RESUMO
ß-Catenin is a bifunctional molecule that is an effector of the wingless-related integration site (Wnt) signaling to control gene expression and contributes to the regulation of cytoskeleton and neurotransmitter vesicle trafficking. In its former role, ß-catenin binds transcription factor 7-like 2 (TCF7L2), which shows strong genetic associations with the pathogenesis of obesity and type-2 diabetes. Here, we sought to determine whether ß-catenin plays a role in the neuroendocrine regulation of body weight and glucose homeostasis. Bilateral injections of adeno-associated virus type-2 (AAV2)-mCherry-Cre were placed into the arcuate nucleus of adult male and female ß-catenin flox mice, to specifically delete ß-catenin expression in the mediobasal hypothalamus (MBH-ß-cat KO). Metabolic parameters were then monitored under conditions of low-fat (LFD) and high-fat diet (HFD). On LFD, MBH-ß-cat KO mice showed minimal metabolic disturbances, but on HFD, despite having only a small difference in weekly caloric intake, the MBH-ß-cat KO mice were significantly heavier than the control mice in both sexes (p < 0.05). This deficit seemed to be due to a failure to show an adaptive increase in energy expenditure seen in controls, which served to offset the increased calories by HFD. Both male and female MBH-ß-cat KO mice were highly glucose intolerant when on HFD and displayed a significant reduction in both leptin and insulin sensitivity compared with controls. This study highlights a critical role for ß-catenin in the hypothalamic circuits regulating body weight and glucose homeostasis and reveals potential mechanisms by which genetic variation in this pathway could impact on development of metabolic disease.
Assuntos
Diabetes Mellitus Tipo 2 , Dieta Hiperlipídica , Animais , Feminino , Masculino , Camundongos , beta Catenina/genética , beta Catenina/metabolismo , Peso Corporal/genética , Diabetes Mellitus Tipo 2/patologia , Dieta Hiperlipídica/efeitos adversos , Metabolismo Energético/genética , Glucose/metabolismo , Hipotálamo/metabolismo , Leptina/metabolismo , Camundongos Endogâmicos C57BL , Camundongos Knockout , Obesidade/genética , Obesidade/metabolismoRESUMO
Replacing one of the morpholine groups of the phosphatidylinositol 3-kinase (PI3K) inhibitor ZSTK474 with a variety of sulfonamide-linked solubilizing substituents produced a new class of active and potent PI3Kα inhibitors, with several derivatives demonstrating high PI3Kα enzyme potency and good cellular potency in two human derived cell lines. The overall results suggest a preference for linear and somewhat flexible solubilizing functions. From this series, compound 16, also known as SN32976, was selected for advanced preclinical evaluation.
Assuntos
Fosfatidilinositol 3-Quinases/química , Inibidores de Fosfoinositídeo-3 Quinase/síntese química , Sulfonamidas/química , Triazinas/química , Animais , Antineoplásicos/síntese química , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Avaliação Pré-Clínica de Medicamentos , Feminino , Humanos , Concentração Inibidora 50 , Camundongos , Neoplasias/tratamento farmacológico , Fosfatidilinositol 3-Quinases/metabolismo , Inibidores de Fosfoinositídeo-3 Quinase/farmacologia , Inibidores de Fosfoinositídeo-3 Quinase/uso terapêutico , Subunidades Proteicas/antagonistas & inibidores , Subunidades Proteicas/metabolismo , Relação Estrutura-Atividade , Sulfonamidas/farmacologia , Sulfonamidas/uso terapêutico , Transplante HeterólogoRESUMO
The WNT pathway has been well characterized in embryogenesis and tumorigenesis. In humans, specific polymorphisms in the T cell-specific transcription factor 7 and the WNT coreceptor, low-density lipoprotein receptor-related protein-6 (LRP-6), both prominent components of this pathway, correlate with a higher incidence of type 2 diabetes, suggesting that the WNT pathway might be involved in the control of adult glucose homeostasis. We previously demonstrated that glycogen-synthase-kinase-3ß (GSK-3ß), the key enzyme of the WNT pathway, is increased in the hypothalamus during obesity and exacerbates high-fat diet-induced weight gain as well as glucose intolerance. These data suggest that WNT action in the hypothalamus might be required for normal glucose homeostasis. Here we characterized whether WNT signaling in general is altered in the hypothalamus of adult obese mice relative to controls. First we identified expression of multiple components of this pathway in the murine arcuate nucleus by in situ hybridization. In this region mRNA of ligands and target genes of the WNT pathway were down-regulated in obese and glucose-intolerant leptin-deficient mice. Similarly, the number of cells immunoreactive for the phosphorylated (active) form of the WNT-coreceptor LRP-6 was also decreased in leptin-deficient mice. Leptin treatment normalized expression of the WNT-target genes Axin-2 and Cylin-D1 and increased the number of phospho-LRP-6-immunoreactive cells reaching levels of lean controls. Leptin also increased the levels of phosphorylated (inactive) GSK-3ß in the arcuate nucleus, and this effect was colocalized to neuropeptide Y neurons, suggesting that inactivation of GSK-3ß may contribute to the neuroendocrine control of energy homeostasis. Taken together our findings identify hypothalamic WNT signaling as an important novel pathway that integrates peripheral information of the body's energy status encoded by leptin.
