RESUMO
AIMS/HYPOTHESIS: We aimed to investigate potential interactions between insulin and glucagon-like peptide (GLP)-1 signalling pathways in the regulation of beta cell-cycle dynamics in vivo, in the context of the therapeutic potential of GLP-1 to modulate impaired beta cell function. METHODS: Beta cell-specific insulin receptor knockout (ßIRKO) mice, which exhibit beta cell dysfunction and an age-dependent decrease in beta cell mass, were treated with the dipeptidyl peptidase-4 inhibitor vildagliptin. Following this, glucose homeostasis and beta cell proliferation were evaluated and underlying molecular mechanisms were investigated. RESULTS: The sustained elevation in circulating GLP-1 levels, caused by treatment of the knockout mice with vildagliptin for 6 weeks, significantly improved glucose tolerance secondary to enhanced insulin secretion and proliferation of beta cells. Treating ßIRKO beta cell lines with the GLP-1 analogue, exendin-4, promoted Akt phosphorylation and protein expression of cyclins A, D1 and E two- to threefold, in addition to cyclin D2. Pancreases from the vildagliptin-treated ßIRKO mice exhibited increased cyclin D1 expression, while cyclin D2 expression was impaired. CONCLUSIONS/INTERPRETATION: Activation of GLP-1 signalling compensates for impaired growth factor (insulin) signalling and enhances expression of cyclins to promote beta cell proliferation. Together, these data indicate the potential of GLP-1-related therapies to enhance beta cell proliferation and promote beneficial outcomes in models with dysfunctional beta cells.
Assuntos
Peptídeo 1 Semelhante ao Glucagon/metabolismo , Células Secretoras de Insulina/metabolismo , Insulina/metabolismo , Receptor de Insulina/metabolismo , Adamantano/análogos & derivados , Adamantano/farmacologia , Animais , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/genética , Ciclina A/metabolismo , Ciclina D/metabolismo , Inibidores da Dipeptidil Peptidase IV/farmacologia , Exenatida , Peptídeo 1 Semelhante ao Glucagon/análogos & derivados , Células Secretoras de Insulina/efeitos dos fármacos , Camundongos , Camundongos Knockout , Nitrilas/farmacologia , Peptídeos/farmacologia , Fosforilação/efeitos dos fármacos , Pirrolidinas/farmacologia , Receptor de Insulina/deficiência , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Peçonhas/farmacologia , VildagliptinaRESUMO
The successful launches of dipeptidyl peptidase IV (DPP IV) inhibitors as oral anti-diabetics warrant and spur the further quest for additional chemical entities in this promising class of therapeutics. Numerous pharmaceutical companies have pursued their proprietary candidates towards the clinic, resulting in a large body of published chemical structures associated with DPP IV. Herein, we report the discovery of a novel chemotype for DPP IV inhibition based on the C-(1-aryl-cyclohexyl)-methylamine scaffold and its optimization to compounds which selectively inhibit DPP IV at low-nM potency and exhibit an excellent oral pharmacokinetic profile in the rat.
Assuntos
Dipeptidil Peptidase 4/metabolismo , Inibidores da Dipeptidil Peptidase IV/síntese química , Inibidores da Dipeptidil Peptidase IV/farmacocinética , Descoberta de Drogas , Metilaminas/síntese química , Metilaminas/farmacocinética , Adamantano/análogos & derivados , Adamantano/química , Adamantano/farmacologia , Administração Oral , Animais , Células CACO-2 , Cristalografia por Raios X , Ciclização , Inibidores da Dipeptidil Peptidase IV/química , Inibidores da Dipeptidil Peptidase IV/farmacologia , Ativação Enzimática/efeitos dos fármacos , Humanos , Concentração Inibidora 50 , Metilaminas/química , Metilaminas/farmacologia , Estrutura Molecular , Nitrilas/química , Nitrilas/farmacologia , Pirazinas/química , Pirazinas/farmacologia , Pirrolidinas/química , Pirrolidinas/farmacologia , Ratos , Fosfato de Sitagliptina , Triazóis/química , Triazóis/farmacologia , VildagliptinaRESUMO
Novel deazaxanthine-based DPP-4 inhibitors have been identified that are potent (IC(50) <10nM) and highly selective versus other dipeptidyl peptidases. Their synthesis and SAR are reported, along with initial efforts to improve the PK profile through decoration of the deazaxanthine core. Optimisation of compound 3a resulted in the identification of compound (S)-4i, which displayed an improved in vitro and ADME profile. Further enhancements to the PK profile were possible by changing from the deazahypoxanthine to the deazaxanthine template, culminating in compound 12g, which displayed good ex vivo DPP-4 inhibition and a superior PK profile in rat, suggestive of once daily dosing in man.
