Partial purification from human mononuclear cells and placental plasma membranes of an insulin mediator which stimulates pyruvate dehydrogenase and suppresses glucose-6-phosphatase.
A substance capable of stimulating pyruvate dehydrogenase (PDH) and suppressing glucose-6-phosphatase (G-6-Pase) in a cell-free system was prepared from insulin-treated human placental plasma membranes and peripheral blood mononuclear cells by formic acid extraction. This material was partially purified by molecular-exclusion chromatography, ion-exchange chromatography, and hydroxylapatite chromatography. This was found to stimulate pyruvate dehydrogenase and inhibit glucose-6-phosphatase in a dose-dependent manner. The amount or ability of this substance to stimulate pyruvate dehydrogenase was increased in the proportion to the concentration of insulin. The stimulation of pyruvate dehydrogenase by the factor was eliminated when sodium fluoride was presented in the assay of the activation. This result implied that the activation of pyruvate dehydrogenase was mediated by the stimulation of the phosphatase of pyruvate dehydrogenase complex. Each material isolated from insulin-treated human placental plasma membranes and mononuclear cells shared a number of important characteristics of putative second messengers of insulin action as follows: (i) heat and acid stability; (ii) a similar molecular weight; (iii) increased activity of pyruvate dehydrogenase in a insulin-dependent manner; and (iv) stimulated pyruvate dehydrogenase by the sodium fluoride-sensitive mechanism. This human putative second messenger of insulin action was eluted from the anion-exchange resin AG1-X8 at an ionic strength of 0.3-0.4 M, as well as from the hydroxylapatite column at a phosphate concentration of 0.2-0.3 M.
D000255:Adenosine Triphosphate / Q000494:pharmacology; D002462:Cell Membrane / Q000032:analysis; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D004789:Enzyme Activation / Q000187:drug effects; D005952:Glucose-6-Phosphatase / Q000037:antagonists & inhibitors*; D006801:Humans; D006882:Hydroxyapatites; D007295:Inositol Phosphates; D007328:Insulin / Q000494:pharmacology; D009000:Monocytes / Q000032:analysis*; D010920:Placenta / Q000032:analysis*; D011134:Polysaccharides; D011768:Pyruvate Dehydrogenase Complex / Q000378:metabolism*; D011972:Receptor, Insulin / Q000302:isolation & purification* / Q000494:pharmacology; D012969:Sodium Fluoride / Q000494:pharmacology
D006882:Hydroxyapatites; D007295:Inositol Phosphates; D007328:Insulin; D011134:Polysaccharides; D011768:Pyruvate Dehydrogenase Complex; C028039:inositol phosphate glycan; D000255:Adenosine Triphosphate; D012969:Sodium Fluoride; D011972:Receptor, Insulin; D005952:Glucose-6-Phosphatase