RESUMEN
Derived from enteroendocrine cells (EECs), glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic peptide (GIP) are pivotal incretin hormones crucial for blood glucose regulation. Medications of GLP-1 analogs and GLP-1 receptor activators are extensively used in the treatment of type 2 diabetes (T2D) and obesity. However, there are currently no agents to stimulate endogenous incretin secretion. Here, we find the pivotal role of KCNH2 potassium channels in the regulation of incretin secretion. Co-localization of KCNH2 with incretin-secreting EECs in the intestinal epithelium of rodents highlights its significance. Gut epithelial cell-specific KCNH2 knockout in mice improves glucose tolerance and increases oral glucose-triggered GLP-1 and GIP secretion, particularly GIP. Furthermore, KCNH2-deficient primary intestinal epithelial cells exhibit heightened incretin, especially GIP secretion upon nutrient stimulation. Mechanistically, KCNH2 knockdown in EECs leads to reduced K+ currents, prolonged action potential duration, and elevated intracellular calcium levels. Finally, we found that dofetilide, a KCNH2-specific inhibitor, could promote incretin secretion in enteroendocrine STC-1 cells in vitro and in hyperglycemic mice in vivo. These findings elucidate, for the first time, the mechanism and application of KCNH2 in regulating incretin secretion by EECs. Given the therapeutic promise of GLP-1 and GIP in diabetes and obesity management, this study advances our understanding of incretin regulation, paving the way for potential incretin secretagogue therapies in the treatment of diabetes and obesity.
Asunto(s)
Células Enteroendocrinas , Péptido 1 Similar al Glucagón , Incretinas , Animales , Ratones , Incretinas/farmacología , Células Enteroendocrinas/metabolismo , Células Enteroendocrinas/efectos de los fármacos , Péptido 1 Similar al Glucagón/genética , Péptido 1 Similar al Glucagón/metabolismo , Humanos , Polipéptido Inhibidor Gástrico/genética , Diabetes Mellitus Tipo 2/genética , Diabetes Mellitus Tipo 2/metabolismo , Diabetes Mellitus Tipo 2/tratamiento farmacológico , Diabetes Mellitus Tipo 2/patología , Secretagogos/farmacología , Ratones Noqueados , Canal de Potasio ERG1RESUMEN
Glucagon-like peptide-1 receptor (GLP1R) and glucose-dependent insulinotropic polypeptide receptor (GIPR) are transmembrane receptors involved in insulin, glucagon and somatostatin secretion from the pancreatic islet. Therapeutic targeting of GLP1R and GIPR restores blood glucose levels in part by influencing beta cell, alpha cell and delta cell function. Despite the importance of the incretin-mimetics for diabetes therapy, our understanding of GLP1R and GIPR expression patterns and signaling within the islet remain incomplete. Here, we present the evidence for GLP1R and GIPR expression in the major islet cell types, before addressing signaling pathway(s) engaged, as well as their influence on cell survival and function. While GLP1R is largely a beta cell-specific marker within the islet, GIPR is expressed in alpha cells, beta cells, and (possibly) delta cells. GLP1R and GIPR engage Gs-coupled pathways in most settings, although the exact outcome on hormone release depends on paracrine communication and promiscuous signaling. Biased agonism away from beta-arrestin is an emerging concept for improving therapeutic efficacy, and is also relevant for GLP1R/GIPR dual agonism. Lastly, dual agonists exert multiple effects on islet function through GIPR > GLP1R imbalance, increased GLP1R surface expression and cAMP signaling, as well as beneficial alpha cell-beta cell-delta cell crosstalk.
Asunto(s)
Células Secretoras de Glucagón , Receptores de la Hormona Gastrointestinal , Células Secretoras de Somatostatina/metabolismo , Células Secretoras de Glucagón/metabolismo , Receptor del Péptido 1 Similar al Glucagón/genética , Receptores de la Hormona Gastrointestinal/metabolismo , Polipéptido Inhibidor Gástrico/genética , Polipéptido Inhibidor Gástrico/metabolismo , Transducción de SeñalRESUMEN
BACKGROUND: In vitro and in vivo studies have shown that certain cytokines and hormones may play a role in the development and progression of type 2 diabetes (T2D). However, studies on their role in T2D in humans are scarce. We evaluated associations between 11 circulating cytokines and hormones with T2D among a population of sub-Saharan Africans and tested for causal relationships using Mendelian randomization (MR) analyses. METHODS: We used logistic regression analysis adjusted for age, sex, body mass index, and recruitment country to regress levels of 11 cytokines and hormones (adipsin, leptin, visfatin, PAI-1, GIP, GLP-1, ghrelin, resistin, IL-6, IL-10, IL-1RA) on T2D among Ghanaians, Nigerians, and Kenyans from the Africa America Diabetes Mellitus study including 2276 individuals with T2D and 2790 non-T2D individuals. Similar linear regression models were fitted with homeostatic modelling assessments of insulin sensitivity (HOMA-S) and ß-cell function (HOMA-B) as dependent variables among non-T2D individuals (n = 2790). We used 35 genetic variants previously associated with at least one of these 11 cytokines and hormones among non-T2D individuals as instrumental variables in univariable and multivariable MR analyses. Statistical significance was set at 0.0045 (0.05/11 cytokines and hormones). RESULTS: Circulating GIP and IL-1RA levels were associated with T2D. Nine of the 11 cytokines and hormones (exceptions GLP-1 and IL-6) were associated with HOMA-S, HOMA-B, or both among non-T2D individuals. Two-stage least squares MR analysis provided evidence for a causal effect of GIP and IL-RA on HOMA-S and HOMA-B in multivariable analyses (GIP ~ HOMA-S ß = - 0.67, P-value = 1.88 × 10-6 and HOMA-B ß = 0.59, P-value = 1.88 × 10-5; IL-1RA ~ HOMA-S ß = - 0.51, P-value = 8.49 × 10-5 and HOMA-B ß = 0.48, P-value = 5.71 × 10-4). IL-RA was partly mediated via BMI (30-34%), but GIP was not. Inverse variance weighted MR analysis provided evidence for a causal effect of adipsin on T2D (multivariable OR = 1.83, P-value = 9.79 × 10-6), though these associations were not consistent in all sensitivity analyses. CONCLUSIONS: The findings of this comprehensive MR analysis indicate that circulating GIP and IL-1RA levels are causal for reduced insulin sensitivity and increased ß-cell function. GIP's effect being independent of BMI suggests that circulating levels of GIP could be a promising early biomarker for T2D risk. Our MR analyses do not provide conclusive evidence for a causal role of other circulating cytokines in T2D among sub-Saharan Africans.
