Assuntos
Asma , Termoplastia Brônquica , Remodelação das Vias Aéreas , Asma/terapia , Brônquios/cirurgia , Broncoscopia , HumanosRESUMO
Glucagon-like peptide 2 (GLP-2) is an enteroendocrine hormone trophic for intestinal mucosa; it has been shown to increase enteric neuronal expression of vasoactive intestinal polypeptide (VIP) in vivo. We hypothesized that GLP-2 would regulate VIP expression in enteric neurons via a phosphatidylinositol-3 kinase-γ (PI3Kγ) pathway. The mechanism of action of GLP-2 was investigated using primary cultures derived from the submucosal plexus (SMP) of the rat and mouse colon. GLP-2 (10(-8) M) stimulation for 24 h increased the proportion of enteric neurons expressing VIP (GLP-2: 40 ± 6% vs. control: 22 ± 5%). GLP-2 receptor expression was identified by immunohistochemistry on neurons (HuC/D+) and glial cells (GFAP+) but not on smooth muscle or fibroblasts in culture. Over 1-4 h, GLP-2 stimulation of SMP increased phosphorylated Akt/Akt ratios 6.1-fold, phosphorylated ERK/ERK 2.5-fold, and p70S6K 2.2-fold but did not affect intracellular cAMP. PI3Kγ gene deletion or pharmacological blockade of PI3Kγ, mammalian target of rapamycin (mTOR), and MEK/ERK pathways blocked the increase in VIP expression by GLP-2. GLP-2 increased the expression of growth factors and their receptors in SMP cells in culture [IGF-1r (3.2-fold increase), EGFr (5-fold), and ErbB-2-4r (6- to 7-fold)] and ligands [IGF-I (1.5-fold), amphiregulin (2.5-fold), epiregulin (3.2-fold), EGF (7.5-fold), heparin-bound EGF (2.0-fold), ß-cellulin (50-fold increase), and neuregulins 2-4 (300-fold increase) (by qRT-PCR)]. We conclude that GLP-2 acts on enteric neurons and glial cells in culture via a PI3Kγ/Akt pathway, stimulating neuronal differentiation via mTOR and ERK pathways, and expression of receptors and ligands for the IGF-I and ErbB pathways.
Assuntos
Sistema Nervoso Entérico/metabolismo , Peptídeo 2 Semelhante ao Glucagon/farmacologia , Neurônios/metabolismo , Fosfatidilinositol 3-Quinase/metabolismo , Peptídeo Intestinal Vasoativo/biossíntese , Animais , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/genética , Células Cultivadas , Proteínas ELAV/metabolismo , Sistema Nervoso Entérico/efeitos dos fármacos , Receptores ErbB/metabolismo , Proteína Glial Fibrilar Ácida/metabolismo , Imuno-Histoquímica , Camundongos , Neurregulinas/metabolismo , Neuroglia/efeitos dos fármacos , Neuroglia/metabolismo , Neurônios/efeitos dos fármacos , Proteínas Oncogênicas v-erbB/biossíntese , Proteínas Oncogênicas v-erbB/genética , Fosfatidilinositol 3-Quinase/fisiologia , Ratos , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologia , Serina-Treonina Quinases TOR/metabolismoRESUMO
Despite numerous advances in the identification of the molecular machinery for clathrin-mediated budding at the plasma membrane, the mechanistic details of this process remain incomplete. Moreover, relatively little is known regarding the regulation of clathrin-mediated budding at other membrane systems. To address these issues, we have utilized the powerful new approach of subcellular proteomics to identify novel proteins present on highly enriched clathrin-coated vesicles (CCVs). Among the ten novel proteins identified is the rat homologue of a predicted gene product from human, mouse, and Drosophila genomics projects, which we named enthoprotin. Enthoprotin is highly enriched on CCVs isolated from rat brain and liver extracts. In cells, enthoprotin demonstrates a punctate staining pattern that is concentrated in a perinuclear compartment where it colocalizes with clathrin and the clathrin adaptor protein (AP)1. Enthoprotin interacts with the clathrin adaptors AP1 and with Golgi-localized, gamma-ear-containing, Arf-binding protein 2. Through its COOH-terminal domain, enthoprotin binds to the terminal domain of the clathrin heavy chain and stimulates clathrin assembly. These data suggest a role for enthoprotin in clathrin-mediated budding on internal membranes. Our study reveals the utility of proteomics in the identification of novel vesicle trafficking proteins.
