RESUMEN
Glucagon-like peptide 1 (GLP1), which is mainly processed and cleaved from proglucagon in enteroendocrine cells (EECs) of the intestinal tract, acts on the GLP1 receptor in pancreatic cells to stimulate insulin secretion and to inhibit glucagon secretion. However, GLP1 processing is not fully understood. Here, we show that reticulon 4B (Nogo-B), an endoplasmic reticulum (ER)-resident protein, interacts with the major proglucagon fragment of proglucagon to retain proglucagon on the ER, thereby inhibiting PCSK1-mediated cleavage of proglucagon in the Golgi. Intestinal Nogo-B knockout in male type 2 diabetes mellitus (T2DM) mice increases GLP1 and insulin levels and decreases glucagon levels, thereby alleviating pancreatic injury and insulin resistance. Finally, we identify aberrantly elevated Nogo-B expression and inhibited proglucagon cleavage in EECs from diabetic patients. Our study reveals the subcellular regulatory processes involving Nogo-B during GLP1 production and suggests intestinal Nogo-B as a potential therapeutic target for T2DM.
Asunto(s)
Diabetes Mellitus Tipo 2 , Retículo Endoplásmico , Péptido 1 Similar al Glucagón , Proteínas Nogo , Proglucagón , Proproteína Convertasa 1 , Animales , Humanos , Masculino , Ratones , Diabetes Mellitus Tipo 2/metabolismo , Diabetes Mellitus Tipo 2/genética , Retículo Endoplásmico/metabolismo , Células Enteroendocrinas/metabolismo , Péptido 1 Similar al Glucagón/metabolismo , Aparato de Golgi/metabolismo , Células HEK293 , Insulina/metabolismo , Resistencia a la Insulina , Intestinos/patología , Ratones Endogámicos C57BL , Ratones Noqueados , Proteínas Nogo/metabolismo , Proteínas Nogo/genética , Proglucagón/metabolismo , Proglucagón/genética , Proproteína Convertasa 1/metabolismo , Proproteína Convertasa 1/genética , Unión Proteica , ProteolisisRESUMEN
(1) Background: Proglucagon-derived peptides (PDGPs) including glucagon (Gcg), GLP-1, and GLP-2 regulate lipid metabolism in the liver, adipocytes, and intestine. However, the mechanism by which PGDPs participate in alterations in lipid metabolism induced by high-fat diet (HFD) feeding has not been elucidated. (2) Methods: Mice deficient in PGDP (GCGKO) and control mice were fed HFD for 7 days and analyzed, and differences in lipid metabolism in the liver, adipose tissue, and duodenum were investigated. (3) Results: GCGKO mice under HFD showed lower expression levels of the genes involved in free fatty acid (FFA) oxidation such as Hsl, Atgl, Cpt1a, Acox1 (p < 0.05), and Pparα (p = 0.05) mRNA in the liver than in control mice, and both FFA and triglycerides content in liver and adipose tissue weight were lower in the GCGKO mice. On the other hand, phosphorylation of hormone-sensitive lipase (HSL) in white adipose tissue did not differ between the two groups. GCGKO mice under HFD exhibited lower expression levels of Pparα and Cd36 mRNA in the duodenum as well as increased fecal cholesterol contents compared to HFD-controls. (4) Conclusions: GCGKO mice fed HFD exhibit a lesser increase in hepatic FFA and triglyceride contents and adipose tissue weight, despite reduced ß-oxidation in the liver, than in control mice. Thus, the absence of PGDP prevents dietary-induced fatty liver development due to decreased lipid uptake in the intestinal tract.
Asunto(s)
Antígenos CD36 , Dieta Alta en Grasa , Absorción Intestinal , Metabolismo de los Lípidos , Hígado , Ratones Noqueados , PPAR alfa , Proglucagón , Animales , Masculino , Dieta Alta en Grasa/efectos adversos , PPAR alfa/metabolismo , PPAR alfa/genética , Hígado/metabolismo , Proglucagón/metabolismo , Proglucagón/genética , Antígenos CD36/metabolismo , Antígenos CD36/genética , Ratones , Esterol Esterasa/metabolismo , Esterol Esterasa/genética , Triglicéridos/metabolismo , Ratones Endogámicos C57BL , Ácidos Grasos no Esterificados/metabolismo , Péptido 1 Similar al Glucagón/metabolismo , Duodeno/metabolismo , Carnitina O-Palmitoiltransferasa/metabolismo , Carnitina O-Palmitoiltransferasa/genética , Tejido Adiposo/metabolismo , Grasas de la Dieta , Péptido 2 Similar al Glucagón/metabolismo , Aciltransferasas , LipasaRESUMEN
Glucagon-like peptide-1 receptor (GLP-1R) agonists are now commonly used to treat type 2 diabetes and obesity. GLP-1R signaling in the spinal cord has been suggested to account for the mild tachycardia caused by GLP-1R agonists, and may also be involved in the therapeutic effects of these drugs. However, the neuroanatomy of the GLP-1/GLP-1R system in the spinal cord is still poorly understood. Here we applied in situ hybridization and immunohistochemistry to characterize this system, and its relation to cholinergic neurons. GLP-1R transcript and protein were expressed in neuronal cell bodies across the gray matter, in matching distribution patterns. GLP-1R-immunolabeling was also robust in dendrites and axons, especially in laminae II-III in the dorsal horn. Cerebrospinal fluid-contacting neurons expressed GLP-1R protein at exceedingly high levels. Only small subpopulations of cholinergic neurons expressed GLP-1R, including a subset of sympathetic preganglionic neurons at the rostral tip of the intermediolateral nucleus. GLP-1 axons innervated all regions where GLP-1R neurons were distributed, except laminae II-III. Scattered preproglucagon (Gcg) mRNA-expressing neurons were identified in the cervical and lumbar enlargements. The results will facilitate further studies on how GLP-1 regulates the sympathetic system and other autonomic and somatic functions via the spinal cord.