Asunto(s)
Diabetes Mellitus Tipo 2 , Polipéptido Inhibidor Gástrico , Resistencia a la Insulina , Proteína Antagonista del Receptor de Interleucina 1 , Humanos , Pueblo Africano , Glucemia , Factor D del Complemento/genética , Diabetes Mellitus Tipo 2/complicaciones , Estudio de Asociación del Genoma Completo , Ghana , Péptido 1 Similar al Glucagón , Insulina/genética , Resistencia a la Insulina/genética , Proteína Antagonista del Receptor de Interleucina 1/genética , Interleucina-6/genética , Kenia , Análisis de la Aleatorización Mendeliana , Factores de Riesgo , Nigeria , Polipéptido Inhibidor Gástrico/genéticaRESUMEN
Glucose-dependent insulinotropic polypeptide receptor (GIPR) is a potential drug target for metabolic disorders. It works with glucagon-like peptide-1 receptor and glucagon receptor in humans to maintain glucose homeostasis. Unlike the other two receptors, GIPR has at least 13 reported splice variants (SVs), more than half of which have sequence variations at either C or N terminus. To explore their roles in endogenous peptide-mediated GIPR signaling, we determined the cryoelectron microscopy (cryo-EM) structures of the two N terminus-altered SVs (referred as GIPR-202 and GIPR-209 in the Ensembl database, SV1 and SV2 here, respectively) and investigated the outcome of coexpressing each of them in question with GIPR in HEK293T cells with respect to ligand binding, receptor expression, cAMP (adenosine 3,5-cyclic monophosphate) accumulation, ß-arrestin recruitment, and cell surface localization. It was found that while both N terminus-altered SVs of GIPR neither bound to the hormone nor elicited signal transduction per se, they suppressed ligand binding and cAMP accumulation of GIPR. Meanwhile, SV1 reduced GIPR-mediated ß-arrestin 2 responses. The cryo-EM structures of SV1 and SV2 showed that they reorganized the extracellular halves of transmembrane helices 1, 6, and 7 and extracellular loops 2 and 3 to adopt a ligand-binding pocket-occupied conformation, thereby losing binding ability to the peptide. The results suggest a form of signal bias that is constitutive and ligand-independent, thus expanding our knowledge of biased signaling beyond pharmacological manipulation (i.e., ligand specific) as well as constitutive and ligand-independent (e.g., SV1 of the growth hormone-releasing hormone receptor).
Asunto(s)
Polipéptido Inhibidor Gástrico , Receptores de la Hormona Gastrointestinal , Humanos , Polipéptido Inhibidor Gástrico/genética , Polipéptido Inhibidor Gástrico/metabolismo , Polipéptido Inhibidor Gástrico/farmacología , Ligandos , Microscopía por Crioelectrón , Células HEK293 , Transducción de Señal/fisiología , Receptores de la Hormona Gastrointestinal/genética , Receptores de la Hormona Gastrointestinal/química , Receptores de la Hormona Gastrointestinal/metabolismo , Péptidos , Receptor del Péptido 1 Similar al Glucagón/metabolismoRESUMEN
Attention deficit/hyperactivity disorder (ADHD) is the most common childhood neurodevelopmental disorder. Single nucleotide polymorphisms (SNPs) in the Adhesion G Protein-Coupled Receptor L3 (ADGRL3) gene are associated with increased susceptibility to developing ADHD worldwide. However, the effect of ADGRL3 non-synonymous SNPs (nsSNPs) on the ADGRL3 protein function is vastly unknown. Using several bioinformatics tools to evaluate the impact of mutations, we found that nsSNPs rs35106420, rs61747658, and rs734644, previously reported to be associated and in linkage with ADHD in disparate populations from the world over, are predicted as pathogenic variants. Docking analysis of rs35106420, harbored in the ADGLR3-hormone receptor domain (HRM, a common extracellular domain of the secretin-like GPCRs family), showed that HRM interacts with the Glucose-dependent insulinotropic polypeptide (GIP), part of the incretin hormones family. GIP has been linked to the pathogenesis of diabetes mellitus, and our analyses suggest a potential link to ADHD. Overall, the comprehensive application of bioinformatics tools showed that functional mutations in the ADGLR3 gene disrupt the standard and wild ADGRL3 structure, most likely affecting its metabolic regulation. Further in vitro experiments are granted to evaluate these in silico predictions of the ADGRL3-GIP interaction and dissect the complexity underlying the development of ADHD.
