RESUMO
GDF15, a hormone acting on the brainstem, has been implicated in the nausea and vomiting of pregnancy, including its most severe form, hyperemesis gravidarum (HG), but a full mechanistic understanding is lacking1-4. Here we report that fetal production of GDF15 and maternal sensitivity to it both contribute substantially to the risk of HG. We confirmed that higher GDF15 levels in maternal blood are associated with vomiting in pregnancy and HG. Using mass spectrometry to detect a naturally labelled GDF15 variant, we demonstrate that the vast majority of GDF15 in the maternal plasma is derived from the feto-placental unit. By studying carriers of rare and common genetic variants, we found that low levels of GDF15 in the non-pregnant state increase the risk of developing HG. Conversely, women with ß-thalassaemia, a condition in which GDF15 levels are chronically high5, report very low levels of nausea and vomiting of pregnancy. In mice, the acute food intake response to a bolus of GDF15 is influenced bi-directionally by prior levels of circulating GDF15 in a manner suggesting that this system is susceptible to desensitization. Our findings support a putative causal role for fetally derived GDF15 in the nausea and vomiting of human pregnancy, with maternal sensitivity, at least partly determined by prepregnancy exposure to the hormone, being a major influence on its severity. They also suggest mechanism-based approaches to the treatment and prevention of HG.
Assuntos
Fator 15 de Diferenciação de Crescimento , Hiperêmese Gravídica , Náusea , Vômito , Animais , Feminino , Humanos , Camundongos , Gravidez , Talassemia beta/sangue , Talassemia beta/metabolismo , Feto/metabolismo , Fator 15 de Diferenciação de Crescimento/sangue , Fator 15 de Diferenciação de Crescimento/metabolismo , Hormônios/sangue , Hormônios/metabolismo , Hiperêmese Gravídica/complicações , Hiperêmese Gravídica/metabolismo , Hiperêmese Gravídica/prevenção & controle , Hiperêmese Gravídica/terapia , Náusea/sangue , Náusea/complicações , Náusea/metabolismo , Placenta/metabolismo , Vômito/sangue , Vômito/complicações , Vômito/metabolismoRESUMO
RATIONALE: Insulin-like peptide 5 (INSL5) is a hormone produced by enteroendocrine L-cells in the colon that has recently been implicated in the control of metabolic homeostasis. However, research into its physiology has been hindered by the reported unreliability of commercially available immunoassays and additional detection assays would benefit this emerging field. METHODS: Peptides from purified murine L-cells and homogenates from both human and mouse colonic tissues were extracted by precipitating larger proteins with acetonitrile. Untargeted liquid chromatography/tandem mass spectrometry (LC/MS/MS) analyses, followed by database searching, were used to detect and identify various INSL5 gene derived peptides and characterise their precise sequence. A similar approach was developed to quantify INSL5 levels in primary intestinal culture supernatants after purification and concentration by solid-phase extraction. RESULTS: Mass spectral analysis of purified enteroendocrine cells and tissue homogenates identified the exact sequence of A and B chains of INSL5 endogenously expressed in L-cells. Differences in the endogenously processed peptide and the Swissprot database entry were observed for murine INSL5, whereas the human sequence matched previous predictions from heterologous expression experiments. INSL5 was detected in the supernatant of human and mouse primary colonic cultures and concentrations increased after treatment with a known L-cell stimulus. CONCLUSIONS: The first LC/MS/MS-based method capable of the detection and semi-quantitative analysis of endogenous INSL5 using MS-based techniques has been demonstrated. The methodology will enable the identification of stimulants for INSL5 secretion from murine and human primary colonic epithelial cultures.
Assuntos
Insulina/análise , Mucosa Intestinal/química , Proteínas/análise , Sequência de Aminoácidos , Animais , Células Cultivadas/química , Cromatografia Líquida/métodos , Colo/citologia , Células Epiteliais/química , Células Epiteliais/citologia , Humanos , Mucosa Intestinal/citologia , Camundongos , Espectrometria de Massas em Tandem/métodosRESUMO
A significant number of pancreases procured for transplantation are deemed unsuitable due to concerns about graft quality and the associated risk of complications. However, this decision is subjective and some declined grafts may be suitable for transplantation. Ex vivo normothermic perfusion (EVNP) prior to transplantation may allow a more objective assessment of graft quality and reduce discard rates. We report ex vivo normothermic perfusion of human pancreases procured but declined for transplantation, with ABO-compatible warm oxygenated packed red blood cells for 1-2 h. Five declined human pancreases were assessed using this technique after a median cold ischemia time of 13 h 19 min. One pancreas, with cold ischemia over 30 h, did not appear viable and was excluded. In the remaining pancreases, blood flow and pH were maintained throughout perfusion. Insulin secretion was observed in all four pancreases, but was lowest in an older donation after cardiac death pancreas. Amylase levels were highest in a gland with significant fat infiltration. This is the first study to assess the perfusion, injury, as measured by amylase, and exocrine function of human pancreases using EVNP and demonstrates the feasibility of the approach, although further refinements are required.