Assuntos
Hipotálamo/fisiologia , Leptina/farmacologia , Obesidade/tratamento farmacológico , Obesidade/metabolismo , Via de Sinalização Wnt/fisiologia , Envelhecimento/fisiologia , Animais , Animais Recém-Nascidos , Cromatina , Feminino , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Masculino , Camundongos , Camundongos Endogâmicos , Miocárdio/metabolismo , Obesidade/genética , Gravidez , Efeitos Tardios da Exposição Pré-Natal , Receptores Androgênicos/genética , Receptores Androgênicos/metabolismo , Testículo/metabolismo , Testosterona/farmacologiaRESUMO
GSK3ß (glycogen synthase kinase 3ß) is a ubiquitous kinase that plays a key role in multiple intracellular signalling pathways, and increased GSK3ß activity is implicated in disorders ranging from cancer to Alzheimer's disease. In the present study, we provide the first evidence of increased hypothalamic signalling via GSK3ß in leptin-deficient Lep(ob/ob) mice and show that intracerebroventricular injection of a GSK3ß inhibitor acutely improves glucose tolerance in these mice. The beneficial effect of the GSK3ß inhibitor was dependent on hypothalamic signalling via PI3K (phosphoinositide 3-kinase), a key intracellular mediator of both leptin and insulin action. Conversely, neuron-specific overexpression of GSK3ß in the mediobasal hypothalamus exacerbated the hyperphagia, obesity and impairment of glucose tolerance induced by a high-fat diet, while having little effect in controls fed standard chow. These results demonstrate that increased hypothalamic GSK3ß signalling contributes to deleterious effects of leptin deficiency and exacerbates high-fat diet-induced weight gain and glucose intolerance.
Assuntos
Ingestão de Alimentos/fisiologia , Glucose/metabolismo , Quinase 3 da Glicogênio Sintase/metabolismo , Hipotálamo/enzimologia , Animais , Núcleo Arqueado do Hipotálamo/enzimologia , Núcleo Arqueado do Hipotálamo/fisiologia , Sequência de Bases , Primers do DNA/genética , Dieta Hiperlipídica/efeitos adversos , Intolerância à Glucose/enzimologia , Intolerância à Glucose/etiologia , Quinase 3 da Glicogênio Sintase/deficiência , Quinase 3 da Glicogênio Sintase/genética , Glicogênio Sintase Quinase 3 beta , Humanos , Hipotálamo/fisiologia , Leptina/deficiência , Leptina/genética , Masculino , Camundongos , Camundongos Knockout , Obesidade/enzimologia , Obesidade/etiologia , Transdução de Sinais , Aumento de Peso/fisiologiaRESUMO
We have made a novel series of pyrazolo[1,5-a]pyridines as PI3 kinase inhibitors, and demonstrated their selectivity for the p110α isoform over the other Class Ia PI3 kinases. We investigated the SAR around the pyrazolo[1,5-a]pyridine ring system, and found compound 5x to be a particularly potent example (p110α IC(50) 0.9nM). This compound inhibits cell proliferation and phosphorylation of Akt/PKB, a downstream marker of PI3 kinase activity, and showed in vivo activity in an HCT-116 human xenograft model.