Asunto(s)
Trastorno por Déficit de Atención con Hiperactividad , Receptores Acoplados a Proteínas G , Trastorno por Déficit de Atención con Hiperactividad/genética , Niño , Polipéptido Inhibidor Gástrico/genética , Polipéptido Inhibidor Gástrico/metabolismo , Genómica , Glucosa , Humanos , Incretinas/genética , Incretinas/metabolismo , Neurogénesis , Receptores Acoplados a Proteínas G/genética , Receptores de Péptidos , SecretinaRESUMEN
OBJECTIVE: The hypothalamus is a key region of the brain implicated in homeostatic regulation, and is an integral centre for the control of feeding behaviour. Glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP) are incretin hormones with potent glucoregulatory function through engagement of their respective cognate receptors, GLP-1R and GIPR. Recent evidence indicates that there is a synergistic effect of combining GIP- and GLP-1-based pharmacology on appetite and body weight. The mechanisms underlying the enhanced weight loss exhibited by GIPR/GLP-1R co-agonism are unknown. Gipr and Glp1r are expressed in the hypothalamus in both rodents and humans. To better understand incretin receptor-expressing cell populations, we compared the cell types and expression profiles of Gipr- and Glp1r-expressing hypothalamic cells using single-cell RNA sequencing. METHODS: Using Glp1r-Cre or Gipr-Cre transgenic mouse lines, fluorescent reporters were introduced into either Glp1r- or Gipr-expressing cells, respectively, upon crossing with a ROSA26-EYFP reporter strain. From the hypothalami of these mice, fluorescent Glp1rEYFP+ or GiprEYFP+ cells were FACS-purified and sequenced using single-cell RNA sequencing. Transcriptomic analysis provided a survey of both non-neuronal and neuronal cells, and comparisons between Glp1rEYFP+ and GiprEYFP + populations were made. RESULTS: A total of 14,091 Glp1rEYFP+ and GiprEYFP+ cells were isolated, sequenced and taken forward for bioinformatic analysis. Both Glp1rEYFP+ and GiprEYFP+ hypothalamic populations were transcriptomically highly heterogeneous, representing vascular cell types, oligodendrocytes, astrocytes, microglia, and neurons. The majority of GiprEYFP+ cells were non-neuronal, whereas the Glp1rEYFP+ population was evenly split between neuronal and non-neuronal cell types. Both Glp1rEYFP+ and GiprEYFP+ oligodendrocytes express markers for mature, myelin-forming oligodendrocytes. While mural cells are represented in both Glp1rEYFP+ and GiprEYFP+ populations, Glp1rEYFP+ mural cells are largely smooth muscle cells, while the majority of GiprEYFP+ mural cells are pericytes. The co-expression of regional markers indicate that clusters of Glp1rEYFP+ and GiprEYFP+ neurons have been isolated from the arcuate, ventromedial, lateral, tuberal, suprachiasmatic, and premammillary nuclei of the hypothalamus. CONCLUSIONS: We have provided a detailed comparison of Glp1r and Gipr cells of the hypothalamus with single-cell resolution. This resource will provide mechanistic insight into how engaging Gipr- and Glp1r-expressing cells of the hypothalamus may result in changes in feeding behaviour and energy balance.
Asunto(s)
Receptor del Péptido 1 Similar al Glucagón , Incretinas , Animales , Polipéptido Inhibidor Gástrico/genética , Polipéptido Inhibidor Gástrico/metabolismo , Péptido 1 Similar al Glucagón/metabolismo , Receptor del Péptido 1 Similar al Glucagón/genética , Receptor del Péptido 1 Similar al Glucagón/metabolismo , Glucosa , Humanos , Hipotálamo/metabolismo , Ratones , TranscriptomaRESUMEN
The improper expression of glucose-dependent insulinotropic polypeptide receptor (GIPR) and the GIP/GIPR axis activation has been increasingly recognized in endocrine tumors, with a potential diagnostic and prognostic value. A high tumor-to-normal tissue ratio (T/N ratio) of GIPR was reported both in humans' and in rats' medullary thyroid cancer (MTC), suggesting a direct link between the neoplastic transformation and the mechanism of receptor overexpression. In this study, we evaluated the potential diagnostic and prognostic significance of GIPR expression in a large cohort of MTC patients by correlating GIPR mRNA steady-state levels to clinical phenotypes. The molecular effect of GIP/GIPR axis stimulation in MTC-derived cells was also determined. We detected GIPR expression in ~80% of tumor specimens, especially in sporadic, larger, advanced-stage cancers with higher Ki-67 values. GIPR stimulation induced cAMP elevation in MTC-derived cells and a small but significant fluctuation in Ca2+, both likely associated with increased calcitonin secretion. On the contrary, the effects on PI3K-Akt and MAPK-ERK1/2 signaling pathways were marginal. To conclude, our data confirm the high T/N GIPR ratio in MTC tumors and suggest that it may represent an index for the degree of advancement of the malignant process. We have also observed a functional coupling between GIP/GIPR axis and calcitonin secretion in MTC models. However, the molecular mechanisms underlying this process and the possible implication of GIP/GIPR axis activation in MTC diagnosis and prognosis need further evaluation.
