RESUMEN
Enteropeptidase, localized into the duodenum brush border, is a key enzyme catalyzing the conversion of pancreatic trypsinogen proenzyme to active trypsin, thereby regulating protein digestion and energy homeostasis. We report the discovery and pharmacological profiles of SCO-792, a novel inhibitor of enteropeptidase. A screen employing fluorescence resonance energy transfer was performed to identify enteropeptidase inhibitors. Inhibitory profiles were determined by in vitro assays. To evaluate the in vivo inhibitory effect on protein digestion, an oral protein challenge test was performed in rats. Our screen identified a series of enteropeptidase inhibitors, and compound optimization resulted in identification of SCO-792, which inhibited enteropeptidase activity in vitro, with IC 50 values of 4.6 and 5.4 nmol/L in rats and humans, respectively. In vitro inhibition of enteropeptidase by SCO-792 was potentiated by increased incubation time, and the calculated Kinact/KI was 82 000/mol/L s. An in vitro dissociation assay showed that SCO-792 had a dissociation half-life of almost 14 hour, with a calculated koff rate of 0.047/hour, which suggested that SCO-792 is a reversible enteropeptidase inhibitor. In normal rats, a ≤4 hour prior oral dose of SCO-792 effectively inhibited plasma elevation of branched-chain amino acids in an oral protein challenge test, which indicated that SCO-792 effectively inhibited protein digestion in vivo. In conclusion, our new screen system identified SCO-792 as a potent and reversible inhibitor against enteropeptidase. SCO-792 slowly dissociated from enteropeptidase in vitro and inhibited protein digestion in vivo. Further study using SCO-792 could reveal the effects of inhibiting enteropeptidase on biological actions.
Asunto(s)
Enteropeptidasa/antagonistas & inhibidores , Inhibidores Enzimáticos/administración & dosificación , Bibliotecas de Moléculas Pequeñas/administración & dosificación , Administración Oral , Animales , Evaluación Preclínica de Medicamentos , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacología , Transferencia Resonante de Energía de Fluorescencia , Humanos , Concentración 50 Inhibidora , Ratas , Bibliotecas de Moléculas Pequeñas/química , Bibliotecas de Moléculas Pequeñas/farmacologíaRESUMEN
We examined the pancreatic function of p13 encoded by 1110001J03Rik, whose expression is decreased in pancreatic islets in high-fat-fed diabetic mice, by generating transgenic mice overexpressing p13 (p13-Tg) in pancreatic ß-cells. p13-Tg mice showed normal basal glucose metabolism; however, under high-fat feeding, these animals showed augmented glucose-induced first-phase and total insulin secretion, improved glucose disposal, greater islet area and increased mitotic insulin-positive cells. In addition, high-fat diet-induced 4-hydroxynonenal immunoreactivity, a reliable marker and causative agent of lipid peroxidative stress, was significantly decreased in p13-Tg mouse islets. These results indicate that p13 is a novel pancreatic factor exerting multiple beneficial effects against type 2 diabetes.
Asunto(s)
Diabetes Mellitus Tipo 2/metabolismo , Dieta Alta en Grasa , Células Secretoras de Insulina/metabolismo , Obesidad/metabolismo , Animales , Ratones , Ratones Transgénicos , Regulación hacia ArribaRESUMEN
In diabetes mellitus, pituitary adenylate cyclase-activating polypeptide (PACAP) has insulinotropic and glucose-lowering properties. We previously demonstrated that transgenic mice overexpressing PACAP in pancreatic ß-cells (PACAP-Tg) show attenuated pancreatic islet hyperplasia and hyperinsulinemia in type 2 diabetic models. To explore the underlying mechanisms, here we crossed PACAP-Tg mice with lethal yellow agouti (KKAy) diabetic mice, and performed gene chip analysis of laser capture microdissected pancreatic islets from four F1 offspring genotypes (wild-type, PACAP-Tg, KKAy, and PACAP-Tg:KKAy). We identified 1371 probes with >16-fold differences between at least one pair of genotypes, and classified the probes into five clusters with characteristic expression patterns. Gene ontology enrichment analysis showed that genes involved in the terms ribosome and intracellular organelles such as ribonucleoprotein complex, mitochondrion, and chromosome organization were significantly enriched in clusters characterized by up-regulated genes in PACAP-Tg:KKAy mice compared with KKAy mice. These results may provide insight into the mechanisms of diabetes that accompany islet hyperplasia and amelioration by PACAP.