Assuntos
Diabetes Mellitus Tipo 2/tratamento farmacológico , Inibidores da Dipeptidil Peptidase IV/química , Inibidores da Dipeptidil Peptidase IV/uso terapêutico , Animais , Células CACO-2 , Cristalografia por Raios X , Inibidores da Dipeptidil Peptidase IV/síntese química , Inibidores da Dipeptidil Peptidase IV/farmacologia , Ativação Enzimática/efeitos dos fármacos , Compostos Heterocíclicos/química , Compostos Heterocíclicos/farmacologia , Compostos Heterocíclicos/uso terapêutico , Humanos , Concentração Inibidora 50 , Masculino , Modelos Moleculares , Estrutura Molecular , Ratos , Relação Estrutura-AtividadeRESUMO
Oscillatory network activity at gamma frequencies is assumed to be of major importance in cortical information processing. Whereas the synaptic mechanisms of gamma oscillations have been studied in detail, the ionic currents involved at the cellular level remain to be elucidated. Here we show that in vitro gamma oscillations induced by muscarine require activation of M1 receptors on hippocampal CA3 pyramidal neurons and are absent in M1 receptor-deficient mice. M1 receptor activation depolarizes pyramidal neurons by increasing the mixed Na(+)/K(+) current I(h) and the Ca(2+)-dependent nonspecific cation current I(cat), but not by modulation of I(M). Our data provide important insight into the molecular basis of gamma oscillations by unequivocally establishing a novel role for muscarinic modulation of I(h) and I(cat) in rhythmic network activity.
Assuntos
Relógios Biológicos/fisiologia , Proteínas de Transporte de Cátions/metabolismo , Hipocampo/metabolismo , Canais Iônicos/metabolismo , Rede Nervosa/metabolismo , Células Piramidais/metabolismo , Receptores Muscarínicos/deficiência , Animais , Relógios Biológicos/efeitos dos fármacos , Fármacos Cardiovasculares/farmacologia , Proteínas de Transporte de Cátions/efeitos dos fármacos , Proteínas Quinases Dependentes de AMP Cíclico/antagonistas & inibidores , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Hipocampo/citologia , Hipocampo/efeitos dos fármacos , Imuno-Histoquímica , Indóis/farmacologia , Interneurônios/citologia , Interneurônios/efeitos dos fármacos , Interneurônios/metabolismo , Canais Iônicos/efeitos dos fármacos , Potenciais da Membrana/efeitos dos fármacos , Potenciais da Membrana/fisiologia , Camundongos , Camundongos Knockout , Muscarina/farmacologia , Agonistas Muscarínicos/farmacologia , Rede Nervosa/citologia , Rede Nervosa/efeitos dos fármacos , Bloqueadores dos Canais de Potássio/farmacologia , Células Piramidais/citologia , Células Piramidais/efeitos dos fármacos , Piridinas/farmacologia , Pirimidinas/farmacologia , Receptor Muscarínico M1 , Receptores Muscarínicos/efeitos dos fármacos , Receptores Muscarínicos/genética , Transmissão Sináptica/efeitos dos fármacos , Transmissão Sináptica/fisiologiaRESUMO
A coordinated functional genomics program was implemented to identify secreted polypeptides with therapeutic applications in the treatment of diabetes. Secreted factors were predicted from a diverse expressed-sequence tags (EST) database, representing >1,000 cDNA libraries, using a combination of bioinformatic algorithms. Subsequently, approximately 8,000 human proteins were screened in high-throughput cell-based assays designed to monitor key physiological transitions known to be centrally involved in the physiology of type 2 diabetes. Bone morphogenetic protein-9 (BMP-9) gave a positive response in two independent assays: reducing phosphoenolpyruvate carboxykinase (PEPCK) expression in hepatocytes and activating Akt kinase in differentiated myotubes. Purified recombinant BMP-9 potently inhibited hepatic glucose production and activated expression of key enzymes of lipid metabolism. In freely fed diabetic mice, a single subcutaneous injection of BMP-9 reduced glycemia to near-normal levels, with maximal reduction observed 30 hours after treatment. BMP-9 represents the first hepatic factor shown to regulate blood glucose concentration. Using a combination of bioinformatic and high-throughput functional analyses, we have identified a factor that may be exploited for the treatment of diabetes.