Assuntos
Proteínas Adaptadoras de Transporte Vesicular/metabolismo , Proteínas de Transporte , Vesículas Revestidas por Clatrina/metabolismo , Clatrina/metabolismo , Proteínas de Membrana/metabolismo , Proteômica , Complexo 1 de Proteínas Adaptadoras/metabolismo , Proteínas Adaptadoras de Transporte Vesicular/química , Animais , Células COS , Proteínas de Fluorescência Verde , Humanos , Proteínas Luminescentes , Espectrometria de Massas , Proteínas de Membrana/química , Camundongos , Ligação Proteica , Proteínas/metabolismo , RatosRESUMO
BACKGROUND AND AIMS: A glucagon-like peptide 2 (GLP-2) analogue is approved for adults with intestinal failure, but no studies of GLP-2 have included children. This study examined the pharmacokinetics, safety, and nutritional effects of GLP-2 in children with intestinal failure. METHODS: Native human GLP-2(1-33) was synthesized following good manufacturing practices. In an open-label trial, with parental consent, 7 parenteral nutrition-dependent pediatric patients were treated with subcutaneous GLP-2 (20 µg/kg/d) for 3 days (phase 1) and, if tolerated, continued for 42 days (phase 2). Nutritional treatment was directed by the primary caregivers. Patients were followed to 1 year. RESULTS: Seven patients were enrolled (age: 4.0 ± 0.8 years; bowel length, mean ± SEM: 24% ± 4% of predicted). All were parenteral nutrition dependent since birth, receiving 44% ± 5% of calories by parenteral nutrition. GLP-2 treatment had no effect on vital signs (blood pressure, heart rate, and temperature) and caused no significant adverse events. Peak GLP-2 levels were 380 pM (day 3) and 295 pM (day 42), with no change in half-life or endogenous GLP-2 levels. Nutritional indices showed a numeric improvement in z scores and citrulline levels; the z score was maintained while citrulline levels returned to baseline once GLP-2 was discontinued. CONCLUSIONS: GLP-2 was well tolerated in children, with a pharmacokinetic profile similar to that of adults. There were no changes in endogenous GLP-2 release or metabolism. These results suggest that GLP-2 ligands may be safely used in pediatric patients; larger trials are suggested to investigate nutritional effects.
Assuntos
Peptídeo 2 Semelhante ao Glucagon/administração & dosagem , Síndrome do Intestino Curto/terapia , Pré-Escolar , Relação Dose-Resposta a Droga , Nutrição Enteral , Seguimentos , Peptídeo 2 Semelhante ao Glucagon/sangue , Peptídeo 2 Semelhante ao Glucagon/farmacocinética , Humanos , Absorção Intestinal/efeitos dos fármacos , Mucosa Intestinal/metabolismo , Nutrição Parenteral , Tamanho da Amostra , Síndrome do Intestino Curto/sangueRESUMO
OBJECTIVE: To determine the effects of exogenous glucagon-like peptide-2 (GLP-2), with or without massive distal bowel resection, on adaptation of jejunal mucosa, enteric neurons, gut hormones and tissue reserves in rats. BACKGROUND: GLP-2 is a gut hormone known to be trophic for small bowel mucosa, and to mimic intestinal adaptation in short bowel syndrome (SBS). However, the effects of exogenous GLP-2 and SBS on enteric neurons are unclear. METHODS: Sprague Dawley rats were randomized to four treatments: Transected Bowel (TB) (n = 8), TB + GLP-2 (2.5 nmol/kg/h, n = 8), SBS (n = 5), or SBS + GLP-2 (2.5 nmol/kg/h, n = 9). SBS groups underwent a 60% jejunoileal resection with cecectomy and jejunocolic anastomosis. All rats were maintained on parenteral nutrition for 7 d. Parameters measured included gut morphometry, qPCR for hexose transporter (SGLT-1, GLUT-2, GLUT-5) and GLP-2 receptor mRNA, whole mount immunohistochemistry for neurons (HuC/D, VIP, nNOS), plasma glucose, gut hormones, and body composition. RESULTS: Resection increased the proportion of nNOS immunopositive myenteric neurons, intestinal muscularis propria thickness and crypt cell proliferation, which were not recapitulated by GLP-2 therapy. Exogenous GLP-2 increased jejunal mucosal surface area without affecting enteric VIP or nNOS neuronal immunopositivity, attenuated resection-induced reductions in jejunal hexose transporter abundance (SGLT-1, GLUT-2), increased plasma amylin and decreased peptide YY concentrations. Exogenous GLP-2 attenuated resection-induced increases in blood glucose and body fat loss. CONCLUSIONS: Exogenous GLP-2 stimulates jejunal adaptation independent of enteric neuronal VIP or nNOS changes, and has divergent effects on plasma amylin and peptide YY concentrations. The novel ability of exogenous GLP-2 to modulate resection-induced changes in peripheral glucose and lipid reserves may be important in understanding the whole-body response following intestinal resection, and is worthy of further study.
