RESUMEN
Peptide YY 3-36 (PYY3-36) is known as a critical satiety factor that reduces food intake both in rodents and humans. Although the anorexic effect of PYY3-36 is assumed to be mediated mainly by the Y2 receptor, the involvement of other Y-receptors in this process has never been conclusively resolved. Amongst them, the Y5 receptor (Y5R) is the most likely candidate to also be a target for PYY3-36, which is considered to counteract the anorectic effects of Y2R activation. In the present study, we show that short-term treatment of diet-induced obese wild-type (WT) and Y5R knockout mice (Y5KO) with PYY3-36 leads to a significantly reduced food intake in both genotypes, which is more pronounced in Y5R KO mice. Interestingly, chronic PYY3-36 infusion via minipumps to WT mice causes an increased cumulative food intake, which is associated with increased body weight gain. By contrast, lack of Y5R reversed this effect. Consistent with the observed increased body weight and fat mass in WT-treated mice, glucose tolerance was also impaired by chronic PYY3-36 treatment. Again, this was less affected in Y5KO mice, suggestive of a role of Y5R in the regulation of glucose homeostasis. Taken together, our data suggest that PYY3-36 mediated signalling via Y5 receptors may counteract the anorectic effects that it mediates via the Y2 receptor (Y2R), consequently lowering bodyweight in the absence of Y5 signalling. These findings open the potential of combination therapy using PYY3-36 and Y5R antagonists to enhance the food intake reducing effects of PYY3-36.
Asunto(s)
Anorexia/metabolismo , Obesidad/metabolismo , Fragmentos de Péptidos/metabolismo , Péptido YY/metabolismo , Receptores de Neuropéptido Y/metabolismo , Animales , Peso Corporal , Huesos/metabolismo , Dieta Alta en Grasa , Ingestión de Alimentos , Glucosa/metabolismo , Homeostasis , Ratones Noqueados , Receptores de Neuropéptido Y/genéticaRESUMEN
OBJECTIVE: The positive metabolic outcome of Roux-en-Y gastric bypass (RYGB) surgery may involve fibroblast growth factor 21 (FGF21), in both the fasting state and postprandially. We measured the fasting levels of FGF21 before and after bariatric surgery as well as the postprandial FGF21 responses after a glucose load and after a mixed meal. DESIGN: Observational intervention trial. PATIENTS AND MEASUREMENTS: Eight obese, nondiabetic patients underwent RYGB. Plasma FGF21 was measured both before and after surgery on three different days during oral glucose loads (25 g or 50 g glucose) or a mixed meal. Blood samples were taken right before the meal and at 15-min intervals until 90 min and at 150 min and 210 min relative to the start of the meal. RESULTS: Overall, fasting plasma FGF21 did not change significantly before and after surgery (262 ± 71 vs 411 ± 119 pg/ml), but for three subjects, fasting plasma FGF21 increased significantly after surgery. Furthermore, FGF21 levels increased significantly at t = 90 and t = 150 min in response to 50 g glucose, but not after a mixed meal. CONCLUSIONS: In conclusion, the observed increase in postprandial plasma FGF21 in response to glucose and the lack of FGF21 response to a mixed meal may have important implications for the physiologic role of FGF21. The increase in postprandial FGF21 in response to glucose in the early postoperative period may contribute to the metabolic improvements observed after gastric bypass.
Asunto(s)
Factores de Crecimiento de Fibroblastos/sangre , Derivación Gástrica , Obesidad/sangre , Adulto , Femenino , Prueba de Tolerancia a la Glucosa , Humanos , Masculino , Periodo PosprandialRESUMEN
Our aim was to study the potential mechanisms responsible for the improvement in glucose control in Type 2 diabetes (T2D) within days after Roux-en-Y gastric bypass (RYGB). Thirteen obese subjects with T2D and twelve matched subjects with normal glucose tolerance (NGT) were examined during a liquid meal before (Pre), 1 wk, 3 mo, and 1 yr after RYGB. Glucose, insulin, C-peptide, glucagon-like peptide-1 (GLP-1), glucose-dependent-insulinotropic polypeptide (GIP), and glucagon concentrations were measured. Insulin resistance (HOMA-IR), ß-cell glucose sensitivity (ß-GS), and disposition index (D(ß-GS): ß-GS × 1/HOMA-IR) were calculated. Within the first week after RYGB, fasting glucose [T2D Pre: 8.8 ± 2.3, 1 wk: 7.0 ± 1.2 (P < 0.001)], and insulin concentrations decreased significantly in both groups. At 129 min, glucose concentrations decreased in T2D [Pre: 11.4 ± 3, 1 wk: 8.2 ± 2 (P = 0.003)] but not in NGT. HOMA-IR decreased by 50% in both groups. ß-GS increased in T2D [Pre: 1.03 ± 0.49, 1 wk: 1.70 ± 1.2, (P = 0.012)] but did not change in NGT. The increase in DI(ß-GS) was 3-fold in T2D and 1.5-fold in NGT. After RYGB, glucagon secretion was increased in response to the meal. GIP secretion was unchanged, while GLP-1 secretion increased more than 10-fold in both groups. The changes induced by RYGB were sustained or further enhanced 3 mo and 1 yr after surgery. Improvement in glycemic control in T2D after RYGB occurs within days after surgery and is associated with increased insulin sensitivity and improved ß-cell function, the latter of which may be explained by dramatic increases in GLP-1 secretion.