Assuntos
Proteínas Morfogenéticas Ósseas/genética , Proteínas Morfogenéticas Ósseas/metabolismo , Diabetes Mellitus/genética , Diabetes Mellitus/metabolismo , Perfilação da Expressão Gênica/métodos , Animais , Proteínas Morfogenéticas Ósseas/química , Proteínas Morfogenéticas Ósseas/uso terapêutico , Células Cultivadas , Diabetes Mellitus/tratamento farmacológico , Desenho de Fármacos , Glucose/metabolismo , Fator 2 de Diferenciação de Crescimento , Fatores de Diferenciação de Crescimento , Humanos , Rim/química , Rim/embriologia , Rim/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Proteínas/química , Proteínas/genética , Proteínas/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Valores de Referência , Alinhamento de Sequência/métodos , Análise de Sequência de Proteína/métodos , Integração de SistemasRESUMO
The primary therapeutic goal for the treatment of diabetes is maintenance of a long-term, near-normoglycemic condition and prevention of the onset or progression of the complications associated with the disease. Although several analogs of human insulin have been developed, the currently prescribed long-acting insulin analogs do not provide a stable basal glycemia for more than a few hours. Here, we report the development of Albulin, a long-acting insulin analog obtained by direct gene fusion of a single-chain human insulin to human serum albumin. Albulin showed an elimination t(1/2) of approximately 7 h in normoglycemic mice. In vitro pharmacodynamic profiles for Albulin characterized by receptor binding, inhibition of gluconeogenesis, induction of glucose uptake, and global regulation of gene expression in relevant cell types showed that Albulin produced similar activity profiles compared with that of recombinant human insulin. A single Albulin administration in vivo normalized blood glucose level in diabetic mice in a relatively peakless and sustained (24-h) fashion. A further reduction in glucose levels was achieved by administering a recombinant human insulin a few hours after Albulin injection in mice, indicating the potential for Albulin therapy in combination with available fast-acting insulin derivatives. In summary, Albulin displays characteristics of a potent long-acting insulin analog that can be evaluated for use as a novel insulin therapy for patients with insulin-dependent diabetes.
Assuntos
Insulina/genética , Insulina/farmacocinética , Proteínas Recombinantes de Fusão/farmacocinética , Albumina Sérica/genética , Albumina Sérica/farmacocinética , Células 3T3 , Adipócitos/efeitos dos fármacos , Adipócitos/metabolismo , Animais , Clonagem Molecular , Escherichia coli , Perfilação da Expressão Gênica , Regulação da Expressão Gênica/efeitos dos fármacos , Genes Sintéticos , Glucose/metabolismo , Humanos , Insulina/farmacologia , Insulina de Ação Prolongada , Insulina Regular Humana , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Reação em Cadeia da Polimerase , Receptor de Insulina/metabolismo , Proteínas Recombinantes de Fusão/farmacologia , Albumina Sérica/farmacologia , Albumina Sérica HumanaRESUMO
The five muscarinic acetylcholine receptors (M1-M5 mAChRs) mediate a very large number of important physiological functions (Caulfield, 1993; Caulfield and Birdsall, 1998; Wess, 2004). Because of the lack of small molecule ligands endowed with a high degree of receptor subtype selectivity and the fact that most tissues or cell types express two or more mAChR subtypes, identification of the physiological and pathophysiological roles of the individual mAChR subtypes has proved to be a challenging task. To overcome these difficulties, we recently generated mutant mouse lines deficient in each of the five mAChR genes (M1R-/- mice, M2R-/- mice, M3R-/- mice, etc. [Wess, 2004]). Phenotyping studies showed that each of the five mutant mouse lines displayed characteristic physiological, pharmacological, behavioral, biochemical, or neurochemical deficits (Wess, 2004). This chapter summarizes recent findings dealing with the importance of the M2mAChR for cognitive processes and the roles of the M1 and M3 mAChRs in mediating stimulation of glandular secretion.