Asunto(s)
Péptido 1 Similar al Glucagón , Receptor del Péptido 1 Similar al Glucagón , Médula Espinal , Animales , Receptor del Péptido 1 Similar al Glucagón/metabolismo , Receptor del Péptido 1 Similar al Glucagón/genética , Masculino , Médula Espinal/metabolismo , Ratones , Péptido 1 Similar al Glucagón/metabolismo , Neuronas Colinérgicas/metabolismo , Proglucagón/metabolismo , Proglucagón/genética , Ratones Endogámicos C57BL , Axones/metabolismoRESUMEN
Proglucagon mRNA expression and GLP-1 secretion by cultured human L-cells (NCI-H716) were inhibited following exposure to λ-carrageenan, a commonly used additive in processed foods. Carrageenan is composed of sulfated or unsulfated galactose residues linked in alternating alpha-1,3 and beta-1,4 bonds and resembles the endogenous sulfated glycosaminoglycans. However, carrageenan has unusual alpha-1,3-galactosidic bonds, which are not innate to human cells and are implicated in immune responses. Exposure to carrageenan predictably causes inflammation, and carrageenan impairs glucose tolerance and contributes to insulin resistance. When cultured human L-cells were deprived overnight of glucose and serum and then exposed to high glucose, 10% FBS, and λ-carrageenan (1 µg/ml) for 10 minutes, 1 h, and 24 h, mRNA expression of proglucagon and secretion of GLP-1 were significantly reduced, compared to control cells not exposed to carrageenan. mRNA expression of proglucagon by mouse L-cells (STC-1) was also significantly reduced and supports the findings in the human cells. Exposure of co-cultured human intestinal epithelial cells (LS174T) to the spent media of the carrageenan-treated L-cells led to a decline in mRNA expression of GLUT-2 at 24 h. These findings suggest that ingestion of carrageenan-containing processed foods may impair the production of GLP-1, counteract the effect of GLP-1 receptor agonists and induce secondary effects on intestinal epithelial cells.
Asunto(s)
Carragenina , Células Enteroendocrinas , Aditivos Alimentarios , Péptido 1 Similar al Glucagón , Proglucagón , Carragenina/farmacología , Humanos , Péptido 1 Similar al Glucagón/metabolismo , Aditivos Alimentarios/farmacología , Proglucagón/metabolismo , Células Enteroendocrinas/metabolismo , Células Enteroendocrinas/efectos de los fármacos , Ratones , Animales , ARN Mensajero/metabolismo , Línea Celular , Glucosa/metabolismoRESUMEN
AIMS/HYPOTHESES: Glucagon and glucagon-like peptide-1 (GLP-1) are derived from the same precursor; proglucagon, and dual agonists of their receptors are currently being explored for the treatment of obesity and metabolic dysfunction-associated steatotic liver disease (MASLD). Elevated levels of endogenous glucagon (hyperglucagonaemia) have been linked with hyperglycaemia in individuals with type 2 diabetes but are also observed in individuals with obesity and MASLD. GLP-1 levels have been reported to be largely unaffected or even reduced in similar conditions. We investigated potential determinants of plasma proglucagon and associations of glucagon receptor signalling with metabolic diseases based on data from the UK Biobank. METHODS: We used exome sequencing data from the UK Biobank for ~410,000 white participants to identify glucagon receptor variants and grouped them based on their known or predicted signalling. Data on plasma levels of proglucagon estimated using Olink technology were available for a subset of the cohort (~40,000). We determined associations of glucagon receptor variants and proglucagon with BMI, type 2 diabetes and liver fat (quantified by liver MRI) and performed survival analyses to investigate if elevated proglucagon predicts type 2 diabetes development. RESULTS: Obesity, MASLD and type 2 diabetes were associated with elevated plasma levels of proglucagon independently of each other. Baseline proglucagon levels were associated with the risk of type 2 diabetes development over a 14 year follow-up period (HR 1.13; 95% CI 1.09, 1.17; n=1562; p=1.3×10-12). This association was of the same magnitude across strata of BMI. Carriers of glucagon receptor variants with reduced cAMP signalling had elevated levels of proglucagon (ß 0.847; 95% CI 0.04, 1.66; n=17; p=0.04), and carriers of variants with a predicted frameshift mutation had higher levels of liver fat compared with the wild-type reference group (ß 0.504; 95% CI 0.03, 0.98; n=11; p=0.04). CONCLUSIONS/INTERPRETATION: Our findings support the suggestion that glucagon receptor signalling is involved in MASLD, that plasma levels of proglucagon are linked to the risk of type 2 diabetes development, and that proglucagon levels are influenced by genetic variation in the glucagon receptor, obesity, type 2 diabetes and MASLD. Determining the molecular signalling pathways downstream of glucagon receptor activation may guide the development of biased GLP-1/glucagon co-agonist with improved metabolic benefits. DATA AVAILABILITY: All coding is available through https://github.com/nicwin98/UK-Biobank-GCG.