RESUMEN
Adiponectin secreted from adipocytes binds to adiponectin receptors AdipoR1 and AdipoR2, and exerts antidiabetic effects via activation of AMPK and PPAR-α pathways, respectively. Levels of adiponectin in plasma are reduced in obesity, which causes insulin resistance and type 2 diabetes. Thus, orally active small molecules that bind to and activate AdipoR1 and AdipoR2 could ameliorate obesity-related diseases such as type 2 diabetes. Here we report the identification of orally active synthetic small-molecule AdipoR agonists. One of these compounds, AdipoR agonist (AdipoRon), bound to both AdipoR1 and AdipoR2 in vitro. AdipoRon showed very similar effects to adiponectin in muscle and liver, such as activation of AMPK and PPAR-α pathways, and ameliorated insulin resistance and glucose intolerance in mice fed a high-fat diet, which was completely obliterated in AdipoR1 and AdipoR2 double-knockout mice. Moreover, AdipoRon ameliorated diabetes of genetically obese rodent model db/db mice, and prolonged the shortened lifespan of db/db mice on a high-fat diet. Thus, orally active AdipoR agonists such as AdipoRon are a promising therapeutic approach for the treatment of obesity-related diseases such as type 2 diabetes.
Asunto(s)
Diabetes Mellitus Tipo 2/tratamiento farmacológico , Longevidad/efectos de los fármacos , Obesidad/fisiopatología , Piperidinas/farmacología , Receptores de Adiponectina/agonistas , Adenilato Quinasa/metabolismo , Adiponectina/metabolismo , Adiponectina/farmacología , Tejido Adiposo Blanco/efectos de los fármacos , Tejido Adiposo Blanco/metabolismo , Tejido Adiposo Blanco/patología , Administración Oral , Animales , Diabetes Mellitus Tipo 2/complicaciones , Diabetes Mellitus Tipo 2/metabolismo , Diabetes Mellitus Tipo 2/prevención & control , Dieta Alta en Grasa , Evaluación Preclínica de Medicamentos , Dislipidemias/tratamiento farmacológico , Activación Enzimática/efectos de los fármacos , Intolerancia a la Glucosa/tratamiento farmacológico , Inflamación/tratamiento farmacológico , Resistencia a la Insulina , Hígado/efectos de los fármacos , Hígado/metabolismo , Hígado/patología , Ratones , Mitocondrias/efectos de los fármacos , Mitocondrias/metabolismo , Fibras Musculares Esqueléticas/citología , Fibras Musculares Esqueléticas/efectos de los fármacos , Músculos/citología , Obesidad/complicaciones , Obesidad/tratamiento farmacológico , Obesidad/genética , Estrés Oxidativo/efectos de los fármacos , PPAR alfa/metabolismo , Coactivador 1-alfa del Receptor Activado por Proliferadores de Peroxisomas gamma , Piperidinas/administración & dosificación , Piperidinas/metabolismo , Piperidinas/uso terapéutico , Receptores de Adiponectina/deficiencia , Receptores de Adiponectina/genética , Receptores de Adiponectina/metabolismo , Transducción de Señal/efectos de los fármacos , Bibliotecas de Moléculas Pequeñas/química , Factores de Transcripción/biosíntesis , Triglicéridos/metabolismoRESUMEN
Inadequate compensatory insulin secretion is observed during the development of type 2 diabetes and deteriorates over time in a manner that is difficult to reverse. Here, we found that plasma glucose levels in genetically diabetic KKA(y) mice fed a high-fat diet were markedly increased in young mice. However, the levels started to decrease at 22 weeks of age and returned to normal levels at around 40 weeks of age. These changes were accompanied by a marked increase in insulin levels from week 25 onwards. Decreased energy intake and suppressed fat pad accumulation were observed at 44-45 weeks of age compared with those at 19-22 weeks of age. ß cell-specific overexpression of pituitary adenylate cyclase-activating polypeptide (PACAP), an insulinotropic neuropeptide, decreased the insulin levels required to compensate for hyperglycemia. Glucose disposal was significantly enhanced despite impaired insulin sensitivity in 41-44-week-old A(y) mice without or with PACAP overexpression. In conclusion, the present results provide further evidence that PACAP is involved in the regulation of hyperinsulinemia and islet hyperplasia in type 2 diabetes. Our results also indicate that A(y) mice fed a high-fat diet constitute an animal model suitable to study compensatory islet hyperplasia.