Assuntos
Cognição/fisiologia , Hormônios/metabolismo , Receptor Muscarínico M1/deficiência , Receptor Muscarínico M2/deficiência , Receptor Muscarínico M3/deficiência , Animais , Carbacol/farmacologia , Insulina/metabolismo , Secreção de Insulina , Ilhotas Pancreáticas/metabolismo , Camundongos , Camundongos Knockout , Fenótipo , Receptor Muscarínico M1/genética , Receptor Muscarínico M2/genética , Receptor Muscarínico M3/genéticaRESUMO
Pancreatic muscarinic acetylcholine receptors play an important role in stimulating insulin and glucagon secretion from islet cells. To study the potential role of the M(3) muscarinic receptor subtype in cholinergic stimulation of insulin release, we initially examined the effect of the muscarinic agonist, oxotremorine-M (Oxo-M), on insulin secretion from isolated pancreatic islets prepared from wild-type (WT) and M(3) receptor-deficient mice (M3(+/-) and M3(-/-) mice). At a stimulatory glucose level (16.7 mmol/l), Oxo-M strongly potentiated insulin output from islets of WT mice. Strikingly, this effect was completely abolished in islets from M3(-/-) mice and significantly reduced in islets from M3(+/-) mice. Additional in vitro studies showed that Oxo-M-mediated glucagon release was also virtually abolished in islets from M3(-/-) mice. Consistent with the in vitro data, in vivo studies showed that M3(-/-) mice displayed reduced serum insulin and plasma glucagon levels and a significantly blunted increase in serum insulin after an oral glucose load. Despite the observed impairments in insulin release, M3(-/-) mice showed significantly reduced blood glucose levels and even improved glucose tolerance, probably due to the reduction in plasma glucagon levels and the fact that M3(-/-) mice are hypophagic and lean. These findings provide important new insights into the metabolic roles of the M(3) muscarinic receptor subtype.
Assuntos
Glucagon/metabolismo , Insulina/metabolismo , Agonistas Muscarínicos/farmacologia , Oxotremorina/análogos & derivados , Oxotremorina/farmacologia , Pâncreas/metabolismo , Receptor Muscarínico M3/fisiologia , Animais , Glicemia/análise , Glucagon/sangue , Insulina/análise , Insulina/sangue , Secreção de Insulina , Camundongos , Camundongos Knockout , Pâncreas/química , Receptor Muscarínico M3/deficiência , Receptor Muscarínico M3/genética , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Until recently, little was known about the possible physiological functions of the M(5) muscarinic acetylcholine receptor subtype, the last member of the muscarinic receptor family (M(1)-M(5)) to be cloned. To learn more about the potential physiological roles of this receptor subtype, we generated and analyzed M(5) receptor-deficient mice (M5 -/- mice). Strikingly, acetylcholine, a potent dilator of most vascular beds, virtually lost the ability to dilate cerebral arteries and arterioles in M5 -/- mice, suggesting that endothelial M(5) receptors mediate this activity in wild-type mice. This effect was specific for cerebral blood vessels, since acetylcholine-mediated dilation of extra-cerebral arteries remained fully intact in M5 -/- mice. In addition, in vitro neurotransmitter release experiments indicated that M(5) receptors located on dopaminergic nerve terminals play a role in facilitating muscarinic agonist-induced dopamine release in the striatum, consistent with the observation that the dopaminergic neurons innervating the striatum almost exclusively express the M(5) receptor subtype. We also found that the rewarding effects of morphine, the prototypical opiate analgesic, were substantially reduced in M5 -/- mice, as measured in the conditioned place preference paradigm. Furthermore, both the somatic and affective components of naloxone-induced morphine withdrawal symptoms were significantly attenuated in M5 -/- mice. It is likely that these behavioral deficits are caused by the lack of mesolimbic M(5) receptors, activation of which is known to stimulate dopamine release in the nucleus accumbens. These results convincingly demonstrate that the M(5) muscarinic receptor is involved in modulating several important pharmacological and behavioral functions. These findings may lead to novel therapeutic strategies for the treatment of drug addiction and certain cerebrovascular disorders.
