RESUMO
The 72âkDa inositol polyphosphate 5-phosphatase E (72k-5ptase) controls signal transduction through the catalytic dephosphorylation of the 5-position of membrane-bound phosphoinositides. The reduction of 72k-5ptase expression in the hypothalamus results in improved hypothalamic insulin signal transduction and reduction of food intake and body mass. Here, we evaluated the tissue distribution and the impact of obesity on the expression of 72k-5ptase in peripheral tissues of experimental animals. In addition, insulin signal transduction and action were determined in an animal model of obesity and insulin resistance treated with an antisense (AS) oligonucleotide that reduces 72k-5ptase expression. In lean Wistar rats, 72k-5ptase mRNA and protein are found in highest levels in heart, skeletal muscle, and white adipose tissue. In three distinct models of obesity, Wistar rats, Swiss mice fed on high-fat diet, and leptin-deficient ob/ob mice, the expression of 72k-5ptase is increased in skeletal muscle and adipose tissue. The treatment of obese Wistar rats with an anti-72k-5ptase AS oligonucleotide results in significant reduction of 72k-5ptase catalytic activity, which is accompanied by reduced food intake and body mass and improved insulin signal transduction and action as determined by immunoblotting and clamp studies respectively. 72k-5ptase expression is increased in obesity and its AS inhibition resulted in a significant improvement in insulin signal transduction and restoration of glucose homeostasis.
Assuntos
Dieta Hiperlipídica/efeitos adversos , Insulina/fisiologia , Obesidade/etiologia , Obesidade/fisiopatologia , Monoéster Fosfórico Hidrolases/antagonistas & inibidores , Transdução de Sinais/fisiologia , Tecido Adiposo Branco/enzimologia , Animais , Modelos Animais de Doenças , Inositol Polifosfato 5-Fosfatases , Resistência à Insulina/fisiologia , Leptina/deficiência , Masculino , Camundongos , Camundongos Obesos , Músculo Esquelético/enzimologia , Miocárdio/enzimologia , Obesidade/metabolismo , Oligorribonucleotídeos Antissenso/farmacologia , Monoéster Fosfórico Hidrolases/efeitos dos fármacos , Monoéster Fosfórico Hidrolases/metabolismo , Ratos , Ratos WistarRESUMO
The triatomines in the tribe Rhodniini are the main vectors of the Trypanosoma cruzi to humans in recent outbreaks of acute Chagas disease in the Amazon. These insects dwelling in palm trees do not colonize the human domicile. Their success to transmit the infection relies partially on the efficacy of their salivary gland apparatuses. Here we show the transcriptome of the Rhodnius brethesi and Rhodnius robustus salivary glands, comprising 56 and 122 clusters, respectively. Approximately one third of these clusters are described for the first time. The LC-MS/MS analysis identified 123 and 111 proteins in R. brethesi and R. robustus sialome, respectively. Noteworthy, lipocalin platelet aggregation inhibitors, inositol polyphosphate 5-phosphatases, and Kazal domain proteins, which are essential for the insect's successful acquisition of blood meals, were found in our analysis. Moreover, glutathione S transferase and antigen-5, which play roles in the insect's defense and resistance against insecticide, were also observed.
Assuntos
Insetos Vetores/química , Rhodnius/química , Glândulas Salivares/química , Venenos de Vespas , Animais , Doença de Chagas/parasitologia , Doença de Chagas/transmissão , Perfilação da Expressão Gênica , Glutationa Transferase/análise , Glutationa Transferase/genética , Humanos , Inositol Polifosfato 5-Fosfatases , Proteínas de Insetos/análise , Insetos Vetores/patogenicidade , Resistência a Inseticidas/genética , Lipocalinas/análise , Lipocalinas/genética , Monoéster Fosfórico Hidrolases/análise , Monoéster Fosfórico Hidrolases/genética , Inibidores da Agregação Plaquetária/análise , Rhodnius/parasitologia , Glândulas Salivares/parasitologia , Venenos de Vespas/análise , Venenos de Vespas/genéticaRESUMO
Insulin-induced glucose uptake by skeletal muscle results from Akt2 activation and is severely impaired during insulin resistance. Recently, we and others have demonstrated that BMP9 improves glucose homeostasis in diabetic and non-diabetic rodents. However, the mechanism by which BMP9 modulates insulin action remains unknown. Here we demonstrate that Smad5, a transcription factor activated by BMP9, and Akt2, are upregulated in differentiated L6 myotubes. Smad5, rather than Smad1/8, is downregulated "in vivo" and "in vitro" by dexamethasone. Smad5 knockdown decreased Akt2 expression and serine phosphorylation and insulin-induced glucose uptake, and increased the expression of the lipid phosphatase Ship2. Additionally, binding of Smad5 to Akt2 gene is decreased in dexamethasone-treated rats and increased in L6 myotubes compared to myoblasts. The present study indicates that Smad5 regulates glucose uptake in skeletal muscle by controlling Akt2 expression and phosphorylation. These finding reveals Smad5 as a potential target for the therapeutic of type 2 diabetes.