Assuntos
Adaptação Fisiológica/efeitos dos fármacos , Peptídeo 2 Semelhante ao Glucagon/farmacocinética , Mucosa Intestinal/efeitos dos fármacos , Animais , Procedimentos Cirúrgicos do Sistema Digestório/métodos , Modelos Animais de Doenças , Peptídeo 2 Semelhante ao Glucagon/metabolismo , Mucosa Intestinal/metabolismo , Jejuno/efeitos dos fármacos , Jejuno/metabolismo , Masculino , Nutrição Parenteral/métodos , Nutrição Parenteral Total/métodos , Ratos , Ratos Sprague-Dawley , Síndrome do Intestino Curto/metabolismoRESUMO
BACKGROUND & AIMS: Glucagon-like peptide 2 (GLP-2) analogues are approved for adults with intestinal failure (IF), but no studies have included infants. This study examined the pharmacokinetics (PK), safety, and nutritional effects of GLP-2 in infants with IF. METHODS: With parental consent (Health Canada Protocol:150,979), parenteral nutrition (PN)-dependent infants were treated with 5-20-µg/kg/day GLP-2 for 3days (phase 1), and if tolerated continued for 42days (phase 2). Nutritional therapy was by primary caregivers, and follow-up was to one year. RESULTS: Six patients were enrolled, age 5.4±3.2months, bowel length: 27±12% of predicted, PN dependent (67±18% of calories). GLP-2 did not affect vital signs, nor were there significant adverse events during the trial. Dosing 5µg/kg/day gave GLP-2 levels of 52-57pmol/L, with no change in half-life or endogenous GLP-2 levels. Enteral feeds, weight, Z scores, stooling frequency, and citrulline levels improved numerically. The trial was discontinued early because of a drop in potency. CONCLUSIONS: GLP-2 was well tolerated in infants, and pK was similar to children with no changes in endogenous GLP-2 release. The findings suggest that GLP-2 ligands may be safely used in infants and may have beneficial effects on nutritional status. Further study is required. LEVEL OF EVIDENCE: 2b Prospective Interventional Study.