Asunto(s)
Diabetes Mellitus Tipo 2/complicaciones , Derivación Gástrica , Resistencia a la Insulina , Células Secretoras de Insulina/metabolismo , Obesidad/metabolismo , Obesidad/cirugía , Adulto , Índice de Masa Corporal , Péptido C/sangre , Péptido C/metabolismo , Femenino , Estudios de Seguimiento , Glucagón/sangre , Glucagón/metabolismo , Péptido 1 Similar al Glucagón/sangre , Péptido 1 Similar al Glucagón/metabolismo , Glucosa/metabolismo , Humanos , Hiperglucemia/prevención & control , Masculino , Persona de Mediana Edad , Obesidad/complicaciones , Obesidad/fisiopatología , Obesidad Mórbida/complicaciones , Obesidad Mórbida/metabolismo , Obesidad Mórbida/fisiopatología , Obesidad Mórbida/cirugía , Periodo Posprandial , Factores de TiempoRESUMEN
BACKGROUND: Roux-en-Y gastric bypass (RYGB) surgery causes profound changes in secretion of gastrointestinal hormones and glucose metabolism. We present a detailed analysis of the early hormone changes after RYGB in response to three different oral test meals designed to provide this information without causing side effects (such as dumping). METHODS: We examined eight obese non-diabetic patients before and within 2 weeks after RYGB. On separate days, oral glucose tolerance tests (25 or 50 g glucose dissolved in 200 mL of water) and a liquid mixed meal test (200 mL 300 kcal) were performed. We measured fasting and postprandial glucose, insulin, C-peptide, glucagon, total and intact glucagon-like peptide-1 (GLP-1), glucose-dependent insulinotropic polypeptide (GIP), glucagon-like peptide-2 (GLP-2), peptide YY(3-36) (PYY), cholecystokinin (CCK), total and active ghrelin, gastrin, somatostatin, pancreatic polypeptide (PP), amylin, leptin, free fatty acids (FFA), and registered postprandial dumping. Insulin sensitivity was measured by homeostasis model assessment of insulin resistance. RESULTS: Fasting glucose, insulin, ghrelin, and PYY were significantly decreased and FFA was elevated postoperatively. Insulin sensitivity increased after surgery. The postprandial response increased for C-peptide, GLP-1, GLP-2, PYY, CCK, and glucagon (in response to the mixed meal) and decreased for total and active ghrelin, leptin, and gastrin, but were unchanged for GIP, amylin, PP, and somatostatin after surgery. Dumping symptoms did not differ before and after the operation or between the tests. CONCLUSIONS: Within 2 weeks after RYGB, we found an increase in insulin secretion and insulin sensitivity. Responses of appetite-regulating intestinal hormones changed dramatically, all in the direction of reducing hunger.
Asunto(s)
Derivación Gástrica , Hormonas Gastrointestinales/sangre , Células Secretoras de Insulina/metabolismo , Obesidad Mórbida/metabolismo , Pérdida de Peso , Adulto , Apetito , Péptido C/sangre , Colecistoquinina/sangre , Factores de Confusión Epidemiológicos , Femenino , Polipéptido Inhibidor Gástrico/sangre , Gastrinas/sangre , Hormonas Gastrointestinales/metabolismo , Ghrelina/sangre , Glucagón/sangre , Péptido 1 Similar al Glucagón/sangre , Péptido 2 Similar al Glucagón/sangre , Prueba de Tolerancia a la Glucosa , Humanos , Insulina/sangre , Insulina/metabolismo , Resistencia a la Insulina , Secreción de Insulina , Polipéptido Amiloide de los Islotes Pancreáticos/sangre , Leptina/sangre , Masculino , Persona de Mediana Edad , Obesidad Mórbida/sangre , Obesidad Mórbida/cirugía , Polipéptido Pancreático/sangre , Péptido YY/sangre , Periodo Posprandial , Somatostatina/sangre , Factores de TiempoRESUMEN
The neuropeptide Y (NPY)/peptide YY (PYY) system has been implicated in the physiology of obesity for several decades. More recently ignited enormous interest in PYY3-36, an endogenous Y2-receptor agonist, as a promising anti-obesity compound. Despite this interest, there have been remarkably few subsequent reports reproducing or extending the initial findings, while at the same time studies finding no anti-obesity effects have surfaced. Out of 41 different rodent studies conducted (in 16 independent labs worldwide), 33 (83%) were unable to reproduce the reported effects and obtained no change or sometimes increased food intake, despite use of the same experimental conditions (i.e. adaptation protocols, routes of drug administration and doses, rodent strains, diets, drug vendors, light cycles, room temperatures). Among studies by authors in the original study, procedural caveats are reported under which positive effects may be obtained. Currently, data speak against a sustained decrease in food intake, body fat, or body weight gain following PYY3-36 administration and make the previously suggested role of the hypothalamic melanocortin system unlikely as is the existence of PYY deficiency in human obesity. We review the studies that are in the public domain which support or challenge PYY3-36 as a potential anti-obesity target.