Asunto(s)
Polipéptido Inhibidor Gástrico , Neoplasias de la Tiroides , Calcitonina , Carcinoma Neuroendocrino , Polipéptido Inhibidor Gástrico/genética , Polipéptido Inhibidor Gástrico/metabolismo , Polipéptido Inhibidor Gástrico/farmacología , Humanos , Fosfatidilinositol 3-Quinasas , Receptores de la Hormona Gastrointestinal , Neoplasias de la Tiroides/genéticaRESUMEN
BACKGROUND: Glucose-dependent insulinotropic polypeptide receptor (Gipr) gene expression has been reported in mouse spermatids and Gipr knockout male mice have previously been reported to have decreased in vitro fertilization, although the role of Gipr signaling in male mouse fertility is not well understood. OBJECTIVES: The purposes of these studies were to determine the role of glucose-dependent insulinotropic polypeptide receptor in male fertility using Gipr knockout mice and anti-glucose-dependent insulinotropic polypeptide receptor antibody-treated wild-type mice and to determine if the expression of Gipr in mouse testes is similar in non-human and human primates. METHODS AND MATERIALS: Adiponectin promoter-driven Gipr knockout male mice (GiprAdipo-/- ) were assessed for in vitro and in vivo fertility, sperm parameters, and testicular histology. CD1 male mice were administered an anti-glucose-dependent insulinotropic polypeptide receptor antibody (muGIPR-Ab) prior to and during mating for assessment of in vivo fertility and sperm parameters. Expression of Gipr/GIPR mRNA in the mouse, cynomolgus monkey, and human testes was assessed by in situ hybridization methods using species-specific probes. RESULTS: GiprAdipo-/- male mice are infertile in vitro and in vivo, despite normal testis morphology, sperm counts, and sperm motility. In contrast, administration of muGIPR-Ab to CD1 male mice did not impact fertility. While Gipr mRNA expression is detectable in the mouse testes, GIPR mRNA expression is not detectable in monkey or human testes. DISCUSSION: The infertility of GiprAdipo-/- male mice correlated with the lack of Gipr expression in the testis and/or adipocyte tissue. However, as administration of muGIPR-Ab did not impact the fertility of adult male mice, it is possible that the observations in genetically deficient male mice are related to Gipr deficiency during development. CONCLUSION: Our data support a role for Gipr expression in the mouse testis during the development of sperm fertilization potential, but based on gene expression data, a similar role for glucose-dependent insulinotropic polypeptide receptor in non-human primate or human male fertility is unlikely.
Asunto(s)
Polipéptido Inhibidor Gástrico , Testículo , Animales , Femenino , Fertilidad , Polipéptido Inhibidor Gástrico/genética , Polipéptido Inhibidor Gástrico/metabolismo , Expresión Génica , Humanos , Macaca fascicularis/genética , Macaca fascicularis/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , ARN Mensajero/metabolismo , Receptores Acoplados a Proteínas G/genética , Receptores de la Hormona Gastrointestinal , Motilidad Espermática , Testículo/metabolismoRESUMEN
AIMS/HYPOTHESIS: The aim of this study was to leverage human genetic data to investigate the cardiometabolic effects of glucose-dependent insulinotropic polypeptide (GIP) signalling. METHODS: Data were obtained from summary statistics of large-scale genome-wide association studies. We examined whether genetic associations for type 2 diabetes liability in the GIP and GIPR genes co-localised with genetic associations for 11 cardiometabolic outcomes. For those outcomes that showed evidence of co-localisation (posterior probability >0.8), we performed Mendelian randomisation analyses to estimate the association of genetically proxied GIP signalling with risk of cardiometabolic outcomes, and to test whether this exceeded the estimate observed when considering type 2 diabetes liability variants from other regions of the genome. RESULTS: Evidence of co-localisation with genetic associations of type 2 diabetes liability at both the GIP and GIPR genes was observed for five outcomes. Mendelian randomisation analyses provided evidence for associations of lower genetically proxied type 2 diabetes liability at the GIP and GIPR genes with lower BMI (estimate in SD units -0.16, 95% CI -0.30, -0.02), C-reactive protein (-0.13, 95% CI -0.19, -0.08) and triacylglycerol levels (-0.17, 95% CI -0.22, -0.12), and higher HDL-cholesterol levels (0.19, 95% CI 0.14, 0.25). For all of these outcomes, the estimates were greater in magnitude than those observed when considering type 2 diabetes liability variants from other regions of the genome. CONCLUSIONS/INTERPRETATION: This study provides genetic evidence to support a beneficial role of sustained GIP signalling on cardiometabolic health greater than that expected from improved glycaemic control alone. Further clinical investigation is warranted. DATA AVAILABILITY: All data used in this study are publicly available. The scripts for the analysis are available at: https://github.com/vkarhune/GeneticallyProxiedGIP .