Assuntos
Receptor Muscarínico M5/genética , Receptor Muscarínico M5/fisiologia , Analgésicos Opioides/farmacologia , Animais , Química Encefálica/genética , Química Encefálica/fisiologia , Condicionamento Operante/efeitos dos fármacos , Dopamina/metabolismo , Marcação de Genes , Camundongos , Camundongos Knockout , Morfina/farmacologia , Neostriado/metabolismo , Sistema Nervoso Parassimpático/fisiologia , Ratos , Recompensa , Síndrome de Abstinência a Substâncias/genética , Síndrome de Abstinência a Substâncias/fisiopatologia , Vasodilatação/fisiologiaRESUMO
To gain new insight into the physiological and pathophysiological roles of the muscarinic cholinergic system, we generated mutant mouse strains deficient in each of the five muscarinic acetylcholine receptor subtypes (M(1)-M(5)). In this chapter, we review a set of recent studies dealing with the identification of the muscarinic receptor subtypes mediating muscarinic agonist-dependent analgesic effects by central and peripheral mechanisms. Most of these studies were carried out with mutant mouse strains lacking M(2) or/and M(4) muscarinic receptors. It is well known that administration of centrally active muscarinic agonists induces pronounced analgesic effects. To identify the muscarinic receptors mediating this activity, wild-type and muscarinic receptor mutant mice were injected with the non-subtype-selective muscarinic agonist, oxotremorine (s.c., i.t., and i.c.v.), and analgesic effects were assessed in the tail-flick and hot-plate tests. These studies showed that M(2) receptors play a key role in mediating the analgesic effects of oxotremorine, both at the spinal and supraspinal level. However, studies with M(2)/M(4) receptor double KO mice indicated that M(4) receptors also contribute to this activity. Recent evidence suggests that activation of muscarinic receptors located in the skin can reduce the sensitivity of peripheral nociceptors. Electrophysiological and neurochemical studies with skin preparations from muscarinic receptor mutant mice indicated that muscarine-induced peripheral antinociception is mediated by M(2) receptors. Since acetylcholine is synthesized and released by different cell types of the skin, it is possible that non-neuronally released acetylcholine plays a role in modulating peripheral nociception. Our results highlight the usefulness of muscarinic receptor mutant mice to shed light on the functional roles of acetylcholine released from both neuronal and non-neuronal cells.
Assuntos
Analgésicos/farmacologia , Sistema Nervoso Central/fisiopatologia , Dor/fisiopatologia , Sistema Nervoso Periférico/fisiopatologia , Receptores Muscarínicos/genética , Receptores Muscarínicos/fisiologia , Analgésicos/efeitos adversos , Animais , Peptídeo Relacionado com Gene de Calcitonina/metabolismo , Sistema Nervoso Central/efeitos dos fármacos , Camundongos , Camundongos Knockout , Terminações Nervosas/efeitos dos fármacos , Dor/tratamento farmacológico , Sistema Nervoso Periférico/efeitos dos fármacos , Receptores Muscarínicos/efeitos dos fármacos , Pele/inervação , Pele/metabolismo , Medula Espinal/efeitos dos fármacos , Medula Espinal/metabolismoRESUMO
Glucokinase (GK) acts as a glucose sensor by facilitating glucose phosphorylation into glucose-6-phosphate (G6P) in the liver and pancreas, the two key target tissues. LCZ960, a glucokinase activator exerts a stimulatory effect on GK activity in hepatocytes in vitro. This study aimed to verify in vivo that LCZ960 stimulates glucose uptake primarily through a mechanism involving hepatic GK activation. Acute and chronic LCZ960 treatment-induced changes in glycemia and hepatic glucose turnover were measured in high fat diet-induced obese (DIO) mice and rats. G6P production and glycogen cycling were quantified by (13)C-MR spectroscopy during a [1-(13)C]glucose infusion, followed by a pulse-chase with [(12)C]glucose to mimic postprandial conditions in rats. Acute treatment with LCZ960 dose-dependently reduced blood glucose without causing hypoglycemia in DIO mice. Chronic LCZ960 treatment maintained normoglycemia and improved glucose tolerance without increased insulin secretion in DIO mice and rats. In rats, LCZ960 stimulated a 240% increase (P<0.05) in the glycogen synthase flux. However, due to a much higher glycogen breakdown (LCZ960: 48 ± 15 vs control: 4 ± 1µmol/kg/min, P<0.05), this translated to only a 46% (ns) increase in glycogen storage (Vsyn net, LCZ960: 64±26 vs control: 43 ± 6 µmol/kg/min). Despite a 4-fold increase in hepatic glycogen turnover (LCZ960: 36.0 ± 5.5% vs control: 8.3 ± 2.0%), LCZ960 did not impact glucose-stimulated G6P accumulation. LCZ960 did not cause hypoglycemia in DIO rodents. Under hyperglycemic conditions, LCZ960 induced a robust increase in hepatic glycogen cycling. Since net hepatic glycogen storage is diminished in type 2 diabetes patients, stimulation of glycogen synthesis may contribute to the anti-hyperglycemic properties of glucokinase activation.
