RESUMO
Bone marrow (BM) long-lived plasma cells (PCs) are essential for long-term protection against infection, and their persistence within this organ relies on interactions with Cxcl12-expressing stromal cells that are still not clearly identified. Here, using single cell RNAseq and in silico transinteractome analyses, we identified Leptin receptor positive (LepR+ ) mesenchymal cells as the stromal cell subset most likely to interact with PCs within the BM. Moreover, we demonstrated that depending on the isotype they express, PCs may use different sets of integrins and adhesion molecules to interact with these stromal cells. Altogether, our results constitute an unprecedented characterization of PC subset stromal niches and open new avenues for the specific targeting of BM PCs based on their isotype.
Assuntos
Medula Óssea , Células-Tronco Mesenquimais , Medula Óssea/metabolismo , Plasmócitos , Células Estromais , Moléculas de Adesão Celular/metabolismo , Células da Medula ÓsseaRESUMO
To characterize the impact of metabolic disease on the peptidome of human and mouse pancreatic islets, LC-MS was used to analyze extracts of human and mouse islets, purified mouse alpha, beta, and delta cells, supernatants from mouse islet incubations, and plasma from patients with type 2 diabetes. Islets were obtained from healthy and type 2 diabetic human donors, and mice on chow or high fat diet. All major islet hormones were detected in lysed islets as well as numerous peptides from vesicular proteins including granins and processing enzymes. Glucose-dependent insulinotropic peptide (GIP) was not detectable. High fat diet modestly increased islet content of proinsulin-derived peptides in mice. Human diabetic islets contained increased content of proglucagon-derived peptides at the expense of insulin, but no evident prohormone processing defects. Diabetic plasma, however, contained increased ratios of proinsulin and des-31,32-proinsulin to insulin. Active GLP-1 was detectable in human and mouse islets but 100-1000-fold less abundant than glucagon. LC-MS offers advantages over antibody-based approaches for identifying exact peptide sequences, and revealed a shift toward islet insulin production in high fat fed mice, and toward proglucagon production in type 2 diabetes, with no evidence of systematic defective prohormone processing.
Assuntos
Diabetes Mellitus Tipo 2 , Ilhotas Pancreáticas , Animais , Glucagon , Peptídeo 1 Semelhante ao Glucagon , Humanos , Insulina , Camundongos , ObesidadeRESUMO
Among bacterial metabolites, hydrogen sulfide (H2S) has received increasing attention. The epithelial cells of the large intestine are exposed to two sources of H2S. The main one is the luminal source that results from specific bacteria metabolic activity toward sulfur-containing substrates. The other source in colonocytes is from the intracellular production mainly through cystathionine ß-synthase (CBS) activity. H2S is oxidized by the mitochondrial sulfide oxidation unit, resulting in ATP synthesis, and, thus, establishing this compound as the first mineral energy substrate in colonocytes. However, when the intracellular H2S concentration exceeds the colonocyte capacity for its oxidation, it inhibits the mitochondrial respiratory chain, thus affecting energy metabolism. Higher luminal H2S concentration affects the integrity of the mucus layer and displays proinflammatory effects. However, a low/minimal amount of endogenous H2S exerts an anti-inflammatory effect on the colon mucosa, pointing out the ambivalent effect of H2S depending on its intracellular concentration. Regarding colorectal carcinogenesis, forced CBS expression in late adenoma-like colonocytes increased their proliferative activity, bioenergetics capacity, and tumorigenicity; whereas, genetic ablation of CBS in mice resulted in a reduced number of mutagen-induced aberrant crypt foci. Activation of endogenous H2S production and low H2S extracellular concentration enhance cancerous colorectal cell proliferation. Higher exogenous H2S concentrations markedly reduce mitochondrial ATP synthesis and proliferative capacity in cancerous cells and enhance glycolysis but do not affect their ATP cell content or viability. Thus, it appears that, notably through an effect on colonocyte energy metabolism, endogenous and microbiota-derived H2S are involved in the host intestinal physiology and physiopathology.