Assuntos
Glucose/metabolismo , Insulina/metabolismo , Fibras Musculares Esqueléticas/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteína Smad5/metabolismo , Análise de Variância , Animais , Western Blotting , Dexametasona/farmacologia , Glucocorticoides/farmacologia , Imunoprecipitação , Inositol Polifosfato 5-Fosfatases , Insulina/farmacologia , Masculino , Fibras Musculares Esqueléticas/efeitos dos fármacos , Fosfatidilinositol-3,4,5-Trifosfato 5-Fosfatases , Monoéster Fosfórico Hidrolases/metabolismo , Fosforilação/fisiologia , Interferência de RNA/fisiologia , Ratos , Ratos Wistar , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais/fisiologia , Proteína Smad5/genética , Proteína Smad8/metabolismo , TransfecçãoRESUMO
The enzyme phosphatidylinositol 3-kinase (PI3-kinase) exerts an important role in the transduction of the anorexigenic and thermogenic signals delivered by insulin and leptin to first-order neurons of the arcuate nucleus in the hypothalamus. The termination of the intracellular signals generated by the activation of PI3-kinase depends on the coordinated activity of specific inositol phosphatases. Here we show that phosphoinositide-specific inositol polyphosphate 5-phosphatase IV (5ptase IV) is highly expressed in neurons of the arcuate and lateral nuclei of the hypothalamus. Upon intracerebroventricular (ICV) treatment with insulin, 5ptase IV undergoes a time-dependent tyrosine phosphorylation, which follows the same patterns of canonical insulin signaling through the insulin receptor, insulin receptor substrate-2, and PI3-kinase. To evaluate the participation of 5ptase IV in insulin action in hypothalamus, we used a phosphorthioate-modified antisense oligonucleotide specific for this enzyme. The treatment of rats with this oligonucleotide for 4 d reduced the hypothalamic expression of 5ptase IV by approximately 80%. This was accompanied by an approximately 70% reduction of insulin-induced tyrosine phosphorylation of 5ptase IV and an increase in basal accumulation of phosphorylated inositols in the hypothalamus. Finally, inhibition of hypothalamic 5ptase IV expression by the antisense approach resulted in reduced daily food intake and body weight loss. Thus, 5ptase IV is a powerful regulator of signaling through PI3-kinase in hypothalamus and may become an interesting target for therapeutics of obesity and related disorders.
Assuntos
Peso Corporal , Ingestão de Alimentos , Hipotálamo/enzimologia , Monoéster Fosfórico Hidrolases/fisiologia , Sequência de Aminoácidos , Animais , Fármacos Antiobesidade/farmacologia , Sequência de Bases , Inibidores Enzimáticos/farmacologia , Inositol Polifosfato 5-Fosfatases , Insulina/farmacologia , Masculino , Dados de Sequência Molecular , Fosfatidilinositol 3-Quinases/fisiologia , Monoéster Fosfórico Hidrolases/antagonistas & inibidores , Fosforilação , Ratos , Transdução de Sinais , Tirosina/metabolismoRESUMO
We have investigated whether IP3 metabolism presents particular changes during critical stages of muscle development. With this aim, we have measured IP3 formation through phospholipase C activity, IP3 removal through IP3 5-phosphatase and IP3 3-kinase activities, as well as IP3 mass, during myogenesis in vivo and in vitro. In developing rat skeletal muscle, both IP3 3-kinase and 5-phosphatase activities were relatively constant from embryonary day 15, the earliest age studied to postnatal day 10; 5-phosphatase decreased upon further development. A transient, major increase in phospholipase C activity was evident at embryonary day 18 while a non-significant increase in IP3 mass was detected at this embrionary age. In rat skeletal muscle in primary culture, all enzyme activities as well as the mass of IP3 increased significantly in myotubes compared to myoblasts. Myotubes incubated with calcitonin gene-related peptide, responded with a transient increase in IP3 mass after 2 to 10 sec; the CGRP-induced increase being completely blocked by U-73122, a phospholipase C inhibitor. Furthermore, IP3 mass increased within 1 hr after exposure to differentiating agents of both RCMH cells, a line derived from normal human skeletal muscle, and C2C12 cells. These results indicate that changes in IP3 metabolism can be correlated to critical stages of muscle development and differentiation, suggesting a possible role for IP3 in these processes.