Assuntos
Amidas/farmacologia , Glucoquinase/metabolismo , Glucose/metabolismo , Glicogênio/metabolismo , Fígado/efeitos dos fármacos , Fígado/metabolismo , Sulfonamidas/farmacologia , Tiazóis/farmacologia , Animais , Transporte Biológico/efeitos dos fármacos , Ativação Enzimática/efeitos dos fármacos , Glucose-6-Fosfato/metabolismo , Cinética , Masculino , Camundongos , RatosRESUMO
The present study addressed the effect of the dipeptidyl peptidase-4 (DPP-4) inhibitor vildagliptin ((1-[[(3-hydroxy-1-adamantyl) amino] acetyl]-2-cyano-(S)-pyrrolidine), LAF237) on pancreatic beta cell mass in neonatal rats. Newborn rats were treated orally with vildagliptin (60 mg/kg) or vehicle once daily for 19 days starting from postnatal day 2. Pancreatic immunohistochemistry and morphometric analysis were performed to evaluate changes in beta cell mass, cell apoptosis (Apoptag stain) and replication (5'-Bromo-2'-deoxyuridine (BrdU)-incorporation) on days 7, 21, and 33. On day 7, an eight-fold increase in BrdU-positive pancreatic beta cells and a 71% decrease in Apoptag-positive cells were observed. On day 21, vildagliptin produced a two-fold increase in pancreatic beta cell mass compared to placebo (0.06±0.01 mg vs 0.11±0.02 mg, P<0.05). Beta cell mass remained elevated (90%, 0.09±0.02 mg vs 0.16±0.03 mg, P<0.05) on day 33, twelve days after discontinuing vildagliptin treatment. These data show that the DPP-4 inhibitor vildagliptin increased pancreatic beta cell mass through enhanced beta cell replication and reduced apoptosis. The increased beta cell mass was sustained for 12 days after vildagliptin washout. This study demonstrates that DPP-4 inhibitors can elicit beneficial effects on beta cell turnover that could help to prevent or retard the progression of type 2 diabetes.
Assuntos
Adamantano/análogos & derivados , Apoptose/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Inibidores da Dipeptidil Peptidase IV/farmacologia , Células Secretoras de Insulina/efeitos dos fármacos , Nitrilas/farmacologia , Pirrolidinas/farmacologia , Adamantano/farmacologia , Animais , Animais Recém-Nascidos , Células Secretoras de Insulina/citologia , Células Secretoras de Insulina/metabolismo , Ratos , Ratos Wistar , VildagliptinaRESUMO
Type 2 diabetes (T2DM) is not only a disorder of impaired insulin secretion but also glucagon oversecretion. However, the link between the two remains unclear. Is it possible that the latter is a consequence of the former? In this issue, Kawamori et al. (2009) have addressed this question by generating alpha cell-specific insulin receptor knockout mice.