Assuntos
Colo/efeitos dos fármacos , Células Epiteliais/efeitos dos fármacos , Microbioma Gastrointestinal/fisiologia , Sulfeto de Hidrogênio/metabolismo , Mucosa Intestinal/efeitos dos fármacos , Reto/efeitos dos fármacos , Animais , Humanos , Sulfeto de Hidrogênio/toxicidade , Mucosa Intestinal/citologiaRESUMO
OBJECTIVE: Food intake normally stimulates release of satiety and insulin-stimulating intestinal hormones, such as glucagon-like peptide (GLP)-1. This response is blunted in obese insulin resistant subjects, but is rapidly restored following Roux-en-Y gastric bypass (RYGB) surgery. We hypothesised this to be a result of the metabolic changes taking place in the small intestinal mucosa following the anatomical rearrangement after RYGB surgery, and aimed at identifying such mechanisms. DESIGN: Jejunal mucosa biopsies from patients undergoing RYGB surgery were retrieved before and after very-low calorie diet, at time of surgery and 6 months postoperatively. Samples were analysed by global protein expression analysis and Western blotting. Biological functionality of these findings was explored in mice and enteroendocrine cells (EECs) primary mouse jejunal cell cultures. RESULTS: The most prominent change found after RYGB was decreased jejunal expression of the rate-limiting ketogenic enzyme mitochondrial 3-hydroxy-3-methylglutaryl-CoA synthase (mHMGCS), corroborated by decreased ketone body levels. In mice, prolonged high-fat feeding induced the expression of mHMGCS and functional ketogenesis in jejunum. The effect of ketone bodies on gut peptide secretion in EECs showed a â¼40% inhibition of GLP-1 release compared with baseline. CONCLUSION: Intestinal ketogenesis is induced by high-fat diet and inhibited by RYGB surgery. In cell culture, ketone bodies inhibited GLP-1 release from EECs. Thus, we suggest that this may be a mechanism by which RYGB can remove the inhibitory effect of ketone bodies on EECs, thereby restituting the responsiveness of EECs resulting in increased meal-stimulated levels of GLP-1 after surgery.
Assuntos
Restrição Calórica , Células Enteroendócrinas/metabolismo , Derivação Gástrica , Peptídeo 1 Semelhante ao Glucagon/metabolismo , Mucosa Intestinal/metabolismo , Jejuno/metabolismo , Corpos Cetônicos/biossíntese , Ácido 3-Hidroxibutírico/sangue , Ácido 3-Hidroxibutírico/farmacologia , Anastomose em-Y de Roux , Animais , Células Cultivadas , Gorduras na Dieta/administração & dosagem , Emulsões/farmacologia , Emulsões Gordurosas Intravenosas/farmacologia , Feminino , Peptídeo 1 Semelhante ao Glucagon/antagonistas & inibidores , Humanos , Hidroximetilglutaril-CoA Sintase/metabolismo , Corpos Cetônicos/metabolismo , Cetonas/metabolismo , Fígado/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Pessoa de Meia-Idade , Fosfolipídeos/farmacologia , Período Pós-Operatório , Período Pré-Operatório , Cultura Primária de Células , Óleo de Soja/farmacologiaRESUMO
OBJECTIVES: Gastrointestinal hormones contribute to the beneficial effects of Roux-en-Y gastric bypass surgery (RYGB) on glycemic control. Secretin is secreted from duodenal S cells in response to low luminal pH, but it is unknown whether its secretion is altered after RYGB and if secretin contributes to the postoperative improvement in glycemic control. We hypothesized that secretin secretion increases after RYGB as a result of the diversion of nutrients to more distal parts of the small intestine, and thereby affects islet hormone release. METHODS: A specific secretin radioimmunoassay was developed, evaluated biochemically, and used to quantify plasma concentrations of secretin in 13 obese individuals before, 1 week after, and 3 months after RYGB. Distribution of secretin and its receptor was assessed by RNA sequencing, mass-spectrometry and in situ hybridization in human and rat tissues. Isolated, perfused rat intestine and pancreas were used to explore the molecular mechanism underlying glucose-induced secretin secretion and to study direct effects of secretin on glucagon, insulin, and somatostatin secretion. Secretin was administered alone or in combination with GLP-1 to non-sedated rats to evaluate effects on glucose regulation. RESULTS: Plasma postprandial secretin was more than doubled in humans after RYGB (P < 0.001). The distal small intestine harbored secretin expressing cells in both rats and humans. Glucose increased the secretion of secretin in a sodium-glucose cotransporter dependent manner when administered to the distal part but not into the proximal part of the rat small intestine. Secretin stimulated somatostatin secretion (fold change: 1.59, P < 0.05) from the perfused rat pancreas but affected neither insulin (P = 0.2) nor glucagon (P = 0.97) secretion. When administered to rats in vivo, insulin secretion was attenuated and glucagon secretion increased (P = 0.04), while blood glucose peak time was delayed (from 15 to 45 min) and gastric emptying time prolonged (P = 0.004). CONCLUSIONS: Glucose-sensing secretin cells located in the distal part of the small intestine may contribute to increased plasma concentrations observed after RYGB. The metabolic role of the distal S cells warrants further studies.