Asunto(s)
Enfermedades Cardiovasculares , Diabetes Mellitus Tipo 2 , Receptores de la Hormona Gastrointestinal , Enfermedades Cardiovasculares/genética , Diabetes Mellitus Tipo 2/genética , Diabetes Mellitus Tipo 2/metabolismo , Polipéptido Inhibidor Gástrico/genética , Polipéptido Inhibidor Gástrico/metabolismo , Estudio de Asociación del Genoma Completo , Glucosa/metabolismo , Genética Humana , Humanos , Receptores de la Hormona Gastrointestinal/genética , Receptores de la Hormona Gastrointestinal/metabolismoRESUMEN
There is considerable interest in GIPR agonism to enhance the insulinotropic and extrapancreatic effects of GIP, thereby improving glycemic and weight control in type 2 diabetes (T2D) and obesity. Recent genetic epidemiological evidence has implicated higher GIPR-mediated GIP levels in raising coronary artery disease (CAD) risk, a potential safety concern for GIPR agonism. We therefore aimed to quantitatively assess whether the association between higher GIPR-mediated fasting GIP levels and CAD risk is mediated via GIPR or is instead the result of linkage disequilibrium (LD) confounding between variants at the GIPR locus. Using Bayesian multitrait colocalization, we identified a GIPR missense variant, rs1800437 (G allele; E354), as the putatively causal variant shared among fasting GIP levels, glycemic traits, and adiposity-related traits (posterior probability for colocalization [PPcoloc] > 0.97; PP explained by the candidate variant [PPexplained] = 1) that was independent from a cluster of CAD and lipid traits driven by a known missense variant in APOE (rs7412; distance to E354 â¼770 Kb; R 2 with E354 = 0.004; PPcoloc > 0.99; PPexplained = 1). Further, conditioning the association between E354 and CAD on the residual LD with rs7412, we observed slight attenuation in association, but it remained significant (odds ratio [OR] per copy of E354 after adjustment 1.03; 95% CI 1.02, 1.04; P = 0.003). Instead, E354's association with CAD was completely attenuated when conditioning on an additional established CAD signal, rs1964272 (R 2 with E354 = 0.27), an intronic variant in SNRPD2 (OR for E354 after adjustment for rs1964272: 1.01; 95% CI 0.99, 1.03; P = 0.06). We demonstrate that associations with GIP and anthropometric and glycemic traits are driven by genetic signals distinct from those driving CAD and lipid traits in the GIPR region and that higher E354-mediated fasting GIP levels are not associated with CAD risk. These findings provide evidence that the inclusion of GIPR agonism in dual GIPR/GLP1R agonists could potentiate the protective effect of GLP-1 agonists on diabetes without undue CAD risk, an aspect that has yet to be assessed in clinical trials.
Asunto(s)
Enfermedades Cardiovasculares/sangre , Diabetes Mellitus Tipo 2/sangre , Polipéptido Inhibidor Gástrico/sangre , Predisposición Genética a la Enfermedad , Receptores de la Hormona Gastrointestinal/metabolismo , Adulto , Anciano , Diabetes Mellitus Tipo 2/genética , Diabetes Mellitus Tipo 2/metabolismo , Femenino , Finlandia , Polipéptido Inhibidor Gástrico/genética , Polipéptido Inhibidor Gástrico/metabolismo , Variación Genética , Estudio de Asociación del Genoma Completo , Genotipo , Humanos , Masculino , Persona de Mediana Edad , Receptores de la Hormona Gastrointestinal/genética , Factores de Riesgo , Reino UnidoRESUMEN
Glucose-dependent insulinotropic polypeptide (GIP) and its receptor (GIPR) are part of the incretin system that regulates glucose homeostasis. A series of GIPR residues putatively important for ligand binding and receptor activation were mutated and pharmacologically evaluated using GIPR selective agonists in cAMP accumulation, ERK1/2 phosphorylation (pERK1/2) and ß-arrestin 2 recruitment assays. The impact of mutation on ligand efficacy was determined by operational modelling of experimental data for each mutant, with results mapped onto the full-length, active-state GIPR structure. Two interaction networks, comprising transmembrane helix (TM) 7, TM1 and TM2, and extracellular loop (ECL) 2, TM5 and ECL3 were revealed, respectively. Both networks were critical for Gαs-mediated cAMP accumulation and the recruitment of ß-arrestin 2, however, cAMP response was more sensitive to alanine substitution, with most mutated residues displaying reduced signaling. Unlike the other two assays, activation of ERK1/2 was largely independent of the network involving ECL2, TM5 and ECL3, indicating that pERK1/2 is at least partially distinct from Gαs or ß-arrestin pathways and this network is also crucial for potential biased agonism at GIPR. Collectively, our work advances understanding of the structure-function relationship of GIPR and provides a framework for the design and/or interpretation of GIP analogues with unique signaling profiles.
