensors include the nuclear farnesoid receptor and the apical sodium-coupled bile acid transporter ASBT. The aim of this study was to identify pathways important for GLP-1 release and to determine whether bile acids target their receptors on GLP-1-secreting L-cells from the apical or basolateral compartment. Using transgenic mice expressing fluorescent sensors specifically in L-cells, we observed that taurodeoxycholate (TDCA) and taurolithocholate (TLCA) increased intracellular cAMP and Ca(2+). In primary intestinal cultures, TDCA was a more potent GLP-1 secretagogue than taurocholate (TCA) and TLCA, correlating with a stronger Ca(2+) response to TDCA. Using small-volume Ussing chambers optimized for measuring GLP-1 secretion, we found that both a GPBAR1 agonist and TDCA stimulated GLP-1 release better when applied from the basolateral than from the luminal direction and that luminal TDCA was ineffective when intestinal tissue was pretreated with an ASBT inhibitor. ASBT inhibition had no significant effect in nonpolarized primary cultures. Studies in the perfused rat gut confirmed that vascularly administered TDCA was more effective than luminal TDCA. Intestinal primary cultures and Ussing chamber-mounted tissues from GPBAR1-knockout mice did not secrete GLP-1 in response to either TLCA or TDCA. We conclude that the action of bile acids on GLP-1 secretion is predominantly mediated by GPBAR1 located on the basolateral L-cell membrane, suggesting that stimulation of gut hormone secretion may include postabsorptive mechanisms.
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Ácidos y Sales Biliares/farmacología , Células Enteroendocrinas/efectos de los fármacos , Péptido 1 Similar al Glucagón/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Animales , Calcio/metabolismo , Células Cultivadas , AMP Cíclico/metabolismo , Células Enteroendocrinas/metabolismo , Intestino Delgado/efectos de los fármacos , Intestino Delgado/metabolismo , Espacio Intracelular/efectos de los fármacos , Espacio Intracelular/metabolismo , Masculino , Ratones Noqueados , Ratones Transgénicos , Microscopía Fluorescente , Ratas Wistar , Receptores Acoplados a Proteínas G/genética , Ácido Taurodesoxicólico/farmacología , Ácido Taurolitocólico/farmacología , Técnicas de Cultivo de TejidosRESUMEN
Common somatic mutations in CACNAID and ATP1A1 may define a subgroup of smaller, zona glomerulosa (ZG)-like aldosterone-producing adenomas. We have therefore sought signature ZG genes, which may provide insight into the frequency and pathogenesis of ZG-like aldosterone-producing adenomas. Twenty-one pairs of zona fasciculata and ZG and 14 paired aldosterone-producing adenomas from 14 patients with Conn's syndrome and 7 patients with pheochromocytoma were assayed by the Affymetrix Human Genome U133 Plus 2.0 Array. Validation by quantitative real-time polymerase chain reaction was performed on genes >10-fold upregulated in ZG (compared with zona fasciculata) and >10-fold upregulated in aldosterone-producing adenomas (compared with ZG). DACH1, a gene associated with tumor progression, was further analyzed. The role of DACH1 on steroidogenesis, transforming growth factor-ß, and Wnt signaling activity was assessed in the human adrenocortical cell line, H295R. Immunohistochemistry confirmed selective expression of DACH1 in human ZG. Silencing of DACH1 in H295R cells increased CYP11B2 mRNA levels and aldosterone production, whereas overexpression of DACH1 decreased aldosterone production. Overexpression of DACH1 in H295R cells activated the transforming growth factor-ß and canonical Wnt signaling pathways but inhibited the noncanonical Wnt signaling pathway. Stimulation of primary human adrenal cells with angiotensin II decreased DACH1 mRNA expression. Interestingly, there was little overlap between our top ZG genes and those in rodent ZG. In conclusion, (1) the transcriptome profile of human ZG differs from rodent ZG, (2) DACH1 inhibits aldosterone secretion in human adrenals, and (3) transforming growth factor-ß signaling pathway is activated in DACH1 overexpressed cells and may mediate inhibition of aldosterone secretion in human adrenals.