Asunto(s)
Glucemia/análisis , Diabetes Mellitus Tipo 2/fisiopatología , Dieta Alta en Grasa , Insulina/metabolismo , Polipéptido Hipofisario Activador de la Adenilato-Ciclasa/fisiología , Proteína de Señalización Agouti/genética , Animales , Peso Corporal , Cruzamientos Genéticos , Diabetes Mellitus Tipo 2/genética , Diabetes Mellitus Tipo 2/metabolismo , Diabetes Mellitus Tipo 2/patología , Modelos Animales de Enfermedad , Ingestión de Energía , Expresión Génica , Hiperinsulinismo/genética , Hiperinsulinismo/metabolismo , Hiperinsulinismo/prevención & control , Hiperfagia/genética , Hiperplasia , Resistencia a la Insulina/genética , Secreción de Insulina , Grasa Intraabdominal/patología , Islotes Pancreáticos/metabolismo , Islotes Pancreáticos/patología , Hígado/patología , Ratones , Ratones Endogámicos C57BL , Ratones Mutantes , Obesidad/genética , Obesidad/prevención & control , Tamaño de los Órganos , Páncreas/patología , Polipéptido Hipofisario Activador de la Adenilato-Ciclasa/biosíntesis , Polipéptido Hipofisario Activador de la Adenilato-Ciclasa/genética , Proteínas Recombinantes de Fusión/biosíntesis , Proteínas Recombinantes de Fusión/fisiologíaRESUMEN
In our previous study, we reported that cerulein-induced acute pancreatitis is aggravated in pancreatic ß-cell-specific pituitary adenylate cyclase-activating polypeptide (PACAP) transgenic mice, showing that an increase in pancreatic PACAP is a risk factor for progression of acute pancreatitis. Accordingly, in this study, we examined the progression of cerulein-induced acute pancreatitis in PACAP knockout (KO) mice. Unexpectedly, after cerulein, about 60% of the KO mice showed severe hypothermia below 30°C by 12 h and most of them died within 72 h. In contrast, the remaining KO and wild-type mice showed normothermia with no mortality. Thus, KO mice could be classified into two groups as hypothermic (HT-KO) and normothermic (NT-KO) to cerulein. Only HT-KO mice subsequently showed severe mortality, although both HT-KO and NT-KO mice exhibited similar susceptibility of lungs to cerulein toxicity, comparable to that in wild-type mice. Regarding pancreatitis, HT-KO mice showed ameliorated pancreatic damage without any rise in serum enzyme activities, whereas NT-KO mice exhibited a similar degree of pancreatitis to wild-type mice. Taken together, the present results indicate that lack of pancreatic PACAP did not aggravate, but rather ameliorated, cerulein-induced pancreatitis. In addition, about half of KO mice showed a novel phenotype in which cerulein caused rapid and severe hypothermia, followed by death.
Asunto(s)
Ceruletida/farmacología , Pancreatitis/inducido químicamente , Polipéptido Hipofisario Activador de la Adenilato-Ciclasa/metabolismo , Animales , Temperatura Corporal/efectos de los fármacos , Hipotermia , Pulmón/efectos de los fármacos , Pulmón/enzimología , Pulmón/patología , Ratones , Ratones Noqueados , Pancreatitis/patología , Pancreatitis/fisiopatología , Peroxidasa/metabolismo , Polipéptido Hipofisario Activador de la Adenilato-Ciclasa/genética , Tasa de SupervivenciaRESUMEN
Development of human chronic pancreatitis is associated with intrapancreatic accumulation of pituitary adenylate cyclase-activating polypeptide (PACAP) accompanied with an altered inflammatory response (Michalski et al., Am J Physiol Gastrointest Liver Physiol. 2008;294:G50-G57). To investigate the role of pancreatic PACAP in the development of acute pancreatitis, we employed transgenic mice over-expressing PACAP in pancreatic beta-cells (PACAP-Tg). In comparison to wild-type mice, PACAP-Tg mice exhibited more severe pathophysiological signs of the cerulein-induced pancreatitis at 12 h, as evidenced by higher serum amylase and lipase levels accompanied by the exacerbation of pancreatic edema, necrosis, and inflammation. Cerulein treatment increased mRNA expression of several proinflammatory cytokines (TNFalpha, IL-1beta, and IL-6) at 12 h with similar magnitude both in wild-type and PACAP-Tg mice. In addition, the mRNA and protein levels of regenerating gene III beta (RegIIIbeta), a key factor in the pancreatic response to acute pancreatitis, were up-regulated at 24 h in wild-type mice upon cerulein administration, whereas they were attenuated in PACAP-Tg mice. These data indicate that over-expressed PACAP in pancreas enhances the cerulein-induced inflammatory response of both acinar cells, leading to aggravated acute pancreatitis, which was accompanied by a down-regulation of RegIIIbeta, an anti-inflammatory factor.