Assuntos
Células Enteroendócrinas , Derivação Gástrica , Glucose/metabolismo , Intestino Delgado/citologia , Animais , Células Enteroendócrinas/metabolismo , Células Enteroendócrinas/fisiologia , Masculino , Período Pós-Prandial/fisiologia , Ratos , Ratos WistarRESUMO
Current fructose consumption levels often overwhelm the intestinal capacity to absorb fructose. We investigated the impact of fructose malabsorption on intestinal endocrine function and addressed the role of the microbiota in this process. To answer this question, a mouse model of moderate fructose malabsorption [ketohexokinase mutant (KHK)-/-] and wild-type (WT) littermate mice were used and received a 20%-fructose (KHK-F and WT-F) or 20%-glucose diet. Cholecystokinin (Cck) mRNA and protein expression in the ileum and cecum, as well as preproglucagon (Gcg) and neurotensin (Nts) mRNA expression in the cecum, increased in KHK-F mice. In KHK-F mice, triple-label immunohistochemistry showed major up-regulation of CCK in enteroendocrine cells (EECs) that were glucagon-like peptide-1 (GLP-1)+/Peptide YY (PYY-) in the ileum and colon and GLP-1-/PYY- in the cecum. The cecal microbiota composition was drastically modified in the KHK-F in association with an increase in glucose, propionate, succinate, and lactate concentrations. Antibiotic treatment abolished fructose malabsorption-dependent induction of cecal Cck mRNA expression and, in mouse GLUTag and human NCI-H716 cells, Cck mRNA expression levels increased in response to propionate, both suggesting a microbiota-dependent process. Fructose reaching the lower intestine can modify the composition and metabolism of the microbiota, thereby stimulating the production of CCK from the EECs possibly in response to propionate.-Zhang, X., Grosfeld, A., Williams, E., Vasiliauskas, D., Barretto, S., Smith, L., Mariadassou, M., Philippe, C., Devime, F., Melchior, C., Gourcerol, G., Dourmap, N., Lapaque, N., Larraufie, P., Blottière, H. M., Herberden, C., Gerard, P., Rehfeld, J. F., Ferraris, R. P., Fritton, J. C., Ellero-Simatos, S., Douard, V. Fructose malabsorption induces cholecystokinin expression in the ileum and cecum by changing microbiota composition and metabolism.
Assuntos
Ceco/metabolismo , Colecistocinina/metabolismo , Frutose/metabolismo , Frutose/farmacologia , Microbioma Gastrointestinal/efeitos dos fármacos , Íleo/metabolismo , Animais , Ceco/efeitos dos fármacos , Linhagem Celular , Frutoquinases/genética , Frutoquinases/metabolismo , Frutose/administração & dosagem , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Íleo/efeitos dos fármacos , Camundongos , Camundongos KnockoutRESUMO
The intestinal epithelium is an active barrier separating the host from its microbiota. It senses microbial compounds through expression of a wide range of receptors including the Toll-like receptors (TLRs). TLRs have been shown to regulate epithelium permeability or secretion of defensin by Paneth cells. However, the expression and function of TLRs in enteroendocrine L-cells, a specific subtype of intestinal cells secreting PYY and GLP-1, have not yet been assessed. PYY and GLP-1 are implicated in regulation of gut motility, food intake and insulin secretion, and are of great interest regarding obesity and type 2 diabetes. Using a cellular model of human L-cells and a reporter system for NF-κB activation pathway, we reported functional expression of TLRs in these cells. Stimulation with specific TLR-agonists increased expression of Pyy but not Proglucagon in an NF-κB-dependent manner. Moreover, the effect of TLR stimulation was additive to butyrate, a product of bacterial fermentation, on Pyy expression. Additionally, butyrate also increased Tlr expression, including Tlr4, and the NF-κB response to TLR stimulation. Altogether, our results demonstrated a role of TLRs in the modulation of Pyy expression and the importance of butyrate, a product of bacterial fermentation in regulation of microbial TLR-dependent sensing.