Asunto(s)
Fármacos Antiobesidad/farmacología , Peso Corporal/efectos de los fármacos , Ingestión de Alimentos/efectos de los fármacos , Péptido YY/farmacología , Animales , Conducta Animal , Interpretación Estadística de Datos , Dipeptidil Peptidasa 4/metabolismo , Humanos , Fragmentos de Péptidos , Péptido YY/administración & dosificación , Receptores de Neuropéptido Y/agonistas , Respuesta de Saciedad/efectos de los fármacos , Especificidad de la Especie , Estrés Fisiológico/fisiopatologíaRESUMEN
OBJECTIVE: This study was conducted to elucidate whether antagonistic targeting of the histamine H3 receptor increases hypothalamic histamine levels, in parallel with decreases in food intake and body weight. METHODS: The competitive antagonist potency of a recently synthesized histamine H3 receptor antagonist, NNC 38-1049, was studied in intact HEK293 cells expressing human or rat histamine H3 receptor, in which NNC 38-1049 was allowed to antagonize the effect of the H3 receptor agonist R-alpha-methylhistamine on isoprenaline-induced accumulation of cAMP. The affinity of NNC 38-1049 for a number of variants of the histamine receptor was also determined. Following single dosing of normal rats with NNC 38-1049, hypothalamic histamine levels were assessed by means of microdialysis. Plasma and brain levels of NNC 38-1049 and acute effects on food intake and energy expenditure were followed after oral doses of 3-60 mg/kg. Potential side effects were examined with rat models of behaviour satiety sequence (BSS), pica behaviour and conditioned taste aversion (CTA). Intakes of food and water together with body weight were recorded for 15 days during daily dosing of dietary obese rats. RESULTS: NNC 38-1049 was found to be a highly specific and competitive antagonist towards both human and rat histamine H3 receptors, and measurable amounts of NNC 38-1049 were found in the plasma of rats following single oral doses of 3-60 mg/kg and in the brain after 15-60 mg/kg. Following single intraperitoneal injections of NNC 38-1049 (20 mg/kg), significant increases in extracellular histamine concentrations were observed. The same dose did not change BSS or pica behaviour acutely, nor did it induce CTA following repeated administration for 7 days. Reductions in food intake were seen very soon after administration, and occurred in a dose-dependent fashion. Energy expenditure was unchanged, but the respiratory quotient (RQ) tended to decrease at higher doses, indicating an increase in lipid oxidation. Twice daily administration of 20 mg/kg of NNC 38-1049 in old and dietary obese rats resulted in sustained reduction of food intake throughout a 2-week study, and was associated with a highly significant (P<0.01) decrease in body weight compared with controls (-18.4+/-3.4 vs +0.4+/-2.7 g). The same dose of NNC 38-1049 produced an acute decrease of water intake, but 24 h intakes were not significantly changed. CONCLUSIONS: The results of this study strongly support the idea that an increase in the hypothalamic concentration of histamine produces a specific reduction of food intake and that this effect can be translated into a decrease in body weight.
Asunto(s)
Antagonistas de los Receptores Histamínicos/farmacología , Hipotálamo/efectos de los fármacos , Receptores Histamínicos H3/efectos de los fármacos , Administración Oral , Animales , Peso Corporal/efectos de los fármacos , Ingestión de Líquidos/efectos de los fármacos , Ingestión de Alimentos/efectos de los fármacos , Antagonistas de los Receptores Histamínicos/administración & dosificación , Hipotálamo/metabolismo , Masculino , Ratones , Ratones Obesos , Actividad Motora/efectos de los fármacos , Piperazinas/administración & dosificación , Piperazinas/farmacología , Ratas , Ratas Sprague-Dawley , Receptores Histamínicos H3/sangreRESUMEN
Batterham et al. report that the gut peptide hormone PYY3-36 decreases food intake and body-weight gain in rodents, a discovery that has been heralded as potentially offering a new therapy for obesity. However, we have been unable to replicate their results. Although the reasons for this discrepancy remain undetermined, an effective anti-obesity drug ultimately must produce its effects across a range of situations. The fact that the findings of Batterham et al. cannot easily be replicated calls into question the potential value of an anti-obesity approach that is based on administration of PYY3-36.