Asunto(s)
Células Secretoras de Insulina/metabolismo , Pancreatitis/fisiopatología , Polipéptido Hipofisario Activador de la Adenilato-Ciclasa/metabolismo , Proteínas/genética , Enfermedad Aguda , Amilasas/sangre , Animales , Ceruletida/toxicidad , Citocinas/metabolismo , Modelos Animales de Enfermedad , Regulación hacia Abajo , Lipasa/sangre , Ratones , Ratones Transgénicos , Proteínas Asociadas a Pancreatitis , ARN Mensajero/metabolismo , Regulación hacia ArribaRESUMEN
Pituitary adenylate cyclase-activating polypeptide (PACAP) is a neuropeptide implicated in several metabolic functions, including insulin secretion and sympathoadrenal activation. To clarify the roles of PACAP in maintenance of whole-body glucose and lipid homeostasis, the impact of the deletion of PACAP on glucose homeostasis, body weight, and adipose tissue mass was examined by comparing mice lacking the Adcyap1 gene encoding PACAP (Adcyap1(-/-)) with wild-type littermate controls. Adcyap1(-/-) mice showed significant hypoinsulinemia, although being normoglycemic, and lower body weight as well as reduced food intake. They also showed greatly reduced white adipose tissue mass, in which the mRNA expression of adipocyte fatty acid-binding protein (aP2), a marker of adipocyte differentiation, was decreased. Glucose and insulin tolerance tests revealed increased insulin sensitivity in Adcyap1(-/-) mice. In accordance with these observations, plasma levels of resistin, an adipocytokine implicated in insulin resistance, were decreased in Adcyap1(-/-) mice. After a high-fat dietary challenge for six weeks, Adcyap1(-/-) mice still showed lower body weights and increased insulin sensitivity. These results indicate the crucial roles of PACAP in energy metabolism, including lipid metabolism, and in the regulation of body weight, raising the possibility that the PACAP-signaling pathway that favors energy storage could be a therapeutic target for obesity.
Asunto(s)
Tejido Adiposo Blanco/metabolismo , Glucemia/metabolismo , Peso Corporal , Metabolismo Energético , Insulina/sangre , Polipéptido Hipofisario Activador de la Adenilato-Ciclasa/metabolismo , Tejido Adiposo Blanco/crecimiento & desarrollo , Animales , Grasas de la Dieta/administración & dosificación , Ingestión de Alimentos , Proteínas de Unión a Ácidos Grasos/genética , Proteínas de Unión a Ácidos Grasos/metabolismo , Prueba de Tolerancia a la Glucosa , Homeostasis , Islotes Pancreáticos/metabolismo , Islotes Pancreáticos/patología , Ratones , Ratones Noqueados , Tamaño de los Órganos , Polipéptido Hipofisario Activador de la Adenilato-Ciclasa/deficiencia , Polipéptido Hipofisario Activador de la Adenilato-Ciclasa/genética , ARN Mensajero/metabolismo , Resistina/sangre , Factores de Tiempo , Triglicéridos/sangreRESUMEN
Pituitary adenylate cyclase-activating polypeptide (PACAP) is an intraislet neuropeptide and shares insulinotropic and insulin-sensitizing properties with glucagon-like peptide-1 (GLP-1); however, the pathophysiological significance of PACAP in diabetes remains largely unknown. To assess this, we crossed our recently developed transgenic mice overexpressing PACAP in pancreatic beta-cells (Tg/+), with lethal yellow agouti (KKA(y)) mice (A(y)/+), a genetic model for obesity-diabetes, and examined the metabolic and morphological phenotypes of F(1) animals. Tg/+ mice with the A(y) allele (Tg/+:A(y)/+) developed maturity-onset obesity and diabetes associated with hyperglycemia, hyperlipidemia, and hyperphagia, similar to those of A(y)/+ mice, but hyperinsulinemia was significantly ameliorated in Tg/+:A(y)/+ mice. Although A(y)/+ mice exhibited a marked increase in islet mass resulting from hyperplasia and hypertrophy, this increase was significantly attenuated in Tg/+:A(y)/+ mice. Size frequency distribution analysis revealed that the very large islets comprising one-fourth of islets of A(y)/+ mice were selectively reduced in Tg/+:A(y)/+ mice. Because functional defects have been demonstrated in the large islets of obese animal models, together these findings suggest that PACAP regulates hyperinsulinemia and the abnormal increase in islet mass that occurs during the diabetic process.