Abstract
The role of adenosine receptor in regulation of insulin-induced activation of phosphoinositide 3-kinase (PI 3-kinase) and protein kinase B was studied in isolated rat adipocytes. Rat adipocytes are known to spontaneously release adenosine, which in turn binds and stimulates the adenosine A1 receptors on the cells. In the present study, we observed that degradation of this adenosine by adenosine deaminase attenuated markedly the insulin-induced accumulation of phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P3), a product of PI 3-kinase. p-Aminophenylacetyl xanthine amine congener (PAPA-XAC), an inhibitor of the adenosine A1 receptor, also inhibited the insulin-induced PtdIns(3,4,5)P3 accumulation. When extracellular adenosine was inactivated by adenosine deaminase, phenylisopropyladenosine, an adenosine A1 receptor agonist, potentiated the insulin-induced accumulation of PtdIns(3,4,5)P3. Insulin-induced activation of protein kinase B, the activity of which is controlled by the lipid products of PI 3-kinase, was also potentiated by adenosine. Prostaglandin E2, another activator of a pertussis toxin-sensitive GTP-binding protein in these cells, potentiated the insulin actions. Thus, the receptors coupling to the GTP-binding protein were found to positively regulate the production of PtdIns(3,4,5)P3, a putative second messenger for insulin actions, in physiological target cells of insulin.
Highlights
Adenosine deaminase has been shown to impair the insulin sensitivity for glucose transport and antilipolysis by inactivating extracellular adenosine, which adipocytes release spontaneously (1–3)
Rat adipocytes spontaneously release adenosine, which in turn binds to the A1 receptors on the cells
Because degradation of this adenosine by addition of adenosine deaminase is reported to modulate the insulin action on glucose uptake (1), we examined whether the insulin-induced production of [32P]PtdIns(3,4,5)P3 is affected by the enzyme
Summary
Materials—The sources of materials used in this work were as follows: 32Pi and [␥-32P]ATP from NEN Life Science Products Inc.; paminophenylacetyl xanthine amine congener (PAPA-XAC) from Research Biochemicals; 2Ј,5Ј-dideoxyadenosine (DDA) from BIOMOL Research Laboratories; (R)-N6-(phenylisopropyl)-adenosine (PIA), adenosine deaminase, and histone H2B from Roche Molecular Biochemicals; anti-PKB␣(Akt-1) antibody from Santa Cruz; protein GSepharose from Pharmacia; and collagenase (type I) from Worthington. Isolation of Rat Adipocytes—Epididymal fat pads from male Wistar rats weighing 100 –120 g were cut into small pieces and incubated at 37 °C for 30 min in a medium consisting of 130 mM NaCl, 4.7 mM KCl, This paper is available on line at http://www.jbc.org. Measurement of the PtdIns(3,4,5)P3 Production—The isolated adipocytes were washed with an incubation medium consisting of 130 mM NaCl, 4.7 mM KCl, 1 mM CaCl2, 1.2 mM MgSO4, 25 mM HEPES-NaOH (pH 7.4), and 0.5% (w/v) bovine serum albumin and suspended at a density of 1 ϫ 106 cells/ml in the same medium supplemented with 0.2 mCi/ml of carrier-free 32Pi. The cells (0.2 ml) were incubated at 37 °C for 10 min with or without adenosine deaminase, PAPA-XAC, or wortmannin and for 10 min with or without insulin. The filters were washed 8 times with 2 ml of 50 mM Tris-HCl (pH 8.0) containing 100 mM NaCl, and radioactivity remained on the filters was determined
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