Assuntos
Tomada de Decisão Clínica , Função Retardada do Enxerto/prevenção & controle , Seleção do Doador , Preservação de Órgãos , Transplante de Pâncreas , Perfusão/métodos , Coleta de Tecidos e Órgãos , Adolescente , Adulto , Amilases/metabolismo , Função Retardada do Enxerto/diagnóstico , Função Retardada do Enxerto/metabolismo , Feminino , Seguimentos , Sobrevivência de Enxerto , Humanos , Masculino , Pessoa de Meia-Idade , Avaliação de Resultados em Cuidados de Saúde , TemperaturaRESUMO
AIMS/HYPOTHESIS: Targeting the secretion of gut peptides such as glucagon-like peptide 1 (GLP-1) and peptide YY (PYY) is a strategy under development for the treatment of diabetes and obesity, aiming to mimic the beneficial alterations in intestinal physiology that follow gastric bypass surgery. In vitro systems are now well established for studying the mouse enteroendocrine system, but whether these accurately model the human gut remains unclear. The aim of this study was to establish and characterise human primary intestinal cultures as a model for assessing GLP-1 and PYY secretion in vitro. METHODS: Fresh surgical biopsies of human colon were digested with collagenase to generate primary cultures from which GLP-1 and PYY secretion were assayed in response to test stimuli. GLP-1 and PYY co-localisation were assessed by flow cytometry and immunofluorescence microscopy. RESULTS: GLP-1 and PYY were found localised in the same cells and the same secretory vesicles in human colonic tissue samples. GLP-1 release was increased to 2.6-fold the control value by forskolin + isobutylmethylxanthine (10 µmol/l each), 2.8-fold by phorbol myristate acetate (1 µmol/l) and 1.4-fold by linoleic acid (100 µmol/l). PYY release was increased to 2.0-, 1.8- and 1.3-fold by the same stimuli, respectively. Agonists of G-protein-coupled receptor (GPR)40/120 and G-protein-coupled bile acid receptor 1 (GPBAR1) each increased GLP-1 release to 1.5-fold, but a GPR119 agonist did not significantly stimulate secretion. CONCLUSIONS/INTERPRETATION: Primary human colonic cultures provide an in vitro model for interrogating the human enteroendocrine system, and co-secrete GLP-1 and PYY. We found no evidence of PYY-specific cells not producing GLP-1. GLP-1 secretion was enhanced by small molecule agonists of GPR40/120 and GPBAR1.
Assuntos
Colo/metabolismo , Peptídeo 1 Semelhante ao Glucagon/metabolismo , Peptídeo YY/metabolismo , Células Cultivadas , Colagenases/metabolismo , Células Enteroendócrinas/metabolismo , Citometria de Fluxo , Humanos , Microscopia de Fluorescência , Receptores Acoplados a Proteínas G/metabolismoRESUMO
Human pregnancy is frequently accompanied by nausea and vomiting that may become severe and life-threatening, as in hyperemesis gravidarum (HG), the cause of which is unknown. Growth Differentiation Factor-15 (GDF15), a hormone known to act on the hindbrain to cause emesis, is highly expressed in the placenta and its levels in maternal blood rise rapidly in pregnancy. Variants in the maternal GDF15 gene are associated with HG. Here we report that fetal production of GDF15, and maternal sensitivity to it, both contribute substantially to the risk of HG. We found that the great majority of GDF15 in maternal circulation is derived from the feto-placental unit and that higher GDF15 levels in maternal blood are associated with vomiting and are further elevated in patients with HG. Conversely, we found that lower levels of GDF15 in the non-pregnant state predispose women to HG. A rare C211G variant in GDF15 which strongly predisposes mothers to HG, particularly when the fetus is wild-type, was found to markedly impair cellular secretion of GDF15 and associate with low circulating levels of GDF15 in the non-pregnant state. Consistent with this, two common GDF15 haplotypes which predispose to HG were associated with lower circulating levels outside pregnancy. The administration of a long-acting form of GDF15 to wild-type mice markedly reduced subsequent responses to an acute dose, establishing that desensitisation is a feature of this system. GDF15 levels are known to be highly and chronically elevated in patients with beta thalassemia. In women with this disorder, reports of symptoms of nausea or vomiting in pregnancy were strikingly diminished. Our findings support a causal role for fetal derived GDF15 in the nausea and vomiting of human pregnancy, with maternal sensitivity, at least partly determined by pre-pregnancy exposure to GDF15, being a major influence on its severity. They also suggest mechanism-based approaches to the treatment and prevention of HG.