Assuntos
Glucagon/metabolismo , Receptor de Insulina/metabolismo , Animais , Glucagon/genética , Células Secretoras de Glucagon/metabolismo , Células Secretoras de Glucagon/patologia , Insulina/genética , Insulina/metabolismo , Células Secretoras de Insulina/metabolismo , Células Secretoras de Insulina/patologia , Camundongos , Especificidade de Órgãos , Receptor de Insulina/deficiência , Transdução de SinaisRESUMO
OBJECTIVE: This study investigated the acute effects of treatment with vildagliptin on dipeptidyl peptidase-4 (DPP-4) activity, glucagon-like peptide 1 (GLP-1) concentration, pancreatic hormone levels, and glucose metabolism. The primary aims were to determine the effects of DPP-4 inhibition on GLP-1 clearance and on hepatic glucose uptake. RESEARCH DESIGN AND METHODS: Fasted conscious dogs were studied in the presence (n = 6) or absence (control, n = 6) of oral vildagliptin (1 mg/kg). In both groups, GLP-1 was infused into the portal vein (1 pmol . kg(-1) . min(-1)) for 240 min. During the same time, glucose was delivered into the portal vein at 4 mg . kg(-1) . min(-1) and into a peripheral vein at a variable rate to maintain the arterial plasma glucose level at 160 mg/dl. RESULTS: Vildagliptin fully inhibited DPP-4 over the 4-h experimental period. GLP-1 concentrations were increased in the vildagliptin-treated group (50 +/- 3 vs. 85 +/- 7 pmol/l in the portal vein in control and vildagliptin-treated dogs, respectively; P < 0.05) as a result of a 40% decrease in GLP-1 clearance (38 +/- 5 and 22 +/- 2 ml . kg(-1) . min(-1), respectively; P < 0.05). Although hepatic insulin and glucagon levels were not significantly altered, there was a tendency for plasma insulin to be greater (hepatic levels were 73 +/- 10 vs. 88 +/- 15 microU/ml, respectively). During vildagliptin treatment, net hepatic glucose uptake was threefold greater than in the control group. This effect was greater than that predicted by the change in insulin. CONCLUSIONS: Vildagliptin fully inhibited DPP-4 activity, reduced GLP-1 clearance by 40%, and increased hepatic glucose disposal by means beyond the effects of GLP-1 on insulin and glucagon secretion.
Assuntos
Adamantano/análogos & derivados , Peptídeo 1 Semelhante ao Glucagon/farmacologia , Glucose/metabolismo , Fígado/efeitos dos fármacos , Nitrilas/farmacologia , Pirrolidinas/farmacologia , Adamantano/administração & dosagem , Adamantano/farmacologia , Animais , Estado de Consciência , Dipeptidil Peptidase 4/metabolismo , Inibidores da Dipeptidil Peptidase IV/farmacologia , Cães , Glucagon/metabolismo , Peptídeo 1 Semelhante ao Glucagon/administração & dosagem , Glucose/farmacocinética , Insulina/metabolismo , Fígado/metabolismo , Nitrilas/administração & dosagem , Pirrolidinas/administração & dosagem , VildagliptinaRESUMO
Type 2 diabetes is a polygenic disease which afflicts nearly 200 million people worldwide and is expected to increase to near epidemic levels over the next 10-15 years. Glucokinase (GK) activators are currently under investigation by a number of pharmaceutical companies with only a few reaching early clinical evaluation. A GK activator has the promise of potentially affecting both the beta-cells of the pancreas, by improving glucose sensitive insulin secretion, as well as the liver, by reducing uncontrolled glucose output and restoring post-prandial glucose uptake and storage as glycogen. Herein, we report our efforts on a sulfonamide chemotype with the aim to generate liver selective GK activators which culminated in the discovery of 3-cyclopentyl-N-(5-methoxy-thiazolo[5,4-b]pyridin-2-yl)-2-[4-(4-methyl-piperazine-1-sulfonyl)-phenyl]-propionamide (17c). This compound activated the GK enzyme (alphaK(a) = 39 nM) in vitro at low nanomolar concentrations and significantly reduced glucose levels during an oral glucose tolerance test in normal mice.