Asunto(s)
Depresores del Apetito/farmacología , Regulación del Apetito/efectos de los fármacos , Conducta Alimentaria/efectos de los fármacos , Péptido YY/farmacología , Animales , Animales Endogámicos , Apetito/efectos de los fármacos , Apetito/fisiología , Depresores del Apetito/uso terapéutico , Conducta Animal/efectos de los fármacos , Peso Corporal/efectos de los fármacos , Ambiente , Humanos , Metaanálisis como Asunto , Ratones , Obesidad/tratamiento farmacológico , Fragmentos de Péptidos , Péptido YY/administración & dosificación , Péptido YY/sangre , Péptido YY/uso terapéutico , Ratas , Reproducibilidad de los Resultados , Estrés Fisiológico/complicaciones , Estrés Fisiológico/fisiopatologíaRESUMEN
(1) Glucagon and glucagon-like peptide-1 (GLP-1) are homologous peptide hormones with important functions in glucose metabolism. The receptors for glucagon and GLP-1 are homologous family B G-protein coupled receptors. The GLP-1 receptor amino-terminal extracellular domain is a major determinant of glucagon/GLP-1 selectivity of the GLP-1 receptor. However, the divergent residues in glucagon and GLP-1 that determine specificity for the GLP-1 receptor amino-terminal extracellular domain are not known. Less is known about how the glucagon receptor distinguishes between glucagon and GLP-1. (2) We analysed chimeric glucagon/GLP-1 peptides for their ability to bind and activate the glucagon receptor, the GLP-1 receptor and chimeric glucagon/GLP-1 receptors. The chimeric peptide GLP-1(7-20)/glucagon(15-29) was unable to bind and activate the glucagon receptor. Substituting the glucagon receptor core domain with the GLP-1 receptor core domain (chimera A) completely rescued the affinity and potency of GLP-1(7-20)/glucagon(15-29) without compromising the affinity and potency of glucagon. Substituting transmembrane segment 1 (TM1), TM6, TM7, the third extracellular loop and the intracellular carboxy-terminus of chimera A with the corresponding glucagon receptor segments re-established the ability to distinguish GLP-1(7-20)/glucagon(15-29) from glucagon. Corroborant results were obtained with the opposite chimeric peptide glucagon(1-14)/GLP-1(21-37). (3) The results suggest that the glucagon and GLP-1 receptor amino-terminal extracellular domains determine specificity for the divergent residues in the glucagon and GLP-1 carboxy-terminals respectively. The GLP-1 receptor core domain is not a critical determinant of glucagon/GLP-1 selectivity. Conversely, the glucagon receptor core domain contains two or more sub-segments which strongly determine specificity for divergent residues in the glucagon amino-terminus.
Asunto(s)
Receptores de Glucagón/química , Receptores de Glucagón/metabolismo , Secuencia de Aminoácidos , Unión Competitiva/genética , Unión Competitiva/fisiología , Línea Celular , Relación Dosis-Respuesta a Droga , Receptor del Péptido 1 Similar al Glucagón , Humanos , Ligandos , Datos de Secuencia Molecular , Unión Proteica/genética , Unión Proteica/fisiología , Estructura Terciaria de Proteína , Receptores de Glucagón/genética , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/metabolismoRESUMEN
Cocaine and amphetamine regulated transcript (CART) peptide has been shown to be an anorectic peptide that inhibits both normal and starvation-induced feeding and completely blocks the feeding response induced by neuropeptide Y and regulated by leptin in the hypothalamus. The C-terminal part containing the three disulfide bridges CART(48-89) is the biologically active part of the molecule affecting food intake. The solution structure of the active part of CART has a fold equivalent to other functionally distinct small proteins. CART consists mainly of turns and loops spanned by a compact framework composed by a few small stretches of antiparallel beta-sheet common to cystine knots.