RESUMO
AIMS/HYPOTHESIS: Several glucose-sensing pathways have been implicated in glucose-triggered secretion of glucagon-like peptide-1 (GLP-1) from intestinal L cells. One involves glucose metabolism and closure of ATP-sensitive K(+) channels, and another exploits the electrogenic nature of Na(+)-coupled glucose transporters (SGLTs). This study aimed to elucidate the role of these distinct mechanisms in glucose-stimulated GLP-1 secretion. METHODS: Glucose uptake into L cells (either GLUTag cells or cells in primary cultures, using a new transgenic mouse model combining proglucagon promoter-driven Cre recombinase with a ROSA26tdRFP reporter) was monitored with the FLII(12)Pglu-700 µÎ´6 glucose sensor. Effects of pharmacological and genetic interference with SGLT1 or facilitative glucose transport (GLUT) on intracellular glucose accumulation and metabolism (measured by NAD(P)H autofluorescence), cytosolic Ca(2+) (monitored with Fura2) and GLP-1 secretion (assayed by ELISA) were assessed. RESULTS: L cell glucose uptake was dominated by GLUT-mediated transport, being abolished by phloretin but not phloridzin. NAD(P)H autofluorescence was glucose dependent and enhanced by a glucokinase activator. In GLUTag cells, but not primary L cells, phloretin partially impaired glucose-dependent secretion, and suppressed an amplifying effect of glucose under depolarising high K(+) conditions. The key importance of SGLT1 in GLUTag and primary cells was evident from the impairment of secretion by phloridzin or Sglt1 knockdown and failure of glucose to trigger cytosolic Ca(2+) elevation in primary L cells from Sglt1 knockout mice. CONCLUSIONS/INTERPRETATION: SGLT1 acts as the luminal glucose sensor in L cells, but intracellular glucose concentrations are largely determined by GLUT activity. Although L cell glucose metabolism depends partially on glucokinase activity, this plays only a minor role in glucose-stimulated GLP-1 secretion.
Assuntos
Células Enteroendócrinas/metabolismo , Peptídeo 1 Semelhante ao Glucagon/metabolismo , Intestinos/patologia , Canais KATP/metabolismo , Floretina/farmacologia , Transportador 1 de Glucose-Sódio/metabolismo , Animais , Transporte Biológico , Linhagem Celular , Células Cultivadas , Citometria de Fluxo , Regulação da Expressão Gênica , Glucagon/metabolismo , Imuno-Histoquímica , Camundongos , Camundongos KnockoutRESUMO
AIMS/HYPOTHESIS: Glucose-dependent insulinotropic polypeptide (GIP) is an enteroendocrine hormone that promotes storage of glucose and fat. Its secretion from intestinal K cells is triggered by nutrient ingestion and is modulated by intracellular cAMP. In view of the proadipogenic actions of GIP, this study aimed to identify pathways in K cells that lower cAMP levels and GIP secretion. METHODS: Murine K cells purified by flow cytometry were analysed for expression of G(αi)-coupled receptors by transcriptomic microarrays. Somatostatin and cannabinoid receptor expression was confirmed by quantitative RT-PCR. Hormone secretion in vitro was measured in GLUTag and primary murine intestinal cultures. cAMP was monitored in GLUTag cells using the genetically encoded sensor Epac2-camps. In vivo tolerance tests were performed in cannulated rats. RESULTS: Purified murine K cells expressed high mRNA levels for somatostatin receptors (Sstrs) Sstr2, Sstr3 and Sstr5, and cannabinoid receptor type 1 (Cnr1, CB1). Somatostatin inhibited GIP and glucagon-like peptide-1 (GLP-1) secretion from primary small intestinal cultures, in part through SSTR5, and reduced cAMP generation in GLUTag cells. Although the CB1 agonist methanandamide (mAEA) inhibited GIP secretion, no significant effect was observed on GLP-1 secretion from primary cultures. In cannulated rats, treatment with mAEA prior to an oral glucose tolerance test suppressed plasma GIP but not GLP-1 levels, whereas the CB1 antagonist AM251 elevated basal GIP concentrations. CONCLUSIONS/INTERPRETATION: GIP release is inhibited by somatostatin and CB1 agonists. The differential effects of CB1 ligands on GIP and GLP-1 release may provide a new tool to dissociate secretion of these incretin hormones and lower GIP but not GLP-1 levels in vivo.