Assuntos
Glucoquinase/efeitos dos fármacos , Sulfonamidas/farmacologia , Animais , Glicemia/efeitos dos fármacos , Diabetes Mellitus Tipo 2/tratamento farmacológico , Teste de Tolerância a Glucose , Hipoglicemiantes/farmacologia , Fígado/efeitos dos fármacos , Fígado/metabolismo , Camundongos , Relação Estrutura-Atividade , Sulfonamidas/uso terapêuticoRESUMO
Little is known about the physiological roles of the M5 muscarinic receptor, the last member of the muscarinic receptor family (M1-M5) to be cloned. In the brain, the M5 receptor subtype is preferentially expressed by dopaminergic neurons of the substantia nigra and the ventral tegmental area. Dopaminergic neurons located in the ventral tegmental area are known to play important roles in mediating both the rewarding effects of opiates and other drugs of abuse and the manifestations of opiate/drug withdrawal symptoms. We therefore speculated that acetylcholine-dependent activation of M5 receptors might modulate the manifestations of opiate reward and withdrawal. This hypothesis was tested in a series of behavioral, biochemical, and neurochemical studies using M5 receptor-deficient mice (M5-/- mice) as novel experimental tools. We found that the rewarding effects of morphine, as measured in the conditioned place preference paradigm, were substantially reduced in M5-/- mice. Furthermore, both the somatic and affective components of naloxone-induced morphine withdrawal symptoms were significantly attenuated in M5-/- mice. In contrast, the analgesic efficacy of morphine and the development of tolerance to the analgesic effects of morphine remained unaltered by the lack of M5 receptors. The finding that M5 receptor activity modulates both morphine reward and withdrawal processes suggests that M5 receptors may represent a novel target for the treatment of opiate addiction.
Assuntos
Analgesia , Morfina/farmacologia , Núcleo Accumbens/fisiologia , Receptores Muscarínicos/fisiologia , Síndrome de Abstinência a Substâncias/fisiopatologia , Acetilcolina/farmacologia , Animais , Condicionamento Psicológico , Dopamina/metabolismo , Camundongos , Camundongos Knockout , Mutagênese , Neurônios/efeitos dos fármacos , Neurônios/fisiologia , Núcleo Accumbens/efeitos dos fármacos , Núcleo Accumbens/fisiopatologia , Receptor Muscarínico M5 , Receptores Muscarínicos/deficiência , Receptores Muscarínicos/genética , Reforço Psicológico , Síndrome de Abstinência a Substâncias/genética , Área Tegmentar Ventral/efeitos dos fármacos , Área Tegmentar Ventral/fisiologia , Área Tegmentar Ventral/fisiopatologiaRESUMO
Centrally active muscarinic agonists display pronounced analgesic effects. Identification of the specific muscarinic acetylcholine receptor (mAChR) subtype(s) mediating this activity is of considerable therapeutic interest. To examine the roles of the M(2) and M(4) receptor subtypes, the two G(i)/G(o)-coupled mAChRs, in mediating agonist-dependent antinociception, we generated a mutant mouse line deficient in both M(2) and M(4) mAChRs [M(2)/M(4) double-knockout (KO) mice]. In wild-type mice, systemic, intrathecal, or intracerebroventricular administration of centrally active muscarinic agonists resulted in robust analgesic effects, indicating that muscarinic analgesia can be mediated by both spinal and supraspinal mechanisms. Strikingly, muscarinic agonist-induced antinociception was totally abolished in M(2)/M(4) double-KO mice, independent of the route of application. The nonselective muscarinic agonist oxotremorine showed reduced analgesic potency in M(2) receptor single-KO mice, but retained full analgesic activity in M(4) receptor single-KO mice. In contrast, two novel muscarinic agonists chemically derived from epibatidine, CMI-936 and CMI-1145, displayed reduced analgesic activity in both M(2) and M(4) receptor single-KO mice, independent of the route of application. Radioligand binding studies indicated that the two CMI compounds, in contrast to oxotremorine, showed >6-fold higher affinity for M(4) than for M(2) receptors, providing a molecular basis for the observed differences in agonist activity profiles. These data provide unambiguous evidence that muscarinic analgesia is exclusively mediated by a combination of M(2) and M(4) mAChRs at both spinal and supraspinal sites. These findings should be of considerable relevance for the development of receptor subtype-selective muscarinic agonists as novel analgesic drugs.