Asunto(s)
Bancos de Muestras Biológicas , Diabetes Mellitus Tipo 2 , Obesidad , Proglucagón , Receptores de Glucagón , Transducción de Señal , Humanos , Receptores de Glucagón/genética , Receptores de Glucagón/metabolismo , Reino Unido , Femenino , Proglucagón/metabolismo , Proglucagón/genética , Masculino , Diabetes Mellitus Tipo 2/sangre , Diabetes Mellitus Tipo 2/metabolismo , Persona de Mediana Edad , Obesidad/sangre , Anciano , Adulto , Índice de Masa Corporal , Glucagón/sangre , Péptido 1 Similar al Glucagón/sangre , Biobanco del Reino UnidoRESUMEN
OBJECTIVE: Glucagon is a critical hormone regulating glucose metabolism. It stimulates the liver to release glucose under low blood sugar conditions, thereby maintaining blood glucose stability. Excessive glucagon secretion and hyperglycemia is observed in individuals with diabetes. Precise modulation of glucagon is significant to maintain glucose homeostasis. Piezo1 is a mechanosensitive ion channel capable of converting extracellular mechanical forces into intracellular signals, thus regulating hormonal synthesis and secretion. This study aims to investigate the role of Piezo1 in regulating glucagon production in α cells. METHODS: The effects of Piezo1 on glucagon production were examined in normal- or high-fat diet fed α cell-specific Piezo1 knockout mice (Gcg-Piezo1-/-), and the murine pancreatic α cell line αTC1-6. Expression of Proglucagon was investigated by real-time PCR and western blotting. Plasma glucagon and insulin were detected by enzyme immunoassay. RESULTS: Under both normal- and high-fat diet conditions, Gcg-Piezo1-/- mice exhibited increased pancreatic α cell proportion, hyperglucagonemia, impaired glucose tolerance, and activated pancreatic mTORC1 signaling. Activation of Piezo1 by its agonist Yoda1 or overexpression of Piezo1 led to decreased glucagon synthesis and suppressed mTOR signaling pathway in αTC1-6 cells. Additionally, the levels of glucagon in the medium were also reduced. Conversely, knockdown of Piezo1 produced opposite effects. CONCLUSION: Our study uncovers the regulatory role of the Piezo1 ion channel in α cells. Piezo1 influences glucagon production by affecting mTOR signaling pathway.
Asunto(s)
Dieta Alta en Grasa , Células Secretoras de Glucagón , Glucagón , Canales Iónicos , Ratones Noqueados , Animales , Células Secretoras de Glucagón/metabolismo , Glucagón/metabolismo , Ratones , Canales Iónicos/metabolismo , Canales Iónicos/genética , Dieta Alta en Grasa/efectos adversos , Masculino , Transducción de Señal , Insulina/metabolismo , Línea Celular , Diana Mecanicista del Complejo 1 de la Rapamicina/metabolismo , Diana Mecanicista del Complejo 1 de la Rapamicina/genética , Mecanotransducción Celular , Ratones Endogámicos C57BL , Proglucagón/metabolismo , Proglucagón/genética , Pirazinas , TiadiazolesRESUMEN
Glucagon like peptide-1 (GLP-1) is a peptide hormone encoded by the pre-proglucagon gene that serves multiple physiological functions, including incretin action. While GLP-1 is primarily synthesized in the L cells of the lower intestine, recent findings indicate its presence in the stomachs of both rats and humans. However, the role of gastric GLP-1 in other species remains unclear. In this study, we aimed to identify GLP-1-producing cells and examine the localization of GLP-1 production in the mouse stomach. We found that pre-proglucagon mRNA was higher in the corpus than that in the antrum of the stomach. In addition, GLP-1 immunoreactive cells were found in the gastric mucosa, and their cell number was higher in the corpus than that in the antrum. Double immunofluorescence showed that some GLP-1 immunoreactive cells displayed somatostatin immunoreactivity, whereas did not co-localize with ghrelin and gastrin. Moreover, transmembrane G protein-coupled Receptor 5 (TGR5) agonist decreased pre-proglucagon mRNA expression in SG-1 cells in a concentration-dependent manner, and in vivo experiments showed a decrease in its mRNA levels in the gastric corpus but not in the antrum. This study marks the first report of GLP-1 production in the mouse stomach. Our findings suggest that gastric pre-proglucagon mRNA expression is regulated by a distinct mechanism compared to the L cells of the lower intestine.