Assuntos
Colo/metabolismo , Polipeptídeo Inibidor Gástrico/metabolismo , Intestino Delgado/metabolismo , Receptor CB1 de Canabinoide/metabolismo , Receptores de Somatostatina/metabolismo , Animais , Colo/citologia , AMP Cíclico/metabolismo , Células Enteroendócrinas/citologia , Células Enteroendócrinas/metabolismo , Peptídeo 1 Semelhante ao Glucagon/metabolismo , Incretinas/metabolismo , Intestino Delgado/citologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Cultura Primária de Células , RNA Mensageiro/metabolismo , Ratos , Ratos Sprague-Dawley , Receptor CB1 de Canabinoide/genética , Receptores de Somatostatina/genéticaRESUMO
AIMS/HYPOTHESIS: We assessed whether per-arnt-sim (PAS) domain-containing protein kinase (PASK) is involved in the regulation of glucagon secretion. METHODS: mRNA levels were measured in islets by quantitative PCR and in pancreatic beta cells obtained by laser capture microdissection. Glucose tolerance, plasma hormone levels and islet hormone secretion were analysed in C57BL/6 Pask homozygote knockout mice (Pask-/-) and control littermates. Alpha-TC1-9 cells, human islets or cultured E13.5 rat pancreatic epithelia were transduced with anti-Pask or control small interfering RNAs, or with adenoviruses encoding enhanced green fluorescent protein or PASK. RESULTS: PASK expression was significantly lower in islets from human type 2 diabetic than control participants. PASK mRNA was present in alpha and beta cells from mouse islets. In Pask-/- mice, fasted blood glucose and plasma glucagon levels were 25 ± 5% and 50 ± 8% (mean ± SE) higher, respectively, than in control mice. At inhibitory glucose concentrations (10 mmol/l), islets from Pask-/- mice secreted 2.04 ± 0.2-fold (p < 0.01) more glucagon and 2.63 ± 0.3-fold (p < 0.01) less insulin than wild-type islets. Glucose failed to inhibit glucagon secretion from PASK-depleted alpha-TC1-9 cells, whereas PASK overexpression inhibited glucagon secretion from these cells and human islets. Extracellular insulin (20 nmol/l) inhibited glucagon secretion from control and PASK-deficient alpha-TC1-9 cells. PASK-depleted alpha-TC1-9 cells and pancreatic embryonic explants displayed increased expression of the preproglucagon (Gcg) and AMP-activated protein kinase (AMPK)-alpha2 (Prkaa2) genes, implying a possible role for AMPK-alpha2 downstream of PASK in the control of glucagon gene expression and release. CONCLUSIONS/INTERPRETATION: PASK is involved in the regulation of glucagon secretion by glucose and may be a useful target for the treatment of type 2 diabetes.
Assuntos
Diabetes Mellitus Tipo 2/enzimologia , Ilhotas Pancreáticas/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Animais , Linhagem Celular , Células Cultivadas , Diabetes Mellitus Tipo 2/metabolismo , Glucagon/metabolismo , Células Secretoras de Glucagon/efeitos dos fármacos , Células Secretoras de Glucagon/metabolismo , Glucose/farmacologia , Humanos , Células Secretoras de Insulina/efeitos dos fármacos , Células Secretoras de Insulina/metabolismo , Ilhotas Pancreáticas/efeitos dos fármacos , Camundongos , Camundongos Mutantes , Modelos Biológicos , Reação em Cadeia da Polimerase , Proteínas Serina-Treonina Quinases/genética , RatosRESUMO
Glucagon like peptide 1 (GLP-1) based therapies are now widely used for the treatment of type 2 diabetes. Developing our understanding of intestinal GLP-1 release may facilitate the development of new therapeutics aimed at targeting the GLP-1 producing L-cells. This study was undertaken to characterise the electrical activity of primary L-cells and the importance of voltage gated sodium and calcium channels for GLP-1 secretion. Primary murine L-cells were identified and purified using transgenic mice expressing a fluorescent protein driven by the proglucagon promoter. Fluorescent L-cells were identified within primary colonic cultures for patch clamp recordings. GLP-1 secretion was measured from primary colonic cultures. L-cells purified by flow cytometry were used to measure gene expression by microarray and quantitative RT-PCR. Electrical activity in L-cells was due to large voltage gated sodium currents, inhibition of which by tetrodotoxin reduced both basal and glutamine-stimulated GLP-1 secretion. Voltage gated calcium channels were predominantly of the L-type, Q-type and T-type, by expression analysis, consistent with the finding that GLP-1 release was blocked both by nifedipine and ω-conotoxin MVIIC. We observed large voltage-dependent potassium currents, but only a small chromanol sensitive current that might be attributable to KCNQ1. GLP-1 release from primary L-cells is linked to electrical activity and activation of L-type and Q-type calcium currents. The concept of an electrically excitable L-cell provides a basis for understanding how GLP-1 release may be modulated by nutrient, hormonal and pharmaceutical stimuli.
