RESUMO
AIM: We recently discovered a glucose-dependent insulinotropic polypeptide (GIP) receptor antagonist, SKL-14959. GIP plays a role in the glucose and lipid metabolism, and is associated with obesity and insulin resistance. Therefore, we aimed to ascertain the inhibitory potency and glucose and lipid metabolism of SKL-14959. METHODS: SKL-14959 was evaluated for its binding affinity to each GIP, glucagon-like peptide-1 (GLP-1) and glucagon receptors by each labelled and non-labelled ligand; GIP-stimulated cyclic AMP (cAMP) production in CHO cells expressing human GIP receptor in vitro. Oral and intraperitoneal glucose tolerance tests (OGTT and IPGTT) were performed to examine the insulinotropic effect on endogenous and exogenous GIP. Oil tolerance tests were also conducted to examine the lipid metabolism and the postheparin plasma lipase activity, lipoprotein lipase (LPL) and hepatic lipase (HL). RESULT: SKL-14959 selectively bound to GIP receptor and inhibited GIP-stimulated cAMP production with the Ki value of 55 nM and an IC(50) value of 2.9 µM, respectively. SKL-14959·Na significantly increased blood glucose levels, inhibited insulin secretion in OGTT and inhibited the plasma glucose lowering of exogenous GIP in IPGTT. Furthermore, SKL-14959 increased plasma triacylglycerol (TG) levels as well as suppressed the postheparin plasma lipase activity in an oil load test. CONCLUSION: These data indicate that SKL-14959 is distinguished in the physiological phenotype of GIP following direct binding to the receptor.
Assuntos
AMP Cíclico/metabolismo , Peptídeo 1 Semelhante ao Glucagon/metabolismo , Lipase/metabolismo , Metabolismo dos Lipídeos/efeitos dos fármacos , Receptores dos Hormônios Gastrointestinais/antagonistas & inibidores , Receptores de Glucagon/metabolismo , Animais , Glicemia/efeitos dos fármacos , Glicemia/metabolismo , Células CHO , Cricetinae , AMP Cíclico/biossíntese , Peptídeo 1 Semelhante ao Glucagon/efeitos dos fármacos , Teste de Tolerância a Glucose , Humanos , Insulina/metabolismo , Secreção de Insulina , Lipase/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos C57BL , Receptores dos Hormônios Gastrointestinais/metabolismo , Receptores de Glucagon/efeitos dos fármacos , Triglicerídeos/sangueRESUMO
Glucose metabolism in glycolysis and in mitochondria is pivotal to glucose-induced insulin secretion from pancreatic beta cells. One or more factors derived from glycolysis other than pyruvate appear to be required for the generation of mitochondrial signals that lead to insulin secretion. The electrons of the glycolysis-derived reduced form of nicotinamide adenine dinucleotide (NADH) are transferred to mitochondria through the NADH shuttle system. By abolishing the NADH shuttle function, glucose-induced increases in NADH autofluorescence, mitochondrial membrane potential, and adenosine triphosphate content were reduced and glucose-induced insulin secretion was abrogated. The NADH shuttle evidently couples glycolysis with activation of mitochondrial energy metabolism to trigger insulin secretion.