Asunto(s)
Proteínas del Tejido Nervioso/química , Proteínas del Tejido Nervioso/fisiología , Pliegue de Proteína , Secuencia de Aminoácidos , Cistina/química , Disulfuros/química , Humanos , Modelos Moleculares , Datos de Secuencia Molecular , Resonancia Magnética Nuclear Biomolecular , Estructura Secundaria de Proteína , Homología de Secuencia de Aminoácido , SolucionesRESUMEN
The hypothalamic satiety peptide CART (cocaine and amphetamine regulated transcript) is expressed at high levels in anorectic rat glucagonomas but not in hypoglycemic insulinomas. However, a non-anorectic metastasis derived from the glucagonoma retained high CART expression levels and produced circulating CART levels comparable to that of the anorectic tumors. Moreover, distinct glucagonoma lines derived by stable HES-1 transfection of the insulinoma caused severe anorexia but retained low circulating levels of CART comparable to that of insulinoma bearing or control rats. Islet tumor associated anorexia and circulating CART levels are thus not correlated, and in line with this peripheral administration of CART (5-50 mg/kg) produced no effect on feeding behavior. In the rat two alternatively spliced forms of CART mRNA exist and quantitative PCR revealed expression of both forms in the hypothalamus, in the different islet tumors, and in the islets of Langerhans. Immunocytochemistry as well as in situ hybridization localized CART expression to the somatostatin producing islet D cell. A potential endocrine/paracrine role of islet CART remains to be clarified.
Asunto(s)
Adenoma de Células de los Islotes Pancreáticos/sangre , Adenoma de Células de los Islotes Pancreáticos/genética , Anorexia/sangre , Anorexia/genética , Islotes Pancreáticos/metabolismo , Proteínas del Tejido Nervioso/sangre , Proteínas del Tejido Nervioso/genética , Neoplasias Pancreáticas/sangre , Neoplasias Pancreáticas/genética , Animales , Secuencia de Bases , Sondas de ADN/genética , Ingestión de Alimentos/efectos de los fármacos , Femenino , Expresión Génica , Glucagonoma/sangre , Glucagonoma/genética , Inmunohistoquímica , Hibridación in Situ , Insulinoma/sangre , Insulinoma/genética , Proteínas del Tejido Nervioso/farmacología , ARN Mensajero/genética , ARN Neoplásico/genética , Ratas , Células Secretoras de Somatostatina/metabolismoRESUMEN
Cocaine- and amphetamine-regulated transcript (CART) is a recently discovered hypothalamic peptide regulated by leptin and with a potent appetite-suppressing activity. In the rat, the CART gene encodes a peptide of 116 amino acid residues (or a splice variant 13 residues longer). The predicted signal sequence is 27 amino acid residues, resulting in a prohormone of 89 residues. The CART prohormone contains several potential posttranslational processing sites in the form of mono- and dibasic sequences. In the present study we have purified CART peptides from extracts of adrenal gland, hypothalamus, nucleus accumbens, and pituitary gland (anterior and neurointermediate lobe) of the rat and determined the peptide structures by using microsequencing and mass spectrometry. In none of the tissues examined the long splice variant was found. From the adrenal gland, the CART(1-89) and CART(10-89) peptides were isolated, in contrast to the hypothalamus and nucleus accumbens, from which the shorter form peptides CART(42-89) and CART(49-89) were purified. From the anterior lobe of the pituitary gland, CART(42-89) was isolated, in contrast to the neurointermediate lobe, which contains only CART(49-89). This tissue-specific processing indicates that CART peptides may have different biological functions in the periphery and in the central nervous system.
Asunto(s)
Proteínas del Tejido Nervioso/biosíntesis , Procesamiento Proteico-Postraduccional , Animales , Cromatografía Líquida de Alta Presión , Femenino , Especificidad de Órganos , Ratas , Ratas WistarRESUMEN
The mammalian hypothalamus strongly influences ingestive behaviour through several different signalling molecules and receptor systems. Here we show that CART (cocaine- and amphetamine-regulated transcript), a brain-located peptide, is a satiety factor and is closely associated with the actions of two important regulators of food intake, leptin and neuropeptide Y. Food-deprived animals show a pronounced decrease in expression of CART messenger RNA in the arcuate nucleus. In animal models of obesity with disrupted leptin signalling, CART mRNA is almost absent from the arcuate nucleus. Peripheral administration of leptin to obese mice stimulates CART mRNA expression. When injected intracerebroventricularly into rats, recombinant CART peptide inhibits both normal and starvation-induced feeding, and completely blocks the feeding response induced by neuropeptide Y. An antiserum against CART increases feeding in normal rats, indicating that CART may be an endogenous inhibitor of food intake in normal animals.