Assuntos
Estimulação Elétrica , Células Enteroendócrinas/metabolismo , Peptídeo 1 Semelhante ao Glucagon/metabolismo , Potenciais de Ação/fisiologia , Animais , Cálcio/metabolismo , Bloqueadores dos Canais de Cálcio/farmacologia , Canais de Cálcio/genética , Canais de Cálcio/metabolismo , Células Cultivadas , Eletrofisiologia , Células Enteroendócrinas/efeitos dos fármacos , Ensaio de Imunoadsorção Enzimática , Citometria de Fluxo , Ativação do Canal Iônico/efeitos dos fármacos , Ativação do Canal Iônico/fisiologia , Camundongos , Camundongos Transgênicos , Nifedipino/farmacologia , Análise de Sequência com Séries de Oligonucleotídeos , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
BACKGROUND: There is an unmet need for suitable ex vivo large animal models in experimental gastroenterology and intestinal transplantation. This study details a reliable and effective technique for ex vivo normothermic perfusion (EVNP) of segmental porcine small intestine. METHODS: Segments of small intestine, 1.5-3.0 m in length, were retrieved from terminally anaesthetized pigs. After a period of cold ischaemia, EVNP was performed for 2 h at 37°C with a mean pressure of 80 mmHg using oxygenated autologous blood diluted with Ringer's solution. The duration of EVNP was extended to 4 h for a second set of experiments in which two segments of proximal to mid-ileum (1.5-3.0 m) were retrieved from each animal and reperfused with whole blood (control) or leucocyte-depleted blood to examine the impact of leucocyte depletion on reperfusion injury. RESULTS: After a mean cold ischaemia time of 5 h and 20 min, EVNP was performed in an initial group of four pigs. In the second set of experiments, five pigs were used in each group. In all experiments bowel segments were well perfused and exhibited peristalsis during EVNP. Venous glucose levels significantly increased following luminal glucose stimulation (mean(s.e.m.) basal level 1.8(0.6) mmol/l versus peak 15.5(5.8) mmol/l; P < 0.001) and glucagon-like peptide 1 (GLP-1) levels increased in all experiments, demonstrating intact absorptive and secretory intestinal functions. There were no significant differences between control and leucocyte-depleted animals regarding blood flow, venous glucose, GLP-1 levels or histopathology at the end of 4 h of EVNP. CONCLUSIONS: This novel model is suitable for the investigation of gastrointestinal physiology, pathology and ischaemia reperfusion injury, along with evaluation of potential therapeutic interventions.
Assuntos
Intestino Delgado/irrigação sanguínea , Intestino Delgado/transplante , Preservação de Órgãos/métodos , Perfusão/métodos , Animais , Intestino Delgado/patologia , Pós-Condicionamento Isquêmico , Precondicionamento Isquêmico , Masculino , SuínosRESUMO
Glucose-dependent insulinotropic polypeptide (GIP) is an incretin hormone released from the epithelium of the upper small intestine. While GIP shares common actions on the pancreatic beta cell with glucagon-like peptide-1 (GLP-1), unlike GLP-1, GIP presents a complex target for the development of diabetes and obesity therapies due to its extra-pancreatic effects on fat mass. Recent pharmacological developments, however, have provided insight into a previously unrecognized role for GIP receptor (GIPR) signaling in regulating appetite. Additionally, GIP-based therapeutics have demonstrated promising neuroprotective properties. Together these observations identify an important central component of the GIP/GIPR signaling axis, and have triggered a resurgence of research interest into the central actions of GIP. In this review, we discuss what is currently known about where GIP may act in the central nervous system (CNS), the characteristics of its target cell populations, and the physiological effects of manipulating the activity Gipr-expressing cells in the brain.
Assuntos
Sistema Nervoso Central/efeitos dos fármacos , Polipeptídeo Inibidor Gástrico/farmacologia , Fármacos Gastrointestinais/farmacologia , Receptores dos Hormônios Gastrointestinais/metabolismo , Animais , Sistema Nervoso Central/metabolismo , Sistema Nervoso Central/patologia , Humanos , Transdução de SinaisRESUMO
AIMS/HYPOTHESIS: Glucose-dependent insulinotropic polypeptide (GIP) is an incretin hormone with anti-apoptotic effects on the pancreatic beta cell. The aim of this study was to generate transgenic mice with fluorescently labelled GIP-secreting K cells and to use these to investigate pathways by which K cells detect nutrients. METHODS: Transgenic mice were generated in which the GIP promoter drives the expression of the yellow fluorescent protein Venus. Fluorescent cells were purified by flow cytometry and analysed by quantitative RT-PCR. GIP secretion was assayed in primary cultures of small intestine. RESULTS: Expression of Venus in transgenic mice was restricted to K cells, as assessed by immunofluorescence and measurements of the Gip mRNA and GIP protein contents of purified cells. K cells expressed high levels of mRNA for Kir6.2 (also known as Kcnj11), Sur1 (also known as Abcc8), Sglt1 (also known as Slc5a1), and of the G-protein-coupled lipid receptors Gpr40 (also known as Ffar1), Gpr119 and Gpr120. In primary cultures, GIP release was stimulated by glucose, glutamine and linoleic acid, and potentiated by forskolin plus 3-isobutyl-1-methylxanthine (IBMX), but was unaffected by the artificial sweetener sucralose. Secretion was half-maximal at 0.6 mmol/l glucose and partially mimicked by alpha-methylglucopyranoside, suggesting the involvement of SGLT1. Tolbutamide triggered secretion under basal conditions, whereas diazoxide suppressed responses in forskolin/IBMX. CONCLUSIONS/INTERPRETATION: These transgenic mice and primary culture techniques provide novel opportunities to interrogate the mechanisms of GIP secretion. Glucose-triggered GIP secretion was SGLT1-dependent and modulated by K(ATP) channel activity but not determined by sweet taste receptors. Synergistic stimulation by elevated cAMP and glucose suggests that targeting appropriate G-protein-coupled receptors may provide opportunities to modulate GIP release in vivo.