Asunto(s)
Polipéptido Inhibidor Gástrico/genética , Polipéptido Inhibidor Gástrico/metabolismo , Receptores de la Hormona Gastrointestinal/genética , Receptores de la Hormona Gastrointestinal/metabolismo , Secuencia de Aminoácidos , Animales , Sitios de Unión/efectos de los fármacos , Sitios de Unión/fisiología , Células COS , Chlorocebus aethiops , Relación Dosis-Respuesta a Droga , Polipéptido Inhibidor Gástrico/química , Polipéptido Inhibidor Gástrico/farmacología , Células HEK293 , Humanos , Mutación/fisiología , Fragmentos de Péptidos/metabolismo , Fragmentos de Péptidos/farmacología , Estructura Secundaria de Proteína , Receptores de la Hormona Gastrointestinal/químicaRESUMEN
The intrinsic capacity of axonal growth is varied among the neurons form different tissues or different developmental stages. In this study, we established an in vitro model to compare the axonal growth of neurons from embryonic 18 days, post-natal 1 day and post-natal 3 days rat. The E18 neurons showed powerful ability of neuritogenensis and axon outgrowth and the ability decreased rapidly along with development. The transcriptome profile of these neurons revealed a set of genes positively correlated with the capacity of neurite outgrowth. Glucose-dependent insulinotropic polypeptide receptor (GIPR) is identified as a gene to promote neurite outgrowth, which was approved by siRNA knock down assay in E18 neuron. Glucose-dependent insulinotropic polypeptide (GIP), a ligand of GIPR secreted from enteroendocrine K cells, is well-known for its role in nutrient sensing and intake. To verify the effect of GIP-GIPR signal on neurite outgrowth, we administrated GIP to stimulate the E18 neurons, the results showed that GIP significantly improved extension of axon. We further revealed that GIP increased Rac1/Cdc42 phosphorylation in Akt dependent manner. In summary, our study established an in vitro model to screen the genes involved in neurite outgrowth, and we provided mechanical insight on the GIP-GIPR axis to promote axonal outgrowth.
Asunto(s)
Polipéptido Inhibidor Gástrico/metabolismo , Proyección Neuronal/fisiología , Neuronas/fisiología , Proteínas Proto-Oncogénicas c-akt/metabolismo , Receptores de la Hormona Gastrointestinal/metabolismo , Animales , Animales Recién Nacidos , Axones/fisiología , Células Cultivadas , Corteza Cerebral/citología , Corteza Cerebral/embriología , Corteza Cerebral/crecimiento & desarrollo , Femenino , Polipéptido Inhibidor Gástrico/genética , Regulación del Desarrollo de la Expresión Génica , Neuronas/citología , Ratas Sprague-Dawley , Receptores de la Hormona Gastrointestinal/genéticaRESUMEN
Roux-en-Y gastric bypass (RYGB) is the most effective treatment for morbid obesity and results in rapid remission of type 2 diabetes (T2D), before significant weight loss occurs. The underlying mechanisms for T2D remission are not fully understood. To gain insight into these mechanisms we used RYGB-operated diabetic GK-rats and Wistar control rats. Twelve adult male Wistar- and twelve adult male GK-rats were subjected to RYGB- or sham-operation. Oral glucose tolerance tests (OGTT) were performed six weeks after surgery. RYGB normalized fasting glucose levels in GK-rats, without affecting fasting insulin levels. In both rat strains, RYGB caused increased postprandial responses in glucose, GLP-1, and GIP. RYGB caused elevated postprandial insulin secretion in Wistar-rats, but had no effect on insulin secretion in GK-rats. In agreement with this, RYGB improved HOMA-IR in GK-rats, but had no effect on HOMA-ß. RYGB-operated GK-rats had an increased number of GIP receptor and GLP-1 receptor immunoreactive islet cells, but RYGB had no major effect on beta or alpha cell mass. Furthermore, in RYGB-operated GK-rats, increased Slc5a1, Pck2 and Pfkfb1 and reduced Fasn hepatic mRNA expression was observed. In summary, our data shows that RYGB induces T2D remission and enhanced postprandial incretin hormone secretion in GK-rats, without affecting insulin secretion or beta cell mass. Thus our data question the dogmatic view of how T2D remission is achieved and instead point at improved insulin sensitivity as the main mechanism of remission.
Asunto(s)
Diabetes Mellitus Tipo 2/genética , Polipéptido Inhibidor Gástrico/genética , Péptido 1 Similar al Glucagón/genética , Obesidad Mórbida/genética , Animales , Diabetes Mellitus Tipo 2/metabolismo , Diabetes Mellitus Tipo 2/patología , Diabetes Mellitus Tipo 2/cirugía , Modelos Animales de Enfermedad , Derivación Gástrica , Prueba de Tolerancia a la Glucosa , Humanos , Insulina/genética , Insulina/metabolismo , Secreción de Insulina/genética , Células Secretoras de Insulina/metabolismo , Células Secretoras de Insulina/patología , Islotes Pancreáticos/metabolismo , Islotes Pancreáticos/patología , Obesidad Mórbida/metabolismo , Obesidad Mórbida/patología , Obesidad Mórbida/cirugía , Ratas , Ratas Wistar , Pérdida de Peso/genética , Pérdida de Peso/fisiologíaRESUMEN
Like other peptide hormones, glucose-dependent insulinotropic polypeptide (GIP) is rapidly cleared from the circulation. Dipeptidyl peptidase-4 (DPP-4) is known to be involved. Information on the overall pharmacokinetics of GIP in rodents is, however, lacking. We investigated the pharmacokinetics of exogenous GIP after intravenous, subcutaneous and intraperitoneal injection with and without DPP-4 inhibition in conscious female C57Bl/6 mice. Secondly, we compared total and intact GIP levels measured by an in-house RIA and commercially available ELISA kits to determine the suitability of these methods for in vivo and in vitro measurements. GIP half-life following intravenous injection amounted to 93 ± 2 s, which was extended to 5 ± 0.6 min by inhibition of DPP-4. Intact GIP levels following subcutaneous and intraperitoneal GIP administration were approximately 15 % of total GIP. The area under the curve of intact GIP (GIP exposure) following GIP injection was significantly increased by DPP-4 inhibition, whereas total GIP levels remained unchanged. We found significant variation between measurements of total, but not intact GIP performed with our in-house RIA and ELISAs in samples obtained after in vivo administration of GIP. Different preanalytical sample preparation (EDTA plasma, heparin plasma, assay buffer and PBS) significantly influenced results for all ELISA kits used. Thus, in experiments involving exogenous GIP(1-42) administration in mice, it is important to consider that this will result in a very low ratio of intact:total peptide but co-administration of a DPP-4 inhibitor greatly elevates this ratio. Furthermore, for comparison of GIP levels, it is essential to maintain uniformity concerning assay methodology and sample preparation.