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Carcinoma Corticosuprarrenal/genética , Aldosterona/metabolismo , Proteínas del Ojo/genética , Regulación Neoplásica de la Expresión Génica , ARN Neoplásico/genética , Factores de Transcripción/genética , Factor de Crecimiento Transformador beta/genética , Zona Glomerular/metabolismo , Carcinoma Corticosuprarrenal/metabolismo , Carcinoma Corticosuprarrenal/patología , Progresión de la Enfermedad , Proteínas del Ojo/biosíntesis , Femenino , Humanos , Inmunohistoquímica , Masculino , Persona de Mediana Edad , Reacción en Cadena en Tiempo Real de la Polimerasa , Transducción de Señal , Factores de Transcripción/biosíntesis , Factor de Crecimiento Transformador beta/biosíntesis , Células Tumorales Cultivadas , Zona Glomerular/patologíaRESUMEN
Bile acids are signalling molecules, which activate the transmembrane receptor TGR5 and the nuclear receptor FXR. BA sequestrants (BAS) complex bile acids in the intestinal lumen and decrease intestinal FXR activity. The BAS-BA complex also induces glucagon-like peptide-1 (GLP-1) production by L cells which potentiates ß-cell glucose-induced insulin secretion. Whether FXR is expressed in L cells and controls GLP-1 production is unknown. Here, we show that FXR activation in L cells decreases proglucagon expression by interfering with the glucose-responsive factor Carbohydrate-Responsive Element Binding Protein (ChREBP) and GLP-1 secretion by inhibiting glycolysis. In vivo, FXR deficiency increases GLP-1 gene expression and secretion in response to glucose hence improving glucose metabolism. Moreover, treatment of ob/ob mice with the BAS colesevelam increases intestinal proglucagon gene expression and improves glycaemia in a FXR-dependent manner. These findings identify the FXR/GLP-1 pathway as a new mechanism of BA control of glucose metabolism and a pharmacological target for type 2 diabetes.
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Células Enteroendocrinas/metabolismo , Péptido 1 Similar al Glucagón/genética , Mucosa Intestinal/metabolismo , ARN Mensajero/metabolismo , Receptores Citoplasmáticos y Nucleares/genética , Animales , Anticolesterolemiantes/farmacología , Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice/metabolismo , Ácidos y Sales Biliares/metabolismo , Glucemia/metabolismo , Clorhidrato de Colesevelam/farmacología , Colon/citología , Colon/metabolismo , Dieta Alta en Grasa , Péptido 1 Similar al Glucagón/metabolismo , Glucólisis , Humanos , Íleon/citología , Íleon/metabolismo , Insulina/metabolismo , Secreción de Insulina , Células Secretoras de Insulina/metabolismo , Intestinos/citología , Yeyuno/citología , Yeyuno/metabolismo , Ratones , Ratones Noqueados , Ratones Obesos , Proteínas Nucleares/metabolismo , Obesidad/genética , Obesidad/metabolismo , Proglucagón/efectos de los fármacos , Proglucagón/genética , Proglucagón/metabolismo , Receptores Acoplados a Proteínas G/genética , Secuestrantes/farmacología , Transducción de Señal , Factores de Transcripción/metabolismoRESUMEN
At least 5% of individuals with hypertension have adrenal aldosterone-producing adenomas (APAs). Gain-of-function mutations in KCNJ5 and apparent loss-of-function mutations in ATP1A1 and ATP2A3 were reported to occur in APAs. We find that KCNJ5 mutations are common in APAs resembling cortisol-secreting cells of the adrenal zona fasciculata but are absent in a subset of APAs resembling the aldosterone-secreting cells of the adrenal zona glomerulosa. We performed exome sequencing of ten zona glomerulosa-like APAs and identified nine with somatic mutations in either ATP1A1, encoding the Na(+)/K(+) ATPase α1 subunit, or CACNA1D, encoding Cav1.3. The ATP1A1 mutations all caused inward leak currents under physiological conditions, and the CACNA1D mutations induced a shift of voltage-dependent gating to more negative voltages, suppressed inactivation or increased currents. Many APAs with these mutations were <1 cm in diameter and had been overlooked on conventional adrenal imaging. Recognition of the distinct genotype and phenotype for this subset of APAs could facilitate diagnosis.