Assuntos
Glucose/farmacologia , Células Matadoras Naturais/metabolismo , Actinas/genética , Animais , Proteínas de Ligação ao Cálcio/genética , Cromossomos Artificiais Bacterianos , AMP Cíclico/metabolismo , Citometria de Fluxo , Polipeptídeo Inibidor Gástrico/genética , Polipeptídeo Inibidor Gástrico/metabolismo , Células Matadoras Naturais/citologia , Células Matadoras Naturais/efeitos dos fármacos , Lipoproteínas/genética , Camundongos , Camundongos Transgênicos , Regiões Promotoras Genéticas , Ratos , Receptores dos Hormônios Gastrointestinais/fisiologia , Acetato de Tetradecanoilforbol/farmacologiaRESUMO
AIMS/HYPOTHESIS: To investigate the mechanism by which human islet amyloid polypeptide (hIAPP) fibril formation results in calcium influx across the plasma membrane of pancreatic beta cells, and its association with apoptosis. METHODS: Cytoplasmic intracellular calcium concentrations ([Ca(2+)](i)) were monitored for 2 h as the 340/380 nm fluorescence ratio in fura-2 loaded cells of the MIN6 mouse pancreatic beta cell line. Cell morphology was evaluated by transmission electron microscopy, and viability by FACS. RESULTS: hIAPP (10 micromol/l) increased [Ca(2+)](i) in 21% of MIN6 cells in standard buffer, and in 8% of cells in Na(+)-free buffer. Transient receptor potential (TRP) channel inhibitors (gadolinium and ruthenium red) prevented the [Ca(2+)](i) rise under both conditions, whilst nifedipine was only effective in the presence of Na(+). hIAPP increased apoptosis in both insulinoma cells and islets in primary culture, and cell viability was partially rescued by ruthenium red (p < 0.001). By RT-PCR, we detected expression of the mechanosensitive TRP cation channel subfamily V member 4 (Trpv4) in MIN6 cells and mouse pancreas. Small interference RNA against Trpv4 prevented hIAPP-induced [Ca(2+)](i) rises, decreased hIAPP-triggered expression of the endoplasmic reticulum (ER) stress response, and reduced hIAPP-triggered cell death by 50% (p < 0.05). CONCLUSIONS/INTERPRETATION: Alterations in [Ca(2+)](i) play a key role in hIAPP-induced beta cell cytotoxicity. By electron microscopy, we detected extracellular hIAPP aggregates adjacent to irregular invaginated regions of the plasma membrane. We propose that TRPV4 channels may sense physical changes in the plasma membrane induced by hIAPP aggregation, enabling Ca(2+) entry, membrane depolarisation and activation of L-type Ca(2+) channels. Decreasing the activity of TRPV4 prevented hIAPP-induced [Ca(2+)](i) changes, reduced hIAPP-triggered ER stress and improved cell viability.
Assuntos
Amiloide/farmacologia , Cálcio/metabolismo , Células Secretoras de Insulina/efeitos dos fármacos , Células Secretoras de Insulina/metabolismo , Canais de Cátion TRPV/metabolismo , Amiloide/ultraestrutura , Animais , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/genética , Humanos , Células Secretoras de Insulina/citologia , Polipeptídeo Amiloide das Ilhotas Pancreáticas , Camundongos , Microscopia Eletrônica de Transmissão , Especificidade por Substrato , Canais de Cátion TRPV/genéticaRESUMO
Glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP) are gastrointestinal hormones that play an important role in stimulating postprandial insulin release from pancreatic beta-cells. Agents that either mimic GLP-1 or prevent its degradation are now available for the treatment of Type 2 diabetes, and strategies to enhance endogenous GLP-1 release are under assessment. As intestinal peptides have a range of actions, including appetite regulation and coordination of fat metabolism, harnessing the enteric endocrine system is a promising new field for drug development.