Asunto(s)
Dipeptidil Peptidasa 4/genética , Inhibidores de la Dipeptidil-Peptidasa IV/farmacología , Polipéptido Inhibidor Gástrico/farmacología , Péptido 1 Similar al Glucagón/farmacología , Hipoglucemia/tratamiento farmacológico , Fragmentos de Péptidos/farmacología , Animales , Glucemia , Inhibidores de la Dipeptidil-Peptidasa IV/farmacocinética , Femenino , Polipéptido Inhibidor Gástrico/genética , Polipéptido Inhibidor Gástrico/farmacocinética , Glucagón/metabolismo , Péptido 1 Similar al Glucagón/genética , Péptido 1 Similar al Glucagón/farmacocinética , Humanos , Hipoglucemia/sangre , Hipoglucemia/genética , Hipoglucemiantes/farmacología , Ratones , Fragmentos de Péptidos/genética , Fragmentos de Péptidos/farmacocinética , Proteolisis/efectos de los fármacos , Receptores de la Hormona Gastrointestinal/genéticaRESUMEN
Receptors for the peptide hormones glucagon-like peptide-1 (GLP-1R), glucose-dependent insulinotropic polypeptide (GIPR), and glucagon (GCGR) are important regulators of insulin secretion and energy metabolism. GLP-1R agonists have been successfully deployed for the treatment of type 2 diabetes, but it has been suggested that their efficacy is limited by target receptor desensitization and downregulation due to recruitment of ß-arrestins. Indeed, recently described GLP-1R agonists with reduced ß-arrestin-2 recruitment have delivered promising results in preclinical and clinical studies. We therefore aimed to determine if the same phenomenon could apply to the closely related GIPR and GCGR. In HEK293 cells depleted of both ß-arrestin isoforms the duration of G protein-dependent cAMP/PKA signaling was increased in response to the endogenous ligand for each receptor. Moreover, in wildtype cells, "biased" GLP-1, GCG, and GIP analogs with selective reductions in ß-arrestin-2 recruitment led to reduced receptor endocytosis and increased insulin secretion over a prolonged stimulation period, although the latter effect was only seen at high agonist concentrations. Biased GCG analogs increased the duration of cAMP signaling, but this did not lead to increased glucose output from hepatocytes. Our study provides a rationale for the development of GLP-1R, GIPR, and GCGR agonists with reduced ß-arrestin recruitment, but further work is needed to maximally exploit this strategy for therapeutic purposes.
Asunto(s)
AMP Cíclico/metabolismo , Polipéptido Inhibidor Gástrico/metabolismo , Péptido 1 Similar al Glucagón/metabolismo , Receptor del Péptido 1 Similar al Glucagón/metabolismo , Incretinas/farmacología , Receptores de la Hormona Gastrointestinal/metabolismo , beta-Arrestinas/metabolismo , Animales , Polipéptido Inhibidor Gástrico/genética , Glucagón/metabolismo , Péptido 1 Similar al Glucagón/genética , Receptor del Péptido 1 Similar al Glucagón/genética , Células HEK293 , Humanos , Secreción de Insulina , Ligandos , Ratones , Ratones Endogámicos C57BL , Receptores de la Hormona Gastrointestinal/genética , Transducción de Señal , beta-Arrestinas/genéticaRESUMEN
Patients with type 2 diabetes (T2D) fail to secrete insulin in response to increased glucose levels that occur with eating. Glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP) are two incretins secreted from gastrointestinal cells that amplify insulin secretion when glucose is high. In this issue of the JCI, Oduori et al. explore the role of ATP-sensitive K+ (KATP) channels in maintaining glucose homeostasis. In persistently depolarized ß cells from KATP channel knockout (KO) mice, the researchers revealed a shift in G protein signaling from the Gs family to the Gq family. This shift explains why GLP-1, which signals via Gq, but not GIP, which signals preferentially via Gs, can effectively potentiate secretion in islets from the KATP channel-deficient mice and in other models of KATP deficiency, including diabetic KK-Ay mice. Their results provide one explanation for differential insulinotropic potential of incretins in human T2D and point to a potentially unifying model for T2D progression itself.