Assuntos
Glucose/farmacologia , Insulina/metabolismo , Ilhotas Pancreáticas/metabolismo , Mitocôndrias/metabolismo , NAD/metabolismo , Trifosfato de Adenosina/metabolismo , Sequência de Aminoácidos , Ácido Amino-Oxiacético/farmacologia , Animais , Aspartato Aminotransferases/antagonistas & inibidores , Cálcio/metabolismo , Ciclo do Ácido Cítrico , Inibidores Enzimáticos/farmacologia , Feminino , Marcação de Genes , Glucose/metabolismo , Glicerolfosfato Desidrogenase/genética , Glicerolfosfato Desidrogenase/metabolismo , Glicólise , Secreção de Insulina , Masculino , Potenciais da Membrana , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Modelos Biológicos , Dados de Sequência Molecular , Ácido Pirúvico/metabolismoRESUMO
Objective: Gastric inhibitory polypeptide plays a role in glucose and lipid metabolism and is associated with obesity and insulin resistance. The objective of this study is to confirm the anti-obesity effects of the gastric inhibitory polypeptide receptor antagonist, SKL-14959, on diet-induced obesity mice. Method: Diet-induced obesity mice at 20 weeks of age were administered with or without SKL-14959 for 96 d. Body weight and food intake were monitored throughout the experiment. Mice were sacrificed, and physiological and biochemical markers were measured, and then histochemical and gene expression analyses were also performed. In further studies, mice were orally gavaged with [14C]-oleic acid to investigate the excursion of digested lipids. Results: SKL-14959 significantly suppressed weight gain without affecting food intake, decreased triacylglycerol contents in the liver and the muscle and the intensity stained with oil-red in the liver. It also improved plasma glutamic pyruvic transaminase and 3-hydroxybutyrate levels in addition to notably down-regulated relative gene expression of srebf1 and dgat1 in the liver despite not altering in the adipose tissue. Furthermore, SKL-14959 showed remarkable inhibition of lipid uptake in the adipose tissue after the oil challenge. Conclusion: SKL-14959 inhibited lipids uptake and improved lipids metabolism, results in suppression of body-weight gain.
RESUMO
To investigate the role of insulin receptor substrate (IRS)-2 in vivo, we generated IRS-2-deficient mice by gene targeting. Although homozygous IRS-2-deficient mice (IRS-2-/- mice) had a body weight similar to wild-type mice, they progressively developed type 2 diabetes at 10 weeks. IRS-2-/- mice showed insulin resistance and a defect in the insulin-stimulated signaling pathway in liver but not in skeletal muscle. Despite insulin resistance, the amount of beta-cells was reduced to 83% of that in wild-type mice, which was in marked contrast to the 85% increase in the amount of beta-cells in IRS-1-deficient mice (IRS-1-/- mice) to compensate for insulin resistance. Thus, IRS-2 plays a crucial role in the regulation of beta-cell mass. On the other hand, insulin secretion by the same number of cells in response to glucose measured ex vivo was significantly increased in IRS-2-/- mice compared with wild-type mice but was decreased in IRS-1-/- mice. These results suggest that IRS-1 and IRS-2 may play different roles in the regulation of beta-cell mass and the function of individual beta-cells.
Assuntos
Diabetes Mellitus Tipo 2/etiologia , Resistência à Insulina , Ilhotas Pancreáticas/patologia , Fígado/efeitos dos fármacos , Fosfoproteínas/deficiência , Fosfoproteínas/fisiologia , Animais , Diabetes Mellitus Tipo 2/metabolismo , Diabetes Mellitus Tipo 2/patologia , Marcação de Genes , Hiperplasia , Insulina/metabolismo , Insulina/farmacologia , Proteínas Substratos do Receptor de Insulina , Secreção de Insulina , Peptídeos e Proteínas de Sinalização Intracelular , Ilhotas Pancreáticas/metabolismo , Fígado/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos CBA , Camundongos Knockout , Músculo Esquelético/efeitos dos fármacos , Músculo Esquelético/metabolismo , Fosfoproteínas/genética , Transdução de Sinais/efeitos dos fármacosRESUMO
Dextran sulfate competes with binding of modified LDL to the scavenger receptor in macrophages. To elucidate the role of dextran sulfate in the atherosclerotic process, 100 mg of dextran sulfate in drinking water was given to 5 Watanabe heritable hyperlipidemic (WHHL) rabbits for 12 months starting at age 4 months. During the experimental period, there were no significant differences in plasma cholesterol levels between dextran sulfate-treated and untreated rabbits. After 12 months' treatment, accumulation of cholesterol ester in total aorta was significantly suppressed in dextran sulfate-treated rabbits as compared with untreated rabbits (71.4 +/- 22.3 vs. 42.7 +/- 16.5 mg/g dry weight, P < 0.05). Furthermore, lesion area with atherosclerotic plaques in treated rabbits was significantly less than that in untreated rabbits (59.7 +/- 24.5 vs. 30.4 +/- 14.4%, P < 0.05). These results indicate that dextran sulfate might prevent the progression of atherosclerosis by competitively inhibiting the binding of modified LDL to scavenger receptors.