Asunto(s)
Anorexia/metabolismo , Hipotálamo/fisiología , Proteínas del Tejido Nervioso/fisiología , Proteínas/fisiología , Animales , Núcleo Arqueado del Hipotálamo/metabolismo , Clonación Molecular , Escherichia coli , Ayuno , Conducta Alimentaria/fisiología , Regulación de la Expresión Génica , Hipotálamo/metabolismo , Técnicas para Inmunoenzimas , Leptina , Ratones , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo , Neuropéptido Y/biosíntesis , Neuropéptido Y/fisiología , Obesidad/metabolismo , ARN Mensajero/metabolismo , Ratas , Ratas Wistar , Ratas Zucker , Proteínas Recombinantes de Fusión , Transducción de SeñalRESUMEN
Cocaine and amphetamine regulated transcript peptide (CART), is a recently discovered hypothalamic peptide with a potent appetite suppressing activity. In the rat the CART gene encodes a peptide of either 129 or 116 amino acid residues whereas only the short form exists in humans. The predicted signal sequence is 27 amino acid residues resulting in a prohormone of 102 or 89 residues. The C-terminal end of CART, consisting of 48 amino acid residues and 3 disulphide bonds, is thought to constitute a biologically active part of the molecule. In the central nervous system CART is highly expressed in many hypothalamic nuclei, some of which are involved in regulating feeding behaviour. The CART mRNA is regulated by leptin, and the expressed CART is a potent inhibitor of feeding that even overrides the feeding response induced by neuropeptide Y. The putative CART receptor is therefore a potential therapeutic target for an anti-obesity drug.
Asunto(s)
Proteínas del Tejido Nervioso/fisiología , Secuencia de Aminoácidos , Animales , Humanos , Leptina , Datos de Secuencia Molecular , Proteínas del Tejido Nervioso/química , Proteínas del Tejido Nervioso/genética , Estructura Secundaria de Proteína , Proteínas/fisiología , ARN Mensajero/biosíntesisRESUMEN
The importance of two highly conserved cysteines in the human pituitary adenylate cyclase activating polypeptide (PACAP)/vasoactive intestinal peptide 1 (VIP1) receptor was examined. Using site-directed mutagenesis, each Cys residue was converted into Ala or Ser. The mutant and wildtype genes were transfected into HEK293 cells and tested for the ability to bind VIP and to activate cAMP production. Cys215Ala/Ser and Cys285Ala/Ser showed at least a tenfold decrease in binding affinity and receptor potency when compared to the wildtype. In contradiction to the wildtype receptor, both mutations were insensitive to dithiothreitol (DTT). The results indicate the existence of a disulfide bond between Cys215 and Cys285, which is important for stabilizing the receptor in the correct conformation for ligand binding and activation.
Asunto(s)
Cisteína , Estructura Secundaria de Proteína , Receptores de la Hormona Hipofisaria/química , Receptores de la Hormona Hipofisaria/fisiología , Receptores de Péptido Intestinal Vasoactivo/química , Receptores de Péptido Intestinal Vasoactivo/fisiología , Péptido Intestinal Vasoactivo/metabolismo , Alanina , Sustitución de Aminoácidos , Unión Competitiva , Línea Celular , Membrana Celular/metabolismo , Secuencia Conservada , AMP Cíclico/metabolismo , Humanos , Modelos Moleculares , Mutagénesis Sitio-Dirigida , Receptores del Polipéptido Activador de la Adenilato-Ciclasa Hipofisaria , Receptores de Tipo I del Polipéptido Intestinal Vasoactivo , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Serina , Transfección , Péptido Intestinal Vasoactivo/farmacologíaRESUMEN
AIM: To study the cellular transport of L68Q cystatin C, the cystatin variant causing amyloidosis and brain haemorrhage in patients suffering from hereditary cystatin C amyloid angiopathy (HCCAA). METHODS: Expression vectors for wild-type and L68Q cystatin C were constructed and used to transfect mouse NIH/3T3 cells. Stable cell clones were isolated after cotransfection with pSV2neo. Clones expressing human wild-type and L68Q cystatin C were compared with respect to secreted cystatin C by enzyme linked immunosorbent assay (ELISA), and for intracellular cystatin C by western blotting and immunofluorescence cytochemistry. Colocalisation studies in cells were performed by double staining with antibodies against human cystatin C and marker proteins for lysosomes, the Golgi apparatus, or the endoplasmic reticulum, and evaluated by confocal microscopy. RESULTS: Concentrations of human cystatin C secreted from transfected NIH/3T3 cells were similar to those secreted from human cells in culture. In general, clones expressing the gene encoding L68Q cystatin C secreted slightly lower amounts of the protein than clones expressing wild-type human cystatin C. Both immunofluorescence cytochemistry and western blotting experiments showed an increased accumulation of cystatin C in cells expressing the gene encoding L68Q cystatin C compared with cells expressing the gene for the wild-type protein. The intracellularly accumulating L68Q cystatin C was insoluble and located mainly in the endoplasmic reticulum. CONCLUSIONS: The cellular transport of human cystatin C is impeded by the pathogenic amino acid substitution Leu68-->Gln. The resulting intracellular accumulation and increased localised concentration of L68Q cystatin C might be an important event in the molecular pathophysiology of amyloid formation and brain haemorrhage in patients with HCCAA.