Asunto(s)
Diabetes Mellitus Tipo 2 , Incretinas , Animales , Glucemia , Diabetes Mellitus Tipo 2/genética , Polipéptido Inhibidor Gástrico/genética , Péptido 1 Similar al Glucagón , Humanos , Insulina , RatonesRESUMEN
Glucagon is an essential regulator of glucose homeostasis, particularly in type 2 diabetes (T2D). Blocking the glucagon receptor (GCGR) in diabetic animals and humans has been shown to alleviate hyperglycemia and increase circulating glucagon-like peptide-1 (GLP-1) levels. However, the origin of the upregulated GLP-1 remains to be clarified. Here, we administered high-fat diet + streptozotocin-induced T2D mice and diabetic db/db mice with REMD 2.59, a fully competitive antagonistic human GCGR monoclonal antibody (mAb) for 12 weeks. GCGR mAb treatment decreased fasting blood glucose levels and increased plasma GLP-1 levels in the T2D mice. In addition, GCGR mAb upregulated preproglucagon gene expression and the contents of gut proglucagon-derived peptides, particularly GLP-1, in the small intestine and colon. Notably, T2D mice treated with GCGR mAb displayed a higher L-cell density in the small intestine and colon, which was associated with increased numbers of LK-cells coexpressing GLP-1 and glucose-dependent insulinotropic polypeptide and reduced L-cell apoptosis. Furthermore, GCGR mAb treatment upregulated GLP-1 production in the pancreas, which was detected at lower levels than in the intestine. Collectively, these results suggest that GCGR mAb can increase intestinal GLP-1 production and L-cell number by enhancing LK-cell expansion and inhibiting L-cell apoptosis in T2D.
Asunto(s)
Anticuerpos Monoclonales/farmacología , Anticuerpos Neutralizantes/farmacología , Diabetes Mellitus Experimental/tratamiento farmacológico , Polipéptido Inhibidor Gástrico/genética , Péptido 1 Similar al Glucagón/genética , Receptores de Glucagón/genética , Animales , Apoptosis/genética , Glucemia/metabolismo , Colon/efectos de los fármacos , Colon/metabolismo , Diabetes Mellitus Experimental/etiología , Diabetes Mellitus Experimental/genética , Diabetes Mellitus Experimental/metabolismo , Dieta Alta en Grasa/efectos adversos , Ayuno/metabolismo , Polipéptido Inhibidor Gástrico/metabolismo , Regulación de la Expresión Génica , Péptido 1 Similar al Glucagón/metabolismo , Humanos , Intestino Delgado/efectos de los fármacos , Intestino Delgado/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Páncreas/efectos de los fármacos , Páncreas/metabolismo , Proglucagón/genética , Proglucagón/metabolismo , Receptores de Glucagón/antagonistas & inhibidores , Receptores de Glucagón/metabolismo , Transducción de Señal , Estreptozocina/administración & dosificaciónRESUMEN
Expressed in the small intestine, retinol-binding protein 2 (RBP2) facilitates dietary retinoid absorption. Rbp2-deficient (Rbp2-/- ) mice fed a chow diet exhibit by 6-7 months-of-age higher body weights, impaired glucose metabolism, and greater hepatic triglyceride levels compared to controls. These phenotypes are also observed when young Rbp2-/- mice are fed a high fat diet. Retinoids do not account for the phenotypes. Rather, RBP2 is a previously unidentified monoacylglycerol (MAG)-binding protein, interacting with the endocannabinoid 2-arachidonoylglycerol (2-AG) and other MAGs with affinities comparable to retinol. X-ray crystallographic studies show that MAGs bind in the retinol binding pocket. When challenged with an oil gavage, Rbp2-/- mice show elevated mucosal levels of 2-MAGs. This is accompanied by significantly elevated blood levels of the gut hormone GIP (glucose-dependent insulinotropic polypeptide). Thus, RBP2, in addition to facilitating dietary retinoid absorption, modulates MAG metabolism and likely signaling, playing a heretofore unknown role in systemic energy balance.
Asunto(s)
Peso Corporal , Polipéptido Inhibidor Gástrico/metabolismo , Mucosa Intestinal/metabolismo , Monoglicéridos/metabolismo , Proteínas Celulares de Unión al Retinol/metabolismo , Transducción de Señal , Animales , Dieta Alta en Grasa , Polipéptido Inhibidor Gástrico/genética , Ratones , Ratones Noqueados , Proteínas Celulares de Unión al Retinol/genéticaRESUMEN
Endocrine cells in the gastrointestinal tract secrete multiple hormones to maintain homeostasis in the body. In the present study, we generated intestinal organoids from the duodenum, jejunum, and ileum of Neurogenin 3 (Ngn3)-EGFP mice and examined how enteroendocrine cells (EECs) within organoid cultures resemble native epithelial cells in the gut. Transcriptome analysis of EGFP-positive cells from Ngn3-EGFP organoids showed gene expression pattern comparable to EECs in vivo. We also compared mRNAs of five major hormones, namely, ghrelin (Ghrl), cholecystokinin (Cck), Gip, secretin (Sct), and glucagon (Gcg) in organoids and small intestine along the longitudinal axis and found that expression patterns of these hormones in organoids were similar to those in native tissues. These findings suggest that an intestinal organoid culture system can be utilized as a suitable model to study enteroendocrine cell functions in vitro.