Asunto(s)
Péptido 1 Similar al Glucagón , Estómago , Animales , Ratones , Péptido 1 Similar al Glucagón/metabolismo , Receptor del Péptido 1 Similar al Glucagón , Intestinos/metabolismo , Proglucagón/metabolismo , ARN Mensajero/genética , Estómago/metabolismoRESUMEN
We investigated the role of dietary carbohydrates in the maintenance of the enterocyte microvillar structure in the chicken ileum. Male chickens were divided into the control and three experimental groups, and the experimental groups were fed diets containing 50%, 25%, and 0% carbohydrates of the control diet. The structural alterations in enterocytes were examined using transmission electron microscopy and immunofluorescent techniques for ß-actin and villin. Glucagon-like peptide (GLP)-2 and proglucagon mRNA were detected by immunohistochemistry and in situ hybridization, respectively. Fragmentation and wide gap spaces were frequently observed in the microvilli of the 25% and 0% groups. The length, width, and density of microvilli were also decreased in the experimental groups. The experimental groups had shorter terminal web extensions, and there were substantial changes in the mitochondrial density between the control and experimental groups. Intensities of ß-actin and villin immunofluorescence observed on the apical surface of enterocytes were lower in the 0% group. The frequency of GLP-2-immunoreactive and proglucagon mRNA-expressing cells decreased with declining dietary carbohydrate levels. This study revealed that dietary carbohydrates contribute to the structural maintenance of enterocyte microvilli in the chicken ileum. The data from immunohistochemistry and in situ hybridization assays suggest the participation of GLP-2 in this maintenance system.
Asunto(s)
Pollos , Enterocitos , Masculino , Animales , Pollos/genética , Proglucagón/genética , Actinas , Carbohidratos de la Dieta , Íleon , Péptido 2 Similar al Glucagón , ARN Mensajero/genética , MicrovellosidadesRESUMEN
BACKGROUND: Bariatric surgery has long-term beneficial effects on body weight and metabolic status, but there is an apparent lack of comprehensive cardiometabolic, renal, liver, and metabolomic/lipidomic panels, whereas the underlying mechanisms driving the observed postoperative ameliorations are still poorly investigated. We aimed to study the long-term effects of bariatric surgery on metabolic profile, cardiorenal and liver outcomes in association with underlying postoperative gut hormone adaptations. METHODS: 28 individuals who underwent bariatric surgery [17 sleeve gastrectomy (SG), 11 Roux-en-Y gastric bypass (RYGB)] were followed up 3, 6 and 12 and at 10 years following surgery. Participants at 10 years were cross-sectionally compared with an age-, sex- and adiposity-matched group of non-operated individuals (n = 9) and an age-matched pilot group of normal-weight individuals (n = 4). RESULTS: There were durable effects of surgery on body weight and composition, with an increase of lean mass percentage persisting despite some weight regain 10 years postoperatively. The improvements in metabolic and lipoprotein profiles, cardiometabolic risk markers, echocardiographic and cardiorenal outcomes persisted over the ten-year observation period. The robust improvements in insulin resistance, adipokines, activin/follistatin components and postprandial gastrointestinal peptide levels persisted 10 years postoperatively. These effects were largely independent of surgery type, except for a lasting reduction of ghrelin in the SG subgroup, and more pronounced increases in proglucagon products, mainly glicentin and oxyntomodulin, and in the cardiovascular risk marker Trimethylamine-N-oxide (TMAO) within the RYGB subgroup. Despite similar demographic and clinical features, participants 10 years after surgery showed a more favorable metabolic profile compared with the control group, in conjunction with a dramatic increase of postprandial proglucagon product secretion. CONCLUSIONS: We demonstrate that cardiorenal and metabolic benefits of bariatric surgery remain robust and largely unchanged ten years postoperatively and are associated with durable effects on gastrointestinal- muscle- and adipose tissue-secreted hormones. TRIAL REGISTRATION: ClinicalTrials.gov: NCT04170010.
Asunto(s)
Cirugía Bariátrica , Enfermedades Cardiovasculares , Derivación Gástrica , Hormonas Gastrointestinales , Obesidad Mórbida , Humanos , Estudios de Casos y Controles , Proglucagón , Obesidad/cirugía , Hígado , Enfermedades Cardiovasculares/prevención & control , Gastrectomía , Obesidad Mórbida/cirugíaRESUMEN
BACKGROUND: Active glucagon-like peptide-1 (aGLP-1) has been implicated in the pathogenesis of equine insulin dysregulation (ID), but its role is unclear. Cleavage of proglucagon (coded by the GCG gene) produces aGLP-1 in enteral L cells. OBJECTIVES: The aim in vivo was to examine the sequence of the exons of GCG in horses with and without ID, where aGLP-1 was higher in the group with ID. The aims in vitro were to identify and quantify the expression of GCG in the equine intestine (as a marker of L cells) and determine intestinal secretion of aGLP-1. STUDY DESIGN: Genomic studies were case-control studies. Expression and secretion studies in vitro were cross-sectional. METHODS: The GCG gene sequence of the exons was determined using a hybridisation capture protocol. Expression and quantification of GCG in samples of stomach duodenum, jejunum, ileum, caecum and ascending and descending colon was achieved with droplet digital PCR. For secretory studies tissue explants were incubated with 12 mM glucose and aGLP-1 secretion was measured with an ELISA. RESULTS: Although the median [IQR] post-prandial aGLP-1 concentrations were higher (p = 0.03) in animals with ID (10.2 [8.79-15.5]), compared with healthy animals (8.47 [6.12-11.7]), there was 100% pairwise identity of the exons of the GCG sequence for the cohort. The mRNA concentrations of GCG and secretion of aGLP-1 differed (p < 0.001) throughout the intestine. MAIN LIMITATIONS: Only the exons of the GCG gene were sequenced and breeds were not compared. The horses used for the study in vitro were not assessed for ID and different horses were used for the small, and large, intestinal studies. CONCLUSIONS: Differences in post-prandial aGLP-1 concentration were not due to a variant in the exons of the GCG gene sequence in this cohort. Both the large and small intestine are sites of GLP-1 secretion.
