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
In this work, we characterized a Mg(2+)-dependent ecto-phosphatase activity present in live Trypanosoma rangeli epimastigotes. This enzyme showed capacity to hydrolyze the artificial substrate for phosphatases, p-nitrophenylphosphate (p-NPP). At saturating concentration of p-NPP, half-maximal p-NPP hydrolysis was obtained with 0.23mM Mg(2+). Ca(2+) had no effect on the basal phosphatase activity, could not substitute Mg(2+) as an activator and in contrast inhibited the p-NPP hydrolysis stimulated by Mg(2+). The dependence on p-NPP concentration showed a normal Michaelis-Menten kinetics for this phosphatase activity with values of V(max) of 8.94+/-0.36 nmol p-NP x h(-1) x 10(-7) cells and apparent K(m) of 1.04+/-0.16 mM p-NPP. Mg(2+)-dependent ecto-phosphatase activity was stimulated by the alkaline pH range. Experiments using inhibitors, such as, sodium fluoride, sodium orthovanadate and ammonium molybdate, inhibited the Mg(2+)-dependent ecto-phosphatase activity. Inorganic phosphate (Pi), a product of phosphatases, inhibited reversibly in 50% this activity. Okadaic acid and microcystin-LR, specific phosphoserine/threonine phosphatase inhibitors, inhibited significantly the Mg(2+)-dependent ecto-phosphatase activity. In addition, this phosphatase activity was able to recognize as substrates only o-phosphoserine and o-phosphothreonine, while o-phosphotyrosine was not a good substrate for this phosphatase. Epimastigote forms of T. rangeli exhibit a typical growth curve, achieving the stationary phase around fifth or sixth day and the Mg(2+)-dependent ecto-phosphatase activity decreased around 10-fold with the cell growth progression. Cells maintained at Pi-deprived medium (2 mM Pi) present Mg(2+)-dependent ecto-phosphatase activity approximately threefold higher than that maintained at Pi-supplemented medium (50 mM Pi).
Assuntos
Regulação Enzimológica da Expressão Gênica , Compostos Inorgânicos/farmacologia , Magnésio/metabolismo , Fosfatos/farmacologia , Monoéster Fosfórico Hidrolases/efeitos dos fármacos , Trypanosoma/enzimologia , Animais , Cálcio/metabolismo , Membrana Celular/enzimologia , Meios de Cultura , Monoéster Fosfórico Hidrolases/metabolismo , Especificidade por Substrato , Trypanosoma/efeitos dos fármacos , Trypanosoma/crescimento & desenvolvimentoRESUMO
The presence of iron in the extracellular medium is essential for both in vivo and in vitro survival of pathogenic microorganisms, including Trichomonas vaginalis and Tritrichomonas foetus. In these parasites, iron is directly involved in the proliferation, protein expression and activation of critical enzymes. The purpose of this study was to investigate the role of iron in ecto-ATPase, ecto-phophatase and secreted phosphatase activities of these trichomonads. We observed that trichomonads grown in iron-depleted medium exhibited a remarkable decrease in both ecto-ATPase and ecto-phosphatase activities, when compared to those cultivated under control conditions (iron-rich medium). Furthermore, parasites grown in iron-depleted medium restored their enzyme activities when they were re-inoculated into fresh iron-rich medium. We demonstrated that modulation of ecto-phosphohydrolase activities is due neither to enzyme-iron nor to substrate-iron complex formation, since iron addition directly to the medium where the enzymatic reactions occurred did not alter their activities. Previously, we had reported that a fresh clinical isolate of T. vaginalis was much more cytotoxic to epithelial cell monolayers than a long-term cultured one. In this study we witnessed that the fresh isolate of T. vaginalis presented higher activities to all herein investigated enzymes than the long-term cultured one. Altogether, our data clearly point out that iron has a pivotal role in the expression of phosphohydrolases in both trichomonads.
Assuntos
Adenosina Trifosfatases/efeitos dos fármacos , Ferro/farmacologia , Monoéster Fosfórico Hidrolases/efeitos dos fármacos , Trichomonas/enzimologia , Adenosina Trifosfatases/análise , Animais , Meios de Cultura , Monoéster Fosfórico Hidrolases/análise , Trichomonas/efeitos dos fármacos , Trichomonas vaginalis/enzimologiaRESUMO
A phosphatase activity of the trypanosomatid parasite Herpetomonas samuelpessoai was characterized using intact living cells. The effects of dimethyl sulfoxide (DMSO) on this activity were investigated. This phosphatase activity (2.53+/-0.01 nmol P(i)/mg protein x min) was linear with cell density and with time for at least 60 min. The optimum pH for the H. samuelpessoai phosphatase lies in the acid range. This phosphatase activity was inhibited by metal chelators and classical phosphatase inhibitors. A robust stimulation of the phosphatase activity was observed when the flagellates were grown in the presence of 4% DMSO, both when intact flagellates and when culture supernatant from those cells were assayed, as observed by biochemical and cytochemical analysis. We also demonstrate that DMSO induced the secretion and/or shedding of this phosphatase to the extracellular medium, with a possible involvement of protein kinase C in this process.