Asunto(s)
Angiopatía Amiloide Cerebral/metabolismo , Cistatinas/metabolismo , Inhibidores de Cisteína Proteinasa/metabolismo , Animales , Transporte Biológico , Western Blotting , Técnicas de Cultivo de Célula , Angiopatía Amiloide Cerebral/genética , Cistatina C , Cistatinas/genética , Inhibidores de Cisteína Proteinasa/genética , Técnica del Anticuerpo Fluorescente , Humanos , Ratones , Ratones Endogámicos , TransfecciónRESUMEN
The importance of two highly conserved cysteines in the human vasoactive intestinal peptide receptor I (hVIPR 1) was examined. By site-directed mutagenesis each Cys residue was converted into Ala or Ser. The mutant and wild-type genes were transfected into HEK293 cells and tested for the ability to bind VIP and to activate cAMP production. Cys215-Ala/Ser and Cys285-Ala/Ser showed at least a 10-fold decrease in binding affinity and receptor potency when compared to the wild type. In contradiction to the wild-type receptor, both mutations were insensitive to dithiothreitol (DTT). The results indicate the existence of a disulfide bond between Cys215 and Cys285, which is important for stabilising the receptor in the correct conformation for ligand binding and activation.
Asunto(s)
Cisteína/metabolismo , Disulfuros/metabolismo , Receptores de Péptido Intestinal Vasoactivo/química , Receptores de Péptido Intestinal Vasoactivo/metabolismo , Unión Competitiva , Línea Celular , AMP Cíclico/metabolismo , Ditiotreitol/farmacología , Humanos , Mutagénesis Sitio-Dirigida , Estructura Secundaria de Proteína , Receptores de Péptido Intestinal Vasoactivo/genética , Relación Estructura-Actividad , Transfección , Péptido Intestinal Vasoactivo/metabolismoRESUMEN
The actions of glucagon-like peptide-1(7-36)amide (GLP-1(7-36)amide) on cellular signalling were studied in human embryonal kidney 293 (HEK 293) cells stably transfected with the cloned human GLP-1 receptor. The cloned GLP-1 receptor showed a single high-affinity binding site (Kd = 0.76 nM). Binding of GLP-1(7-36)amide stimulated cAMP production in a dose-dependent manner (EC50 = 0.015 nM) and caused an increase in the intracellular free Ca2+ concentration ([Ca2+]i). The latter effect reflected Ca(2+)-induced Ca2+ release and was suppressed by ryanodine. We propose that the ability of GLP-1(7-36)amide to increase [Ca2+]i results from sensitization of the ryanodine receptors by a protein kinase A dependent mechanism.
Asunto(s)
Calcio/metabolismo , AMP Cíclico/metabolismo , Receptores de Glucagón/fisiología , Acetilcolina/farmacología , Calcio/farmacología , Bloqueadores de los Canales de Calcio/farmacología , Línea Celular , Clonación Molecular , AMP Cíclico/análogos & derivados , AMP Cíclico/farmacología , Embrión de Mamíferos , Glucagón/farmacología , Péptido 1 Similar al Glucagón , Receptor del Péptido 1 Similar al Glucagón , Péptidos Similares al Glucagón , Humanos , Ionomicina/farmacología , Riñón , Fragmentos de Péptidos/farmacología , Receptores de Glucagón/biosíntesis , Receptores de Glucagón/efectos de los fármacos , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/efectos de los fármacos , Proteínas Recombinantes/metabolismo , Rianodina/farmacología , Tionucleótidos/farmacologíaRESUMEN
In the insulin-secreting beta-cell line beta TC3, stimulation with 11.2 mmol/l glucose caused a rise in the intracellular free Ca2+ concentration ([Ca2+]i) in only 18% of the tested cells. The number of glucose-responsive cells increased after pretreatment of the cells with glucagon-like peptide I (GLP-I)(7-36)amide and at 10(-11) mol/l; 84% of the cells responded to glucose with a rise in [Ca2+]i. GLP-I(7-36)amide induces a rapid increase in [Ca2+]i only in cells exposed to elevated glucose concentrations (> or = 5.6 mmol/l). The action of GLP-I(7-36)amide and forskolin involved a 10-fold increase in cytoplasmic cAMP concentration and was mediated by activation of protein kinase A. It was not associated with an effect on the membrane potential but required some (small) initial entry of Ca2+ through voltage-dependent L-type Ca2+ channels, which then produced a further increase in [Ca2+]i by mobilization from intracellular stores. The latter effect reflected Ca(2+)-induced Ca2+ release and was blocked by ryanodine. Similar increases in [Ca2+]i were also observed in voltage-clamped cells, although there was neither activation of a background (Ca(2+)-permeable) inward current nor enhancement of the voltage-dependent L-type Ca2+ current. These observations are consistent with GLP-I(7-36) amide inducing glucose sensitivity by promoting mobilization of Ca2+ from intracellular stores. We propose that this novel action of GLP-I(7-36)amide represents an important factor contributing to its insulinotropic action.