Asunto(s)
Péptido 1 Similar al Glucagón , Insulina , Humanos , Animales , Caballos/genética , Péptido 1 Similar al Glucagón/genética , Insulina/metabolismo , Intestino Delgado/metabolismo , Proglucagón/genética , Proglucagón/análisis , Proglucagón/metabolismo , Reacción en Cadena de la Polimerasa/veterinariaRESUMEN
The biological activity of glucagon has recently been proposed to both stimulate hepatic glucose production and also include a paradoxical insulinotropic effect, which could suggest a new role of glucagon in the pathophysiology type 2 diabetes mellitus (T2DM). An insulinotropic role of glucagon has been observed after bariatric/metabolic surgery that is mediated through the GLP-1 receptor on pancreatic beta cells. This effect appears to be modulated by other members of the proglucagon family, playing a key role in the beneficial effects and complications of bariatric/metabolic surgery. Glucagon serves a dual role after sleeve gastrectomy (SG) and Roux-en-Y gastric bypass (RYGB). In addition to maintaining blood glucose levels, glucagon exhibits an insulinotropic effect, suggesting that glucagon has a more complex function than simply an "anti-insulin hormone".
Asunto(s)
Cirugía Bariátrica , Diabetes Mellitus Tipo 2 , Humanos , Glucagón , Insulina , Diabetes Mellitus Tipo 2/cirugía , ProglucagónRESUMEN
The family of proglucagon peptides Includes glucagon and glucagon-like peptide 1 (GLP-1), two unique peptides derived from the same prohormone. Despite numerous similarities between the peptides, these have long been viewed as having opposing actions on metabolism. GLP-1 is described as a postprandial hormone that stimulates anabolic actions via insulin, while glucagon is viewed as a fasting hormone that drives catabolic actions to maintain euglycemia. Here, we revisit a classic article in Diabetes that first established that glucagon and GLP-1 have more in common than previously appreciated, including actions at the same receptor. Furthermore, we discuss how the impact of this observation has guided research decades later that has reshaped the view of how proglucagon hormones regulate metabolism.
Asunto(s)
Glucagón , Insulina , Proglucagón , Péptido 1 Similar al Glucagón , AyunoRESUMEN
BACKGROUND & AIMS: We measured all proglucagon-derived peptides (PGDPs) levels in response to administration of three mixed meal tolerance tests (MMTs), examining differences in postprandial PGDP responses in subjects with leanness and obesity or between high-fat vs. high carbohydrate meals. METHODS: We designed three physiology interventional studies, administering MMTs over a 180-min period to individuals without diabetes after an overnight fast. In Study 1, a 450 kcal MMT was administered to n = 4 normal weight and n = 9 individuals with obesity. In Study 2, a 600 kcal high-fat MMT was administered to n = 15 normal-weight and n = 15 individuals with obesity. In Study 3, n = 32 participants with obesity were assigned to receive a 600-kcal high-fat (n = 15) or an isocaloric high-carbohydrate MMT (n = 17). Fasting and postprandial levels of c-peptide and PGDPs (proglucagon, GLP-1, GLP-2, glicentin, oxyntomodulin, glucagon, major proglucagon fragment [MPGF]) were assessed. RESULTS: In study 1, individuals with normal weight displayed elevated glicentin postprandial secretion compared with people with obesity (p = 0.002). Following a high-fat MMT with 33% higher energy content in study 2, all postprandial PGDPs levels were elevated (p-time<0.001), irrespective of weight status. In study 3, a prolonged postprandial upregulation of PGDPs during the high-fat MMT was observed in contrast with the acute, short-term (max 60 min) PGDP responses to a high-carbohydrate MMT (p-time∗meal<0.001). Across both studies 2 and 3, the postprandial responses of glucagon and MPGF were higher in subjects with male sex whereas glicentin was higher in subjects with female sex. CONCLUSIONS: Fat and carbohydrate content of a meal can substantially affect the postprandial levels of PGDPs. Circulating levels of PGDPs are influenced by the energy content of the meal, and additionally, the presence of leanness or obesity affects circulating levels of select PGDPs. These results, which are to be confirmed by additional studies, expand our understanding of PGDP physiology in leanness and obesity. CLINICALTRIALS: GOV REGISTRATION NUMBERS: (NCT04170010, NCT04430946, NCT04575194).