Assuntos
Diabetes Mellitus Tipo 2/tratamento farmacológico , Polipeptídeo Inibidor Gástrico/uso terapêutico , Peptídeo 1 Semelhante ao Glucagon/uso terapêutico , Incretinas/metabolismo , Células Secretoras de Insulina/metabolismo , Insulina/metabolismo , Diabetes Mellitus Tipo 2/metabolismo , Polipeptídeo Inibidor Gástrico/metabolismo , Peptídeo 1 Semelhante ao Glucagon/metabolismo , HumanosRESUMO
The intestinal epithelium must balance efficient absorption of nutrients with partitioning commensals and pathogens from the bodies' largest immune system. If this crucial barrier fails, inappropriate immune responses can result in inflammatory bowel disease or chronic infection. Enteroendocrine cells represent 1% of this epithelium and have classically been studied for their detection of nutrients and release of peptide hormones to mediate digestion. Intriguingly, enteroendocrine cells are the key sensors of microbial metabolites, can release cytokines in response to pathogen associated molecules and peptide hormone receptors are expressed on numerous intestinal immune cells; thus enteroendocrine cells are uniquely equipped to be crucial and novel orchestrators of intestinal inflammation. In this review, we introduce enteroendocrine chemosensory roles, summarize studies correlating enteroendocrine perturbations with intestinal inflammation and describe the mechanistic interactions by which enteroendocrine and mucosal immune cells interact during disease; highlighting this immunoendocrine axis as a key aspect of innate immunity.
Assuntos
Células Enteroendócrinas/imunologia , Inflamação/imunologia , Mucosa Intestinal/imunologia , Animais , Citocinas/metabolismo , Digestão , Homeostase/imunologia , Humanos , Imunidade nas Mucosas , Moléculas com Motivos Associados a Patógenos/imunologia , Hormônios Peptídicos/metabolismo , Vigilância de Evento SentinelaRESUMO
Peptide-YY (PYY) and Glucagon-Like Peptide-1 (GLP-1) play important roles in the regulation of food intake and insulin secretion, and are of translational interest in the field of obesity and diabetes. PYY production is highest in enteroendocrine cells located in the distal intestine, mirroring the sites where high concentrations of short chain fatty acids (SCFAs) are produced by gut microbiota. We show here that propionate and butyrate strongly increased expression of PYY but not GCG in human cell line and intestinal primary culture models. The effect was predominantly attributable to the histone deacetylase inhibitory activity of SCFA and minor, but significant contributions of FFA2 (GPR43). Consistent with the SCFA-dependent elevation of PYY gene expression, we also observed increased basal and stimulated PYY hormone secretion. Interestingly, the transcriptional stimulation of PYY was specific to human-derived cell models and not reproduced in murine primary cultures. This is likely due to substantial differences in PYY gene structure between mouse and human. In summary, this study revealed a strong regulation of PYY production by SCFA that was evident in humans but not mice, and suggests that high fibre diets elevate plasma concentrations of the anorexigenic hormone PYY, both by targeting gene expression and hormone secretion.
Assuntos
Células Enteroendócrinas/metabolismo , Ácidos Graxos Voláteis/metabolismo , Peptídeo YY/metabolismo , Linhagem Celular , Células Cultivadas , Células Enteroendócrinas/efeitos dos fármacos , Ácidos Graxos Voláteis/farmacologia , Expressão Gênica , Peptídeo 1 Semelhante ao Glucagon/genética , Peptídeo 1 Semelhante ao Glucagon/metabolismo , Humanos , Peptídeo YY/genéticaRESUMO
ATP-sensitive K+ channels couple cell metabolism to electrical activity in nerve, muscle and endocrine cells, and play important roles in these tissues under both physiological and pathological conditions. The KATP channel is an octameric complex of two unrelated types of subunit: a pore-forming subunit (Kir6.2) and a regulatory subunit, the sulphonylurea receptor (SUR). This review focuses on the regulation of KATP channel activity by nucleotides and cell metabolism and considers which regulatory mechanisms are intrinsic to Kir6.2 and which are conferred by association with SUR.
Assuntos
Transportadores de Cassetes de Ligação de ATP , Canais de Potássio Corretores do Fluxo de Internalização , Canais de Potássio/fisiologia , ATPase Trocadora de Sódio-Potássio/metabolismo , Trifosfato de Adenosina/metabolismo , Células/metabolismo , Diabetes Mellitus Tipo 2/genética , Genes Reguladores/genética , Ligação Genética , Hiperinsulinismo/genética , Técnicas In Vitro , Modelos Biológicos , Nucleotídeos/metabolismo , Canais de Potássio/metabolismo , Receptores de Droga/metabolismo , Compostos de Sulfonilureia/metabolismo , Receptores de SulfonilureiasRESUMO
K(ATP) channel openers are a diverse group of drugs with a wide range of potential therapeutic uses. Their molecular targets, the K(ATP) channels, exhibit tissue-specific responses because they possess different types of regulatory sulfonylurea receptor subunits. It is well recognized that complex interactions occur between K(ATP) channel openers and nucleotides, but the cloning of the K(ATP) channel has introduced a new dimension to the study of these events and has furthered our understanding of the molecular basis of the action of K(ATP) channel openers.