Assuntos
Arteriosclerose/prevenção & controle , Sulfato de Dextrana/farmacologia , Hiperlipidemias/metabolismo , Proteínas de Membrana , Receptores Imunológicos/antagonistas & inibidores , Receptores de Lipoproteínas , Animais , Aorta/efeitos dos fármacos , Aorta/metabolismo , Aorta/patologia , Arteriosclerose/etiologia , Arteriosclerose/patologia , Ligação Competitiva/efeitos dos fármacos , Colesterol/sangue , Ésteres do Colesterol/metabolismo , Hiperlipidemias/complicações , Hiperlipidemias/genética , Lipoproteínas LDL/metabolismo , Macrófagos Peritoneais/efeitos dos fármacos , Macrófagos Peritoneais/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos , Oxirredução , Coelhos , Receptores Imunológicos/metabolismo , Receptores Depuradores , Receptores Depuradores Classe BRESUMO
Native oxymyoglobin (MbO2) was isolated directly from the cells of Paramecium caudatum with complete separation from metmyoglobin (metMb) on a DEAE-cellulose column. It was examined for its spectral and stability properties. When compared with sperm whale MbO2 used as a reference, Paramecium MbO2 was found to be much more susceptible to autoxidation over a wide range of pH (4-11) in 0.1 M buffer at 25 degrees C. Kinetic analysis has revealed that a proton-catalyzed displacement of O2- from MbO2 by an entering water molecule can play a dominant role in the autoxidation reaction of Paramecium MbO2 to metMb, as in the case of sperm whale MbO2 involving the distal histidine as its catalytic residue. At pH values higher than 9.5, however, Paramecium MbO2 was found to be oxidized to yield a hemichrome. The spontaneous formation of hemichromes is at variance with the other known myoglobins and is therefore discussed in relation to the unusual amino acid sequence of Paramecium myoglobin having a large number of deletion.
Assuntos
Mioglobina/química , Paramecium/fisiologia , Animais , Heme/química , Concentração de Íons de Hidrogênio , Técnicas In Vitro , Cinética , Oxirredução , Análise Espectral , BaleiasRESUMO
Hexamminecobalt(III) (HAC) chloride was found to have a potent inhibitory effect on glucose-induced insulin secretion from pancreatic islets. HAC at 2 mm inhibited the secretion in response to 22.2 mm glucose by 90% in mouse islets. Perifusion experiments revealed that the first phase of insulin secretion was severely suppressed and that the second phase of secretion was completely abrogated. Removal of HAC from the perifusate immediately restored insulin secretion with a transient overshooting above the normal level. However, HAC failed to affect glucose-induced changes in d-[6-(14)C]glucose oxidation, levels of reduced forms of NAD and NADP, mitochondrial membrane potential, ATP content, cytosolic calcium concentration, or calcium influx into mitochondria. Furthermore, HAC inhibited 50 mm potassium-stimulated insulin secretion by 77% and 10 microm mastoparan-stimulated insulin secretion in the absence of extracellular Ca(2+) by 80%. The results of a co-immunoprecipitation study of lysates from insulin-secreting betaHC9 cells using anti-syntaxin and anti-vesicle-associated membrane protein antibodies for immunoprecipitation or Western blotting suggested that HAC inhibited disruption of the SNARE complex, which is normally observed upon glucose challenge. These results suggest that the inhibitory effect of HAC on glucose-induced insulin secretion is exerted at a site(s) distal to the elevation of cytosolic [Ca(2+)], possibly in the exocytotic machinery per se; and thus, HAC may serve as a useful tool for dissecting the molecular mechanism of insulin exocytotic processes.