Assuntos
Fármacos Gastrointestinais/farmacologia , Fármacos Gastrointestinais/farmacocinética , Peptídeo 2 Semelhante ao Glucagon/farmacologia , Peptídeo 2 Semelhante ao Glucagon/farmacocinética , Enteropatias/tratamento farmacológico , Terapia Combinada , Relação Dose-Resposta a Droga , Esquema de Medicação , Feminino , Seguimentos , Fármacos Gastrointestinais/uso terapêutico , Peptídeo 2 Semelhante ao Glucagon/uso terapêutico , Meia-Vida , Humanos , Lactente , Recém-Nascido , Enteropatias/terapia , Masculino , Estado Nutricional/efeitos dos fármacos , Nutrição Parenteral , Estudos Prospectivos , Resultado do TratamentoRESUMO
Aftiphilin was identified through a database search for proteins containing binding motifs for the gamma-ear domain of clathrin adaptor protein 1 (AP-1). Here, we demonstrate that aftiphilin is expressed predominantly in brain where it is enriched on clathrin-coated vesicles. In addition to eight gamma-ear-binding motifs, aftiphilin contains two WXXF-acidic motifs that mediate binding to the alpha-ear of clathrin adaptor protein 2 (AP-2) and three FXXFXXF/L motifs that mediate binding to the alpha- and beta2-ear. We demonstrate that aftiphilin uses these motifs for interactions with AP-1 and AP-2 and that it immunoprecipitates these APs but not AP-3 or AP-4 from brain extracts. Aftiphilin demonstrates a brefeldin A sensitive localization to the trans-Golgi network in hippocampal neurons where it co-localizes with AP-1. Aftiphilin is also found at synapses where it co-localizes with synaptophysin and AP-2. Our data suggest a role for aftiphilin in clathrin-mediated trafficking in neurons.
Assuntos
Proteínas de Transporte/fisiologia , Clatrina/fisiologia , Proteínas do Tecido Nervoso/fisiologia , Neurônios/fisiologia , Sequência de Aminoácidos , Animais , Proteínas de Transporte/química , Meios de Cultivo Condicionados , Dados de Sequência Molecular , Proteínas do Tecido Nervoso/química , Ratos , Homologia de Sequência de AminoácidosAssuntos
Doenças Autoimunes/tratamento farmacológico , Doenças Autoimunes/imunologia , Hipoglicemiantes/uso terapêutico , Macrófagos Alveolares/efeitos dos fármacos , Pioglitazona/uso terapêutico , Proteinose Alveolar Pulmonar/tratamento farmacológico , Proteinose Alveolar Pulmonar/imunologia , Doenças Autoimunes/sangue , Doenças Autoimunes/patologia , Líquido da Lavagem Broncoalveolar/imunologia , Fibrose/diagnóstico por imagem , Fator Estimulador de Colônias de Granulócitos e Macrófagos/antagonistas & inibidores , Humanos , Hipoglicemiantes/administração & dosagem , Consentimento Livre e Esclarecido/legislação & jurisprudência , Masculino , Pessoa de Meia-Idade , Uso Off-Label/legislação & jurisprudência , Pioglitazona/administração & dosagem , Proteinose Alveolar Pulmonar/sangue , Proteinose Alveolar Pulmonar/patologia , Resultado do TratamentoRESUMO
Enthoprotin, a newly identified component of clathrin-coated vesicles, interacts with the trans-Golgi network (TGN) clathrin adapters AP-1 and GGA2. Here we perform a multi-faceted analysis of the site in enthoprotin that is responsible for the binding to the gamma-adaptin ear (gamma-ear) domain of AP-1. Alanine scan mutagenesis and nuclear magnetic resonance (NMR) studies reveal the full extent of the site as well as critical residues for this interaction. NMR studies of the gamma-ear in complex with a synthetic peptide from enthoprotin provide structural details of the binding site for TGN accessory proteins within the gamma-ear.