Assuntos
Butiratos/metabolismo , Células Enteroendócrinas/metabolismo , Regulação da Expressão Gênica , Peptídeo YY/metabolismo , Receptores Toll-Like/agonistas , Linhagem Celular , Peptídeo 1 Semelhante ao Glucagon/biossíntese , Humanos , NF-kappa B/metabolismoRESUMO
AIMS/HYPOTHESIS: Lipids are a potent stimulus for the secretion of glucagon-like peptide (GLP)-1 and glucose-dependent insulinotropic peptide (GIP). Traditionally, this effect was thought to involve the sensing of lipid digestion products by free fatty acid receptor 1 (FFA1) and G-protein coupled receptor 119 (GPR119) on the apical surface of enteroendocrine cells. However, recent evidence suggests that lipids may in fact be sensed basolaterally, and that fatty acid absorption and chylomicron synthesis may be a prerequisite for their stimulatory effect on gut peptide release. Therefore, we investigated the effect of chylomicrons on GLP-1 and GIP secretion in vitro. METHODS: The effect of chylomicrons on incretin secretion was investigated using GLUTag cells and duodenal cultures of both murine and human origin. The role of lipoprotein lipase (LPL) and FFA1 in GLUTag cells was assessed by pharmacological inhibition and small (short) interfering RNA (siRNA)-mediated knockdown. The effect of chylomicrons on intracellular calcium concentration ([Ca2+]i) was determined by imaging GLUTag cells loaded with Fura-2. In the primary setting, the contributions of FFA1 and GPR119 were investigated using L cell-specific Gpr119 knockout cultures treated with the FFA1 antagonist GW1100. RESULTS: Chylomicrons stimulated GLP-1 release from GLUTag cells, and both GLP-1 and GIP secretion from human and murine duodenal cultures. Chylomicron-triggered GLP-1 secretion from GLUTag cells was largely abolished following lipase inhibition with orlistat or siRNA-mediated knockdown of Lpl. In GLUTag cells, both GW1100 and siRNA-mediated Ffar1 knockdown reduced GLP-1 secretion in response to chylomicrons, and, consistent with FFA1 Gq-coupling, chylomicrons triggered an increase in [Ca2+]i. However, LPL and FFA1 inhibition had no significant effect on chylomicron-mediated incretin secretion in murine cultures. Furthermore, the loss of GPR119 had no impact on GLP-1 secretion in response to chylomicrons, even in the presence of GW1100. CONCLUSIONS/INTERPRETATION: Chylomicrons stimulate incretin hormone secretion from GLUTag cells as well as from human and murine duodenal cultures. In GLUTag cells, the molecular pathway was found to involve LPL-mediated lipolysis, leading to the release of lipid species that activated FFA1 and elevated intracellular calcium.
Assuntos
Quilomícrons/farmacologia , Incretinas/metabolismo , Animais , Células Cultivadas , Células Enteroendócrinas/metabolismo , Polipeptídeo Inibidor Gástrico/metabolismo , Peptídeo 1 Semelhante ao Glucagon/metabolismo , Humanos , Lipase Lipoproteica/genética , Lipase Lipoproteica/metabolismo , Camundongos , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismoRESUMO
Endocrine functions of the gut are supported by a scattered population of cells, the enteroendocrine cells (EECs). EECs sense their environment to secrete hormones in a regulated manner. Distal EECs are in contact with various microbial compounds including hydrogen sulfide (H2S) which modulate cell respiration with potential consequences on EEC physiology. However, the effect of H2S on gut hormone secretion remains discussed and the importance of the modulation of cell metabolism on EEC functions remains to be deciphered. The aim of this project was to characterize the metabolic response of EECs to H2S and the consequences on GLP-1 secretion. We used cell line models of EECs to assess their capacity to metabolize H2S at low concentration and the associated modulation of cell respiration. We confirmed that like what is observed in colonocytes, colonic EEC model, NCI-h716 cell line rapidly metabolizes H2S at low concentrations, resulting in transient increased respiration. Higher concentrations of H2S inhibited this respiration, with the concentration threshold for inhibition depending on cell density. However, increased or inhibited oxidative respiration had little effect on acute GLP-1 secretion. Overall, we present here a first study showing the EEC capacity to detoxify low concentrations of H2S and used this model to acutely address the importance of cell respiration on secretory activity.
Assuntos
Sulfeto de Hidrogênio , Sulfeto de Hidrogênio/metabolismo , Células Enteroendócrinas/metabolismo , Colo/metabolismo , Fatores de Transcrição/metabolismo , Peptídeo 1 Semelhante ao Glucagon/metabolismo , RespiraçãoRESUMO
The commensal bacteria that make up the gut microbiota impact the health of their host on multiple levels. In particular, the interactions taking place between the microbe-associated molecule patterns (MAMPs) and pattern recognition receptors (PRRs), expressed by intestinal epithelial cells (IECs), are crucial for maintaining intestinal homeostasis. While numerous studies showed that TLRs and NLRs are involved in the control of gut homeostasis by commensal bacteria, the role of additional innate immune receptors remains unclear. Here, we seek for novel MAMP-PRR interactions involved in the beneficial effect of the commensal bacterium Akkermansia muciniphila on intestinal homeostasis. We show that A. muciniphila strongly activates NF-κB in IECs by releasing one or more potent activating metabolites into the microenvironment. By using drugs, chemical and gene-editing tools, we found that the released metabolite(s) enter(s) epithelial cells and activate(s) NF-κB via an ALPK1, TIFA and TRAF6-dependent pathway. Furthermore, we show that the released molecule has the biological characteristics of the ALPK1 ligand ADP-heptose. Finally, we show that A. muciniphila induces the expression of the MUC2, BIRC3 and TNFAIP3 genes involved in the maintenance of the intestinal barrier function and that this process is dependent on TIFA. Altogether, our data strongly suggest that the commensal A. muciniphila promotes intestinal homeostasis by activating the ALPK1/TIFA/TRAF6 axis, an innate immune pathway exclusively described so far in the context of Gram-negative bacterial infections.