Asunto(s)
Calcio/metabolismo , Glucosa/farmacología , Insulina/metabolismo , Islotes Pancreáticos/metabolismo , Fragmentos de Péptidos/farmacología , Animales , Canales de Calcio/efectos de los fármacos , Canales de Calcio/fisiología , Línea Celular , Colforsina/farmacología , AMP Cíclico/metabolismo , Citoplasma/metabolismo , Relación Dosis-Respuesta a Droga , Glucagón , Péptido 1 Similar al Glucagón , Péptidos Similares al Glucagón , Fosfatos de Inositol/metabolismo , Secreción de Insulina , Insulinoma , Islotes Pancreáticos/efectos de los fármacos , Cinética , Ratones , Ratones Transgénicos , Neoplasias Pancreáticas , Rianodina/farmacología , Factores de Tiempo , Células Tumorales CultivadasRESUMEN
The chromaffin cells of the adult rat adrenal medulla are essentially growth arrested in situ, but can proliferate in vitro, suggesting the existence of growth inhibitory factors in the adrenal gland. We have investigated whether pituitary adenylate cyclase-activating polypeptide 38 (PACAP38) could be involved in the growth arrest of adrenal chromaffin cells. In adult rat adrenal gland, PACAP38 was detected by radioimmunoassay and high-performance liquid chromatography and its concentration in the medulla was estimated as 24 nmol/kg wet tissue. Immunohistochemistry of the neonatal and adult rat adrenal medulla showed PACAP38 immunoreactivity in a widely distributed network of delicate nerve fibers surrounding the chromaffin cells. In a primary culture system, PACAP38 inhibited growth factor-stimulated DNA synthesis by 90% in neonatal and adult rat chromaffin cells with half-maximal inhibition at 4 and 0.5 nM, respectively, as demonstrated by bromodeoxyuridine pulse-labeling and immunocytochemical staining of cell nuclei. In comparison, corticosterone inhibited neonatal and adult chromaffin cell proliferation by 70% and 95%, respectively, with half-maximal effect at 100 nM. In neonatal chromaffin cells, 100 nM PACAP38 and 1 microM corticosterone added together abolished proliferation completely (99.8% inhibition). Finally, PACAP38 increased cell survival but showed little neurite-promoting activity in the chromaffin cells. Our data suggest that neurally derived PACAP38, in conjunction with glucocorticoids, may override growth factor mitogenic signals, leading to the postmitotic state of chromaffin cells in the adult adrenal medulla.
Asunto(s)
Médula Suprarrenal/citología , Médula Suprarrenal/metabolismo , Sistema Cromafín/citología , Sistema Cromafín/metabolismo , Neuronas/metabolismo , Neuropéptidos/metabolismo , Médula Suprarrenal/crecimiento & desarrollo , Envejecimiento/metabolismo , Animales , Animales Recién Nacidos/metabolismo , División Celular/efectos de los fármacos , Células Cultivadas , Sistema Cromafín/crecimiento & desarrollo , Cromatografía Líquida de Alta Presión , Corticosterona/farmacología , ADN/biosíntesis , Inmunohistoquímica , Masculino , Fibras Nerviosas/metabolismo , Neuronas/ultraestructura , Neuropéptidos/biosíntesis , Neuropéptidos/fisiología , Polipéptido Hipofisario Activador de la Adenilato-Ciclasa , Radioinmunoensayo , Ratas , Ratas WistarRESUMEN
We report here data on the expression of the various sequences derived from the prepro-vasoactive intestinal polypeptide (VIP) precursor and VIP mRNA in the anterior pituitary gland and cerebral cortex of hypothyroid and hyperthyroid rats. Using specific antisera to each of the prepro-VIP sequences we demonstrated an increase of all prepro-VIP derived sequences, and accordingly, found that the number of cells expressing each of these sequences were markedly augmented in the anterior pituitary of the hypothyroid rats. This was accompanied by a marked increase in VIP mRNA. In the cerebral cortex of the hypothyroid rats no changes were observed. In the pituitary of hyperthyroid animals a significant decrease was seen for prepro-VIP 22-79, VIP and prepro-VIP 156-170, whereas in the cerebral cortex a significant increase was observed for prepro-VIP 22-79, PHI and VIP. We were not able to demonstrate any changes in VIP mRNA in the cerebral cortex or pituitary of the hyperthyroid rats. Gel permeation chromatography and reverse-phase HPLC of extracts from cerebral cortex showed elution profiles identical to the synthetic counterparts. The reported data provide further evidence of a tissue-specific expression and regulation of the VIP gene products.