Assuntos
Subunidades gama do Complexo de Proteínas Adaptadoras/metabolismo , Motivos de Aminoácidos/genética , Alanina/genética , Alanina/metabolismo , Sequência de Aminoácidos , Substituição de Aminoácidos , Animais , Sítios de Ligação , Linhagem Celular , Humanos , Camundongos , Modelos Moleculares , Dados de Sequência Molecular , Ressonância Magnética Nuclear Biomolecular , Ligação Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Alinhamento de Sequência , Fator de Transcrição AP-1/química , Fator de Transcrição AP-1/metabolismo , TransfecçãoRESUMO
BACKGROUND: The enteroendocrine hormone glucagon like peptide-2 (GLP-2) and its ligands are under development as therapeutic agents for a variety of intestinal pathologies. A number of these conditions occur in neonates and infants, and thus a detailed understanding of the effects of GLP-2 during the phase of rapid growth during infancy is required to guide the development of therapeutic applications. We studied the effects of GLP-2 in the neonatal pig to determine the potential effects of exogenous administration. METHODS: Two day old newborn domestic piglets were treated with GLP-2 (1-33) at 40 µg/kg/day or control drug vehicle (saline), by subcutaneous injection, given in two doses per day, (n=6/group) for 42 days. Animals were weaned normally, over days 21-25. In the fifth week of life, they underwent neuro-developmental testing, and a pharmacokinetic study. On day 42, they were euthanized, and a complete necropsy performed, with histological assessment of tissues from all major organs. RESULTS: GLP-2 treatment was well tolerated, one control animal died from unrelated causes. There were no effects of GLP-2 on weight gain, feed intake, or behavior. In the treated animals, GLP-2 levels were significantly elevated at 2400±600 pM while at necropsy, organ weights and histology were not affected except in the intestine, where the villus height in the small intestine and the crypt depth, throughout the small intestine and colon, were increased. Similarly, the rate of crypt cell proliferation (Ki-67 staining) was increased in the GLP-2 treated animals and the rate of apoptosis (Caspase-3) was decreased, the depth of the microvilli was increased and the expression of the mRNA for the GLP-2 receptor was decreased throughout the small and large intestine. CONCLUSIONS: In these growing animals, exogenous GLP-2 at pharmacologic doses was well tolerated, with effects confined to the gastrointestinal tract.
Assuntos
Fármacos Gastrointestinais/administração & dosagem , Peptídeo 2 Semelhante ao Glucagon/administração & dosagem , Animais , Animais Recém-Nascidos , Avaliação Pré-Clínica de Medicamentos , Fármacos Gastrointestinais/farmacocinética , Fármacos Gastrointestinais/toxicidade , Trato Gastrointestinal/efeitos dos fármacos , Trato Gastrointestinal/patologia , Peptídeo 2 Semelhante ao Glucagon/farmacocinética , Peptídeo 2 Semelhante ao Glucagon/toxicidade , Tamanho do Órgão/efeitos dos fármacos , Sus scrofa , Desmame , Aumento de Peso/efeitos dos fármacosRESUMO
PURPOSE: The signals that govern the upregulation of nutrient absorption (adaptation) after intestinal resection are not well understood. A Gastric Roux-en-Y bypass (GRYB) model was used to isolate the relative contributions of direct mucosal stimulation by nutrients, biliary-pancreatic secretions, and systemic enteric hormones on intestinal adaptation in short bowel syndrome. METHODS: Male rats (350-400 g; n = 8/group) underwent sham or GRYB with pair feeding and were observed for 14 days. Weight and serum hormonal levels (glucagon-like peptide-2 [GLP-2], PYY) were quantified. Adaptation was assessed by intestinal morphology and crypt cell kinetics in each intestinal limb of the bypass and the equivalent points in the sham intestine. Mucosal growth factors and expression of transporter proteins were measured in each limb of the model. RESULTS: The GRYB animals lost weight compared to controls and exhibited significant adaptive changes with increased bowel width, villus height, crypt depth, and proliferation indices in the alimentary and common intestinal limbs. Although the biliary limb did not adapt at the mucosa, it did show an increased bowel width and crypt cell proliferation rate. The bypass animals had elevated levels of systemic PYY and GLP-2. At the mucosal level, insulin-like growth factor-1 (IGF-1) and basic fibroblast growth factor (bFGF) increased in all limbs of the bypass animals, whereas keratinocyte growth factor (KGF) and epidermal growth factor (EGF) had variable responses. The expression of the passive transporter of glucose, GLUT-2, expression was increased, whereas GLUT-5 was unchanged in all limbs of the bypass groups. Expression of the active mucosal transporter of glucose, SGLT-1 was decreased in the alimentary limb. CONCLUSIONS: Adaptation occurred maximally in intestinal segments stimulated by nutrients. Partial adaptation in the biliary limb may reflect the effects of systemic hormones. Mucosal content of IGF-1, bFGF, and EGF appear to be stimulated by systemic hormones, potentially GLP-2, whereas KGF may be locally regulated. Further studies to examine the relationships between the factors controlling nutrient-induced adaptation are suggested. Direct contact with nutrients appears to be the most potent factor in inducing mucosal adaptation.