Asunto(s)
Glucagón , Delgadez , Masculino , Humanos , Femenino , Proglucagón , Glicentina , Obesidad , Péptidos , Glucemia , Comidas , Periodo PosprandialRESUMEN
AIMS/INTRODUCTION: Glucagon is secreted from pancreatic α-cells and plays an important role in amino acid metabolism in liver. Various animal models deficient in glucagon action show hyper-amino acidemia and α-cell hyperplasia, indicating that glucagon contributes to feedback regulation between the liver and the α-cells. In addition, both insulin and various amino acids, including branched-chain amino acids and alanine, participate in protein synthesis in skeletal muscle. However, the effect of hyperaminoacidemia on skeletal muscle has not been investigated. In the present study, we examined the effect of blockade of glucagon action on skeletal muscle using mice deficient in proglucagon-derived peptides (GCGKO mice). MATERIALS AND METHODS: Muscles isolated from GCGKO and control mice were analyzed for their morphology, gene expression and metabolites. RESULTS: GCGKO mice showed muscle fiber hypertrophy, and a decreased ratio of type IIA and an increased ratio of type IIB fibers in the tibialis anterior. The expression levels of myosin heavy chain (Myh) 7, 2, 1 and myoglobin messenger ribonucleic acid were significantly lower in GCGKO mice than those in control mice in the tibialis anterior. GCGKO mice showed a significantly higher concentration of arginine, asparagine, serine and threonine in the quadriceps femoris muscles, and also alanine, aspartic acid, cysteine, glutamine, glycine and lysine, as well as four amino acids in gastrocnemius muscles. CONCLUSIONS: These results show that hyperaminoacidemia induced by blockade of glucagon action in mice increases skeletal muscle weight and stimulates slow-to-fast transition in type II fibers of skeletal muscle, mimicking the phenotype of a high-protein diet.
Asunto(s)
Glucagón , Músculo Esquelético , Proglucagón , Animales , Ratones , Aminoácidos , Glucagón/metabolismo , Músculo Esquelético/metabolismo , Proglucagón/genética , Proglucagón/metabolismoRESUMEN
A relatively new pharmacological target in obesity treatment has been the preproglucagon (PPG) signalling, predominantly with glucagon-like peptide (GLP) 1 receptor agonists. As far as the PPG role within the digestive system is well recognised, its actions in the brain remain understudied. Here, we investigated PPG signalling in the Dorsomedial Hypothalamus (DMH), a structure involved in feeding regulation and metabolism, using in situ hybridisation, electrophysiology, and immunohistochemistry. Our experiments were performed on animals fed both control, and high-fat diet (HFD), uncovering HFD-mediated alterations. First, sensitivity to exendin-4 (Exn4, a GLP1R agonist) was shown to increase under HFD, with a higher number of responsive neurons. The amplitude of the response to both Exn4 and oxyntomodulin (Oxm) was also altered, diminishing its relationship with the cells' spontaneous firing rate. Not only neuronal sensitivity, but also GLP1 presence, and therefore possibly release, was influenced by HFD. Immunofluorescent labelling of the GLP1 showed changes in its density depending on the metabolic state (fasted/fed), but this effect was eliminated by HFD feeding. Interestingly, these dietary differences were absent after a period of restricted feeding, allowing for an anticipation of the alternating metabolic states, which suggests possible prevention of such outcome.
Asunto(s)
Dieta Alta en Grasa , Hipotálamo , Proglucagón , Transducción de Señal , Animales , Ratas , Hipotálamo/fisiología , Proglucagón/metabolismo , Ratas Sprague-Dawley , Masculino , Receptor del Péptido 1 Similar al Glucagón/genética , Receptor del Péptido 1 Similar al Glucagón/metabolismo , Receptor del Péptido 2 Similar al Glucagón/genética , Receptor del Péptido 2 Similar al Glucagón/metabolismo , ARN Mensajero/metabolismo , Neuronas/metabolismo , Sinapsis , Fibras Nerviosas/metabolismo , Electrofisiología , Proteínas Proto-Oncogénicas c-fos/metabolismo , Respuesta de Saciedad , Conducta AlimentariaRESUMEN
AIM: To investigate the changes of circulating levels of all proglucagon-derived peptides (PGDPs) in individuals with overweight or obesity receiving liraglutide (3 mg) or naltrexone/bupropion (32/360 mg), and to explore the association between induced changes in postprandial PGDP levels and body composition, as well as metabolic variables, after 3 and 6 months on treatment. MATERIALS AND METHODS: Seventeen patients with obesity or with overweight and co-morbidities, but without diabetes, were assigned to receive once-daily oral naltrexone/bupropion 32/360 mg (n = 8) or once-daily subcutaneous liraglutide 3 mg (n = 9). Participants were assessed before treatment initiation and after 3 and 6 months on treatment. At the baseline and 3-month visits, participants underwent a 3-hour mixed meal tolerance test to measure fasting and postprandial levels of PGDPs, C-peptide, hunger and satiety. Clinical and biochemical indices of metabolic function, magnetic resonance-assessed liver steatosis and ultrasound-assessed liver stiffness were measured at each visit. RESULTS: Both medications improved body weight and composition, carbohydrate and lipid metabolism, and liver fat and function. Naltrexone/bupropion produced a weight-independent increase in the levels of proglucagon (P < .001) and decreases in glucagon-like peptide-2 (GLP-2), glucagon and the major proglucagon fragment (P ≤ .01), whereas liraglutide markedly upregulated total glucagon-like peptide-1 (GLP-1) levels in a weight-independent manner (P = .04), and similarly downregulated the major proglucagon fragment, GLP-2 and glucagon (P < .01). PGDP levels at the 3-month visit were positively and independently correlated with improvements in fat mass, glycaemia, lipaemia and liver function, and negatively with reductions in fat-free mass, at both the 3- and 6-month visits. CONCLUSIONS: PGDP levels in response to liraglutide and naltrexone/bupropion are associated with improvements in metabolism. Our study provides support for the administration of the downregulated members of the PGDP family as replacement therapy (e.g. glucagon), in addition to the medications currently in use that induced their downregulation (e.g. GLP-1), and future studies should explore whether the addition of other PGDPs (e.g. GLP-2) could offer additional benefits.