Assuntos
Minoxidil/análogos & derivados , Canais de Potássio/efeitos dos fármacos , Trifosfato de Adenosina/fisiologia , Animais , Sítios de Ligação , Cromakalim/metabolismo , Cromakalim/farmacologia , Diazóxido/metabolismo , Diazóxido/farmacologia , Humanos , Minoxidil/metabolismo , Minoxidil/farmacologia , Nicorandil/metabolismo , Nicorandil/farmacologia , Pinacidil/metabolismo , Pinacidil/farmacologia , Canais de Potássio/metabolismo , Canais de Potássio/fisiologiaRESUMO
Nicorandil is a new antianginal agent that potentially may be used to treat the cardiovascular side effects of diabetes. It is both a nitric oxide donor and an opener of ATP-sensitive K(+) (K(ATP)) channels in muscle and thereby causes vasodilation of the coronary vasculature. The aim of this study was to investigate the domains of the K(ATP) channel involved in nicorandil activity and to determine whether nicorandil interacts with hypoglycemic sulfonylureas that target K(ATP) channels in pancreatic beta-cells. K(ATP) channels in muscle and beta-cells share a common pore-forming subunit, Kir6.2, but possess alternative sulfonylurea receptors (SURs; SUR1 in beta-cells, SUR2A in cardiac muscle, and SUR2B in smooth muscle). We expressed recombinant K(ATP) channels in Xenopus oocytes and measured the effects of drugs and nucleotides by recording macroscopic currents in excised membrane patches. Nicorandil activated Kir6.2/SUR2A and Kir6.2/SUR2B but not Kir6.2/SUR1 currents, consistent with its specificity for cardiac and smooth muscle K(ATP) channels. Drug activity depended on the presence of intracellular nucleotides and was impaired when the Walker A lysine residues were mutated in either nucleotide-binding domain of SUR2. Chimeric studies showed that the COOH-terminal group of transmembrane helices (TMs), especially TM 17, is responsible for the specificity of nicorandil for channels containing SUR2. The splice variation between SUR2A and SUR2B altered the off-rate of the nicorandil response. Finally, we showed that nicorandil activity was unaffected by gliclazide, which specifically blocks SUR1-type K(ATP) channels, but was severely impaired by glibenclamide and glimepiride, which target both SUR1 and SUR2-type K(ATP) channels.
Assuntos
Nicorandil/farmacologia , Canais de Potássio Corretores do Fluxo de Internalização , Compostos de Sulfonilureia/farmacologia , Trifosfato de Adenosina/fisiologia , Animais , Interações Medicamentosas , Eletrofisiologia , Feminino , Oxigenases de Função Mista/química , Oxigenases de Função Mista/efeitos dos fármacos , Oxigenases de Função Mista/genética , Mutação/fisiologia , Oócitos , Canais de Potássio/efeitos dos fármacos , Canais de Potássio/fisiologia , Xenopus laevisRESUMO
Sulfonylureas stimulate insulin secretion from pancreatic beta-cells by closing ATP-sensitive K+ (K(ATP)). The beta-cell and cardiac muscle K(ATP) channels have recently been cloned and shown to possess a common pore-forming subunit (Kir6.2) but different sulfonylurea receptor subunits (SUR1 and SUR2A, respectively). We examined the mechanism underlying the tissue specificity of the sulfonylureas tolbutamide and glibenclamide, and the benzamido-derivative meglitinide, using cloned beta-cell (Kir6.2/SUR1) and cardiac (Kir6.2/SUR2A) K(ATP) channels expressed in Xenopus oocytes. Tolbutamide inhibited Kir6.2/SUR1 (Ki approximately 5 micromol/l), but not Kir6.2/SUR2A, currents with high affinity. Meglitinide produced high-affinity inhibition of both Kir6.2/SUR1 and Kir6.2/SUR2A currents (Kis approximately 0.3 micromol/l and approximately 0.5 micromol/l, respectively). Glibenclamide also blocked Kir6.2/SUR1 and Kir6.2/SUR2A currents with high affinity (Kis approximately 4 nmol/l and approximately 27 nmol/l, respectively); however, only for cardiac-type K(ATP) channels was this block reversible. Physiological concentrations of MgADP (100 micromol/l) enhanced glibenclamide inhibition of Kir6.2/SUR1 currents but reduced that of Kir6.2/SUR2A currents. The results suggest that SUR1 may possess separate high-affinity binding sites for sulfonylurea and benzamido groups. SUR2A, however, either does not possess a binding site for the sulfonylurea group or is unable to translate the binding at this site into channel inhibition. Although MgADP reduces the inhibitory effect of glibenclamide on cardiac-type K(ATP) channels, drugs that bind to the common benzamido site have the potential to cause side effects on the heart.