Assuntos
Microbioma Gastrointestinal , NF-kappa B , Difosfato de Adenosina , Akkermansia , Heptoses , Imunidade Inata , Fator 6 Associado a Receptor de TNF , VerrucomicrobiaRESUMO
In recent years, the importance of the gut microbiota in human health has been revealed and many publications have highlighted its role as a key component of human physiology. Owing to the use of modern sequencing approaches, the characterisation of the microbiome in healthy individuals and in disease has demonstrated a disturbance of the microbiota, or dysbiosis, associated with pathological conditions. The microbiota establishes a symbiotic crosstalk with their host: commensal microbes benefit from the nutrient-rich environment provided by the gut and the microbiota produces hundreds of proteins and metabolites that modulate key functions of the host, including nutrient processing, maintenance of energy homoeostasis and immune system development. Many bacteria-derived metabolites originate from dietary sources. Among them, an important role has been attributed to the metabolites derived from the bacterial fermentation of dietary fibres, namely SCFA linking host nutrition to intestinal homoeostasis maintenance. SCFA are important fuels for intestinal epithelial cells (IEC) and regulate IEC functions through different mechanisms to modulate their proliferation, differentiation as well as functions of subpopulations such as enteroendocrine cells, to impact gut motility and to strengthen the gut barrier functions as well as host metabolism. Recent findings show that SCFA, and in particular butyrate, also have important intestinal and immuno-modulatory functions. In this review, we discuss the mechanisms and the impact of SCFA on gut functions and host immunity and consequently on human health.
Assuntos
Metabolismo Energético/fisiologia , Ácidos Graxos Voláteis/metabolismo , Microbioma Gastrointestinal/fisiologia , Mucosa Intestinal/metabolismo , Disbiose/metabolismo , Homeostase , Humanos , Intestinos , Fenômenos Fisiológicos da NutriçãoRESUMO
OBJECTIVE: Motilin is a proximal small intestinal hormone with roles in gastrointestinal motility, gallbladder emptying, and hunger initiation. In vivo motilin release is stimulated by fats, bile, and duodenal acidification but the underlying molecular mechanisms of motilin secretion remain poorly understood. This study aimed to establish the key signaling pathways involved in the regulation of secretion from human motilin-expressing M-cells. METHODS: Human duodenal organoids were CRISPR-Cas9 modified to express the fluorescent protein Venus or the Ca2+ sensor GCaMP7s under control of the endogenous motilin promoter. This enabled the identification and purification of M-cells for bulk RNA sequencing, peptidomics, calcium imaging, and electrophysiology. Motilin secretion from 2D organoid-derived cultures was measured by liquid chromatography-tandem mass spectrometry (LC-MS/MS), in parallel with other gut hormones. RESULTS: Human duodenal M-cells synthesize active forms of motilin and acyl-ghrelin in organoid culture, and also co-express cholecystokinin (CCK). Activation of the bile acid receptor GPBAR1 stimulated a 3.4-fold increase in motilin secretion and increased action potential firing. Agonists of the long-chain fatty acid receptor FFA1 and monoacylglycerol receptor GPR119 stimulated secretion by 2.4-fold and 1.5-fold, respectively. Acidification (pH 5.0) was a potent stimulus of M-cell calcium elevation and electrical activity, an effect attributable to acid-sensing ion channels, and a modest inducer of motilin release. CONCLUSIONS: This study presents the first in-depth transcriptomic and functional characterization of human duodenal motilin-expressing cells. We identify several receptors important for the postprandial and interdigestive regulation of motilin release.
Assuntos
Bile/metabolismo , Duodeno/metabolismo , Ácidos Graxos não Esterificados/metabolismo , Motilina/metabolismo , Organoides/metabolismo , Células Cultivadas , Humanos , Concentração de Íons de HidrogênioRESUMO
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.