Asunto(s)
Péptido 1 Similar al Glucagón , Glucagón , Humanos , Proglucagón , Glucagón/metabolismo , Liraglutida/farmacología , Liraglutida/uso terapéutico , Bupropión/uso terapéutico , Naltrexona/uso terapéutico , Sobrepeso , Péptidos/farmacología , Pérdida de Peso , Péptido 2 Similar al Glucagón , Obesidad/tratamiento farmacológico , Péptidos Similares al Glucagón/farmacologíaRESUMEN
AIMS/INTRODUCTION: Glucagon, a peptide hormone produced from proglucagon, is involved in the pathophysiology of diabetes. Plasma glucagon levels are currently measured by sandwich enzyme-linked immunosorbent assay (ELISA), but the currently used sandwich ELISA cross-reacts with proglucagon-derived peptides, thereby providing incorrect results in subjects with elevated plasma proglucagon-derived peptide levels. We aimed to develop a more broadly reliable ELISA for measuring plasma glucagon levels. MATERIALS AND METHODS: A new sandwich ELISA was developed using newly generated monoclonal antibodies against glucagon. After its validation, plasma glucagon levels were measured with the new ELISA and the currently used ELISA in subjects who underwent laparoscopic sleeve gastrectomy (LSG) and in outpatients with suspected glucose intolerance. The ELISA results were compared with those from liquid chromatography-high resolution mass (LC-HRMS) analysis, which we previously established as the most accurate measuring system. RESULTS: The new ELISA has high specificity (<1% cross-reactivities) and high sensitivity (a lower range of 0.31 pmol/L). Plasma glucagon values in the subjects who underwent laparoscopic sleeve gastrectomy and some outpatients with suspected glucose intolerance differed between the new ELISA and the currently used ELISA. These subjects also showed markedly high plasma glicentin levels. Despite the elevated plasma glicentin levels, the new ELISA showed better positive correlation with LC-HRMS than did the currently used ELISA. CONCLUSIONS: The new ELISA enables more accurate measurement of plasma glucagon than the currently used ELISA, even in subjects with elevated proglucagon-derived peptide levels. It should be clinically useful in elucidating the pathophysiology of individual diabetic patients.
Asunto(s)
Diabetes Mellitus , Intolerancia a la Glucosa , Hormonas Peptídicas , Humanos , Glucagón , Proglucagón , Glicentina , Intolerancia a la Glucosa/diagnóstico , Glucosa , Ensayo de Inmunoadsorción Enzimática/métodosRESUMEN
Insulin secretion from ß-cells is tightly regulated by local signaling from preproglucagon (Gcg) products from neighboring α-cells. Physiological paracrine signaling within the microenvironment of the ß-cell is altered after metabolic stress, such as high-fat diet or the ß-cell toxin, streptozotocin (STZ). Here, we examined the role and source of Gcg peptides in ß-cell function and in response to STZ-induced hyperglycemia. We used whole body Gcg null (GcgNull) mice and mice with Gcg expression either specifically within the pancreas (GcgΔPanc) or the intestine (GcgΔIntest). With lower doses of STZ exposure, insulin levels were greater and glucose levels were lower in GcgNull mice compared with wild-type mice. When Gcg was functional only in the intestine, plasma glucagon-like peptide-1 (GLP-1) levels were fully restored but these mice did not have any additional protection from STZ-induced diabetes. Pancreatic Gcg reactivation normalized the hyperglycemic response to STZ. In animals not treated with STZ, GcgNull mice had increased pancreas mass via both α- and ß-cell hyperplasia and reactivation of Gcg in the intestine normalized ß- but not α-cell mass, whereas pancreatic reactivation normalized both ß- and α-cell mass. GcgNull and GcgΔIntest mice maintained higher ß-cell mass after treatment with STZ compared with control and GcgΔPanc mice. Although in vivo insulin response to glucose was normal, global lack of Gcg impaired glucose-stimulated insulin secretion in isolated islets. Congenital replacement of Gcg either in the pancreas or intestine normalized glucose-stimulated insulin secretion. Interestingly, mice that had intestinal Gcg reactivated in adulthood had impaired insulin response to KCl. We surmise that the expansion of ß-cell mass in the GcgNull mice compensated for decreased individual ß-cell insulin secretion, which is sufficient to normalize glucose under physiological conditions and conferred some protection after STZ-induced diabetes.NEW & NOTEWORTHY We examined the role of Gcg on ß-cell function under normal and high glucose conditions. GcgNull mice had decreased glucose-stimulated insulin secretion, increased ß-cell mass, and partial protection against STZ-induced hyperglycemia. Expression of Gcg within the pancreas normalized these endpoints. Intestinal expression of Gcg only normalized ß-cell mass and glucose-stimulated insulin secretion. Increased ß-cell mass in GcgNull mice likely compensated for decreased insulin secretion normalizing physiological glucose levels and conferring some protection after STZ-induced diabetes.