Assuntos
Inibidores de Fosfoinositídeo-3 Quinase , Inibidores de Proteínas Quinases/química , Pirazóis/química , Piridinas/química , Proteínas Quinases Dependentes de 3-Fosfoinositídeo , Animais , Antineoplásicos/química , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Sítios de Ligação , Linhagem Celular Tumoral , Simulação por Computador , Avaliação Pré-Clínica de Medicamentos , Ativação Enzimática/efeitos dos fármacos , Humanos , Camundongos , Neoplasias/tratamento farmacológico , Fosfatidilinositol 3-Quinases/metabolismo , Fosforilação , Inibidores de Proteínas Quinases/farmacologia , Inibidores de Proteínas Quinases/uso terapêutico , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-akt/metabolismo , Piridinas/síntese química , Piridinas/farmacologia , Transplante HeterólogoRESUMO
Obesity is associated with resistance to the actions of both leptin and insulin via mechanisms that remain incompletely understood. To investigate whether leptin resistance per se contributes to insulin resistance and impaired glucose homeostasis, we investigated the effect of acute leptin administration on glucose homeostasis in normal as well as leptin- or leptin receptor-deficient mice. In hyperglycemic, leptin-deficient Lep(ob/ob) mice, leptin acutely and potently improved glucose metabolism, before any change of body fat mass, via a mechanism involving the p110α and ß isoforms of phosphatidylinositol-3-kinase (PI3K). Unlike insulin, however, the anti-diabetic effect of leptin occurred independently of phospho-AKT, a major downstream target of PI3K, and instead involved enhanced sensitivity of the hypothalamus to insulin action upstream of PI3K, through modulation of IRS1 (insulin receptor substrate 1) phosphorylation. These data suggest that leptin resistance, as occurs in obesity, reduces the hypothalamic response to insulin and thereby impairs peripheral glucose homeostasis, contributing to the development of type 2 diabetes.
Assuntos
Glucose/metabolismo , Homeostase/fisiologia , Hipotálamo/metabolismo , Resistência à Insulina/fisiologia , Leptina/deficiência , Obesidade/metabolismo , Tecido Adiposo/enzimologia , Tecido Adiposo/fisiopatologia , Animais , Glicemia/metabolismo , Classe Ia de Fosfatidilinositol 3-Quinase/genética , Classe Ia de Fosfatidilinositol 3-Quinase/fisiologia , Classe Ib de Fosfatidilinositol 3-Quinase/genética , Classe Ib de Fosfatidilinositol 3-Quinase/fisiologia , Homeostase/genética , Hipotálamo/enzimologia , Resistência à Insulina/genética , Isoenzimas/genética , Isoenzimas/fisiologia , Leptina/genética , Masculino , Camundongos , Camundongos Knockout , Camundongos Obesos , Obesidade/enzimologia , Obesidade/genética , Ratos , Ratos Sprague-Dawley , Fatores de TempoRESUMO
In the current drug discovery paradigm, validated recombinant targets form the basis of in vitro high-throughput screening (HTS) assays. Isolated proteins cannot, however, be regarded as representative of complex biological systems; hence, cell-based systems can be employed to complement in vitro data, providing greater confidence in compound activity in an intact biological system. The scarcity of human material and the lack of proliferative capacity of primary cell cultures, combined with problems associated with the use of stem cells, mean that immortalized cell lines are generally used as a source of cells for HTS. While such cell lines have improved proliferative capacity, they often display aberrant genetic and functional characteristics. Consequently, interest has focused on creating new cell lines using defined molecular strategies that overcome senescence signals and telomere shortening. These strategies include expression of viral oncogenes and the catalytic subunit of telomerase (TERT) to generate immortalized cells. However, persistent proliferation induced by oncogenes can have undesirable effects, particularly on differentiation; hence, conditional immortalization approaches have been developed to provide more suitable cell models. The present review discusses the advantages of different technologies available for the generation of cell lines for use in in vitro biology, and describes a representative range of cell lines currently used in drug discovery.