Assuntos
Glucose/metabolismo , Fator 15 de Diferenciação de Crescimento/biossíntese , Metformina/farmacologia , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Animais , Transporte Biológico , Respiração Celular/efeitos dos fármacos , Células Cultivadas , Biologia Computacional/métodos , Perfilação da Expressão Gênica , Proteínas Facilitadoras de Transporte de Glucose/genética , Proteínas Facilitadoras de Transporte de Glucose/metabolismo , Glicólise/efeitos dos fármacos , Fator 15 de Diferenciação de Crescimento/genética , Mucosa Intestinal/efeitos dos fármacos , Mucosa Intestinal/metabolismo , Masculino , Camundongos , Mitocôndrias/genética , Fosforilação Oxidativa/efeitos dos fármacos , TranscriptomaRESUMO
OBJECTIVES: To analyse the peptidomics of mouse enteroendocrine cells (EECs) and human gastrointestinal (GI) tissue and identify novel gut derived peptides. METHODS: High resolution nano-flow liquid chromatography mass spectrometry (LC-MS/MS) was performed on (i) flow-cytometry purified NeuroD1 positive cells from mouse and homogenised human intestinal biopsies, (ii) supernatants from primary murine intestinal cultures, (iii) intestinal homogenates from mice fed high fat diet. Candidate bioactive peptides were selected on the basis of species conservation, high expression/biosynthesis in EECs and evidence of regulated secretionin vitro. Candidate novel gut-derived peptides were chronically administered to mice to assess effects on food intake and glucose tolerance. RESULTS: A large number of peptide fragments were identified from human and mouse, including known full-length gut hormones and enzymatic degradation products. EEC-specific peptides were largely from vesicular proteins, particularly prohormones, granins and processing enzymes, of which several exhibited regulated secretion in vitro. No regulated peptides were identified from previously unknown genes. High fat feeding particularly affected the distal colon, resulting in reduced peptide levels from GCG, PYY and INSL5. Of the two candidate novel peptides tested in vivo, a peptide from Chromogranin A (ChgA 435-462a) had no measurable effect, but a progastrin-derived peptide (Gast p59-79), modestly improved glucose tolerance in lean mice. CONCLUSION: LC-MS/MS peptidomic analysis of murine EECs and human GI tissue identified the spectrum of peptides produced by EECs, including a potential novel gut hormone, Gast p59-79, with minor effects on glucose tolerance.
Assuntos
Células Enteroendócrinas/metabolismo , Gastrinas/farmacologia , Trato Gastrointestinal/metabolismo , Teste de Tolerância a Glucose/métodos , Peptídeos/metabolismo , Precursores de Proteínas/farmacologia , Proteoma/metabolismo , Magreza/tratamento farmacológico , Animais , Células Cultivadas , Glucose/metabolismo , Humanos , Masculino , Camundongos , Modelos Animais , Peptídeos/química , Proteoma/análise , Magreza/metabolismoRESUMO
Neuronatin (Nnat) has previously been reported to be part of a network of imprinted genes downstream of the chromatin regulator Trim28. Disruption of Trim28 or of members of this network, including neuronatin, results in an unusual phenotype of a bimodal body weight. To better characterise this variability, we examined the key contributors to energy balance in Nnat+/-p mice that carry a paternal null allele and do not express Nnat. Consistent with our previous studies, Nnat deficient mice on chow diet displayed a bimodal body weight phenotype with more than 30% of Nnat+/-p mice developing obesity. In response to both a 45% high fat diet and exposure to thermoneutrality (30 °C) Nnat deficient mice maintained the hypervariable body weight phenotype. Within a calorimetry system, food intake in Nnat+/-p mice was hypervariable, with some mice consuming more than twice the intake seen in wild type littermates. A hyperphagic response was also seen in Nnat+/-p mice in a second, non-home cage environment. An expected correlation between body weight and energy expenditure was seen, but corrections for the effects of positive energy balance and body weight greatly diminished the effect of neuronatin deficiency on energy expenditure. Male and female Nnat+/-p mice displayed subtle distinctions in the degree of variance body weight phenotype and food intake and further sexual dimorphism was reflected in different patterns of hypothalamic gene expression in Nnat+/-p mice. Loss of the imprinted gene Nnat is associated with a highly variable food intake, with the impact of this phenotype varying between genetically identical individuals.
Assuntos
Ingestão de Alimentos/fisiologia , Proteínas de Membrana/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Obesidade/metabolismo , Animais , Biomarcadores/metabolismo , Peso Corporal , Dieta Hiperlipídica , Metabolismo Energético , Feminino , Perfilação da Expressão Gênica , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Obesidade/etiologia , Obesidade/patologiaRESUMO
This protocol describes the peptidomic analysis of organoid lysates, FACS-purified cell populations, and 2D culture secretions by liquid chromatography mass spectrometry (LC-MS). Currently, most peptides are quantified by ELISA, limiting the peptides that can be studied. However, an LC-MS-based approach allows more peptides to be monitored. Our group has previously used LC-MS for tissue peptidomics and secretion of enteroendocrine peptides from primary culture. Now, we extend the use to organoid models. For complete details on the use and execution of this protocol, please refer to Goldspink et al. (2020).