Asunto(s)
Diabetes Mellitus Experimental , Células Secretoras de Glucagón , Hiperglucemia , Ratones , Animales , Proglucagón/genética , Proglucagón/metabolismo , Estreptozocina , Insulina/metabolismo , Diabetes Mellitus Experimental/metabolismo , Glucosa/farmacología , Ratones Noqueados , Células Secretoras de Glucagón/metabolismo , Glucemia/metabolismoRESUMEN
As action potentials propagate along an axon, pulsed extracellular electric fields (E-fields) are induced. We investigated the role of E-fields in activating microglia cells and affecting capillary function and found that E-fields control human microglia secretions in concert with purinergic factors. We generated E-fields by applying transcranial pulsed electromagnetic fields (T-PEMF) identical to those appearing outside neurons as action potentials propagate. T-PEMF alone enhanced mRNA synthesis for VEGF, IL-8, IL-6 and the proglucagon gene as well as the PC1/3 enzyme that cleaves the proglucagon protein to glucagon and GLP-1 proteins. We found that T-PEMF enhanced secretion from microglia of VEGF, IL-8 and GLP-1 proteins having angiogenic and proliferative profiles. Interestingly, T-PEMF and purinergic transmitters together enhanced secretions confirming synergy between their actions. ATP also induced nitric oxide (NO) syntheses in distinct locations in the nucleus and the mRNA synthesis for the responsible iNOS was reduced by T-PEMF. When the microglia-secretory fluid was added to brain endothelial cells we saw vivid Ca2+ signaling and enhanced transcription of mRNA for IL-8 and VEGF. Our previous work shows that applying T-PEMF to the human brain provides up to 60% remission for patients with refractory depressions within 8 weeks and improvements for Parkinson patients. Thus, physiological E-fields activate microglia, work synergistically with neurotransmitters, and cause paracrine secretions which cause activation of capillaries. Application of these E-Fields is effective for treating refractory depressions and appear promising for treating neurodegenerative brain diseases.
Asunto(s)
Microglía , Factor A de Crecimiento Endotelial Vascular , Humanos , Microglía/metabolismo , Factor A de Crecimiento Endotelial Vascular/metabolismo , Interleucina-8 , Células Endoteliales/metabolismo , Péptido 1 Similar al Glucagón , Comunicación Paracrina , Proglucagón , Factores de Transcripción , ARN Mensajero , Campos ElectromagnéticosRESUMEN
Toll-like receptors (TLRs)-mediated host-bacterial interactions participate in the microbial regulation of gastrointestinal functions, including the epithelial barrier function (EBF). We evaluated the effects of TLR7 stimulation on the colonic EBF in rats. TLR7 was stimulated with the selective agonist imiquimod (100/300 µg/rat, intracolonic), with or without the intracolonic administration of dimethyl sulfoxide (DMSO). Colonic EBF was assessed in vitro (electrophysiology and permeability to macromolecules, Ussing chamber) and in vivo (passage of macromolecules to blood and urine). Changes in the expression (RT-qPCR) and distribution (immunohistochemistry) of tight junction-related proteins were determined. Expression of proglucagon, precursor of the barrier-enhancer factor glucagon-like peptide 2 (GLP-2) was also assessed (RT-qPCR). Intracolonic imiquimod enhanced the EBF in vitro, reducing the epithelial conductance and the passage of macromolecules, thus indicating a pro-barrier effect of TLR7. However, the combination of TLR7 stimulation and DMSO had a detrimental effect on the EBF, which manifested as an increased passage of macromolecules. DMSO alone had no effect. The modulation of the EBF (imiquimod alone or with DMSO) was not associated with changes in gene expression or the epithelial distribution of the main tight junction-related proteins (occludin, tricellulin, claudin-2, claudin-3, junctional adhesion molecule 1 and Zonula occludens-1). No changes in the proglucagon expression were observed. These results show that TLR7 stimulation leads to the modulation of the colonic EBF, having beneficial or detrimental effects depending upon the state of the epithelium. The underlying mechanisms remain elusive, but seem independent of the modulation of the main tight junction-related proteins or the barrier-enhancer factor GLP-2.