Assuntos
Cálcio/metabolismo , Cloretos/farmacologia , Cobalto/farmacologia , Exocitose/efeitos dos fármacos , Glucose/farmacologia , Insulina/metabolismo , Trifosfato de Adenosina/metabolismo , Animais , Transporte Biológico/efeitos dos fármacos , Células Cultivadas , AMP Cíclico/metabolismo , Citosol/efeitos dos fármacos , Citosol/metabolismo , Interações Medicamentosas , Exocitose/fisiologia , Glucose/metabolismo , Secreção de Insulina , Peptídeos e Proteínas de Sinalização Intercelular , Ilhotas Pancreáticas/efeitos dos fármacos , Ilhotas Pancreáticas/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Oxirredução/efeitos dos fármacos , Peptídeos , Cloreto de Potássio/farmacologia , Venenos de Vespas/farmacologiaRESUMO
The NADH shuttle system is composed of the glycerol phosphate and malate-aspartate shuttles. We generated mice that lack mitochondrial glycerol-3-phosphate dehydrogenase (mGPDH), a rate-limiting enzyme of the glycerol phosphate shuttle. Application of aminooxyacetate, an inhibitor of the malate-aspartate shuttle, to mGPDH-deficient islets demonstrated that the NADH shuttle system was essential for coupling glycolysis with activation of mitochondrial ATP generation to trigger glucose-induced insulin secretion. The present study revealed that blocking the NADH shuttle system severely suppressed closure of the ATP-sensitive potassium (K(ATP)) channel and depolarization of the plasma membrane in response to glucose in beta cells, although properties of the K(ATP) channel on the excised beta cell membrane were unaffected. In mGPDH-deficient islets treated with aminooxyacetate, Ca(2+) influx through the plasma membrane induced by a depolarizing concentration of KCl in the presence of the K(ATP) channel opener diazoxide restored insulin secretion. However, the level of the secretion was only approximately 40% of wild-type controls. Thus, glucose metabolism through the NADH shuttle system leading to efficient ATP generation is pivotal to activation of both the K(ATP) channel-dependent pathway and steps distal to an elevation of cytosolic Ca(2+) concentration in glucose-induced insulin secretion.
Assuntos
Cálcio/metabolismo , Insulina/metabolismo , Ilhotas Pancreáticas/fisiologia , NAD/metabolismo , Canais de Potássio/fisiologia , Animais , Citosol/metabolismo , Glucose/farmacologia , Glicerolfosfato Desidrogenase/genética , Glicerolfosfato Desidrogenase/metabolismo , Secreção de Insulina , Potenciais da Membrana , Camundongos , Camundongos KnockoutRESUMO
Agonist-induced activation of peroxisome proliferator-activated receptor gamma (PPAR gamma) is known to cause adipocyte differentiation and insulin sensitivity. The biological role of PPAR gamma was investigated by gene targeting. Homozygous PPAR gamma-deficient embryos died at 10.5-11.5 dpc due to placental dysfunction. Quite unexpectedly, heterozygous PPAR gamma-deficient mice were protected from the development of insulin resistance due to adipocyte hypertrophy under a high-fat diet. These phenotypes were abrogated by PPAR gamma agonist treatment. Heterozygous PPAR gamma-deficient mice showed overexpression and hypersecretion of leptin despite the smaller size of adipocytes and decreased fat mass, which may explain these phenotypes at least in part. This study reveals a hitherto unpredicted role for PPAR gamma in high-fat diet-induced obesity due to adipocyte hypertrophy and insulin resistance, which requires both alleles of PPAR gamma.