Assuntos
Organoides/metabolismo , Peptídeos/metabolismo , Proteômica/métodos , Espectrometria de Massas em Tandem , Sequência de Aminoácidos , Cromatografia Líquida , Citometria de Fluxo , Humanos , Peptídeos/química , Peptídeos/isolamento & purificaçãoRESUMO
CONTEXT: The gastrointestinal hormone ghrelin stimulates growth hormone secretion and appetite, but recent studies indicate that ghrelin also stimulates the secretion of the appetite-inhibiting and insulinotropic hormone glucagon-like peptide-1 (GLP-1). OBJECTIVE: To investigate the putative effect of ghrelin on GLP-1 secretion in vivo and in vitro. SUBJECTS AND METHODS: A randomized placebo-controlled crossover study was performed in eight hypopituitary subjects. Ghrelin or saline was infused intravenously (1 pmol/min × kg) after collection of baseline sample (0 min), and blood was subsequently collected at time 30, 60, 90, and 120 minutes. Mouse small intestine was perfused (n = 6) and GLP-1 output from perfused mouse small intestine was investigated in response to vascular ghrelin administration in the presence and absence of a simultaneous luminal glucose stimulus. Ghrelin receptor expression was quantified in human (n = 11) and mouse L-cells (n = 3) by RNA sequencing and RT-qPCR, respectively. RESULTS: Ghrelin did not affect GLP-1 secretion in humans (area under the curve [AUC; 0-120 min]: ghrelin infusion = 1.37 ± 0.05 min × nmol vs. saline infusion = 1.40 ± 0.06 min × nmol [P = 0.63]), but induced peripheral insulin resistance. Likewise, ghrelin did not stimulate GLP-1 secretion from the perfused mouse small intestine model (mean outputs during baseline/ghrelin infusion = 19.3 ± 1.6/25.5 ± 2.0 fmol/min, n = 6, P = 0.16), whereas glucose-dependent insulinotropic polypeptide administration, used as a positive control, doubled GLP-1 secretion (P < 0.001). Intraluminal glucose increased GLP-1 secretion by 4-fold (P < 0.001), which was not potentiated by ghrelin. Finally, gene expression of the ghrelin receptor was undetectable in mouse L-cells and marginal in human L-cells. CONCLUSIONS: Ghrelin does not interact directly with the L-cell and does not directly affect GLP-1 secretion.
Assuntos
Grelina/farmacologia , Peptídeo 1 Semelhante ao Glucagon/efeitos dos fármacos , Peptídeo 1 Semelhante ao Glucagon/metabolismo , Administração Intravenosa , Adulto , Idoso , Animais , Células Cultivadas , Estudos Cross-Over , Dinamarca , Método Duplo-Cego , Grelina/administração & dosagem , Grelina/sangue , Peptídeo 1 Semelhante ao Glucagon/sangue , Humanos , Hipopituitarismo/sangue , Hipopituitarismo/metabolismo , Mucosa Intestinal/efeitos dos fármacos , Mucosa Intestinal/metabolismo , Intestinos/efeitos dos fármacos , Células L , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Pessoa de Meia-Idade , Placebos , Via Secretória/efeitos dos fármacos , Regulação para Cima/efeitos dos fármacosRESUMO
CONTEXT: Glucagon-like peptide-1 (GLP-1) is an incretin hormone used therapeutically in type 2 diabetes and obesity. The interplay between ambient free fatty acids (FFAs) and GLP-1 remains unclear. Acipimox suppresses adipose tissue lipolysis via activation of the PUMA-G (also known as HCA2 and GPR109a) receptor. OBJECTIVE: To investigate whether lowering of serum FFA level with acipimox affects GLP-1 secretion. DESIGN: Two randomized crossover studies were performed in human subjects. Rat intestine was perfused intra-arterially and intraluminally, and l-cells were incubated with acipimox. PARTICIPANTS: The participants were healthy overweight subjects and hypopituitary adult patients. INTERVENTIONS: The overweight participants received acipimox 250 mg 60 minutes before an oral glucose test. The hypopituitary patients received acipimox 250 mg 12, 9, and 2 hours before and during the metabolic study day, when they were studied in the basal state and during a hyperinsulinemic euglycemic clamp. RESULTS: Acipimox suppressed FFA but did not affect insulin in the clinical trials. In overweight subjects, the GLP-1 increase after the oral glucose tolerance test (area under the curve) was more than doubled [4119 ± 607 pmol/L × min (Acipimox) vs 1973 ± 375 pmol/L × min (control), P = 0.004]. In hypopituitary patients, acipimox improved insulin sensitivity (4.7 ± 0.8 mg glucose/kg/min (Acipimox) vs 3.1 ± 0.5 mg glucose/kg/min (control), P = 0.005], and GLP-1 concentrations increased ~40%. An inverse correlation between FFA and GLP-1 concentrations existed in both trials. In rat intestine, acipimox did not affect GLP-1 secretion, and l-cells did not consistently express the putative receptor for acipimox. CONCLUSIONS: Acipimox treatment increases systemic GLP-1 levels in both obese subjects and hypopituitary patients. Our in vitro data indicate that the underlying mechanisms are indirect.
Assuntos
Peptídeo 1 Semelhante ao Glucagon/metabolismo , Hipolipemiantes/administração & dosagem , Hipopituitarismo/tratamento farmacológico , Sobrepeso/tratamento farmacológico , Pirazinas/administração & dosagem , Adulto , Animais , Glicemia/análise , Glicemia/metabolismo , Células Cultivadas , Estudos Cross-Over , Células Enteroendócrinas/efeitos dos fármacos , Células Enteroendócrinas/metabolismo , Ácidos Graxos não Esterificados/metabolismo , Teste de Tolerância a Glucose , Humanos , Hipopituitarismo/sangue , Hipopituitarismo/metabolismo , Resistência à Insulina , Mucosa Intestinal/citologia , Lipólise/efeitos dos fármacos , Masculino , Pessoa de Meia-Idade , Sobrepeso/sangue , Sobrepeso/metabolismo , Projetos Piloto , Cultura Primária de Células , RatosRESUMO
OBJECTIVE: Enteroendocrine cells (EECs) of the large intestine, found scattered in the epithelial layer, are known to express different hormones, with at least partial co-expression of different hormones in the same cell. Here we aimed to categorize colonic EECs and to identify possible targets for selective recruitment of hormones. METHODS: Single cell RNA-sequencing of sorted enteroendocrine cells, using NeuroD1-Cre x Rosa26-EYFP mice, was used to cluster EECs from the colon and rectum according to their transcriptome. G-protein coupled receptors differentially expressed across clusters were identified, and, as a proof of principle, agonists of Agtr1a and Avpr1b were tested as candidate EEC secretagogues in vitro and in vivo. RESULTS: EECs from the large intestine separated into 7 clear clusters, 4 expressing higher levels of Tph1 (enzyme required for serotonin (5-HT) synthesis; enterochromaffin cells), 2 enriched for Gcg (encoding glucagon-like peptide-1, GLP-1, L-cells), and the 7th expressing somatostatin (D-cells). Restricted analysis of L-cells identified 4 L-cell sub-clusters, exhibiting differential expression of Gcg, Pyy (Peptide YY), Nts (neurotensin), Insl5 (insulin-like peptide 5), Cck (cholecystokinin), and Sct (secretin). Expression profiles of L- and enterochromaffin cells revealed the clustering to represent gradients along the crypt-surface (cell maturation) and proximal-distal gut axes. Distal colonic/rectal L-cells differentially expressed Agtr1a and the ligand angiotensin II was shown to selectively increase GLP-1 and PYY release in vitro and GLP-1 in vivo. CONCLUSION: EECs in the large intestine exhibit differential expression gradients along the crypt-surface and proximal-distal axes. Distal L-cells can be differentially stimulated by targeting receptors such as Agtr1a.
Assuntos
Células Enteroendócrinas/metabolismo , Peptídeo 1 Semelhante ao Glucagon/metabolismo , Insulina/metabolismo , Proteínas/metabolismo , Transcriptoma , Animais , Células Enteroendócrinas/citologia , Feminino , Peptídeo 1 Semelhante ao Glucagon/genética , Insulina/genética , Intestino Grosso/citologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Peptídeo YY/genética , Peptídeo YY/metabolismo , Proteínas/genética , Receptor Tipo 1 de Angiotensina/genética , Receptor Tipo 1 de Angiotensina/metabolismo , Análise de Célula Única , Triptofano Hidroxilase/genética , Triptofano Hidroxilase/metabolismoRESUMO
Bariatric surgery is widely used to treat obesity and improves type 2 diabetes beyond expectations from the degree of weight loss. Elevated post-prandial concentrations of glucagon-like peptide 1 (GLP-1), peptide YY (PYY), and insulin are widely reported, but the importance of GLP-1 in post-bariatric physiology remains debated. Here, we show that GLP-1 is a major driver of insulin secretion after bariatric surgery, as demonstrated by blocking GLP-1 receptors (GLP1Rs) post-gastrectomy in lean humans using Exendin-9 or in mice using an anti-GLP1R antibody. Transcriptomics and peptidomics analyses revealed that human and mouse enteroendocrine cells were unaltered post-surgery; instead, we found that elevated plasma GLP-1 and PYY correlated with increased nutrient delivery to the distal gut in mice. We conclude that increased GLP-1 secretion after bariatric surgery arises from rapid nutrient delivery to the distal gut and is a key driver of enhanced insulin secretion.