To determine whether downregulation of Gi proteins is associated with insulin resistance, we incubated isolated adipocytes with N6-(2-phenylisopropyl)adenosine (PIA; an A1-adenosine receptor agonist; 300 nM), prostaglandin E1 (PGE1; 3 microM), or nicotinic acid (1 mM) for 4 days in primary culture. The cells were washed, and the rate of glucose transport (2-deoxy-[3H]glucose uptake) was measured after incubation with various concentrations of insulin for 45 min. Both PIA and PGE1 (which downregulate Gi) decreased the maximal responsiveness of the cells to insulin by approximately 30% and caused a rightward shift in the dose-response curve. By contrast, nicotinic acid (which does not downregulate Gi) did not alter the insulin sensitivity of the cells. Prolonged treatment of adipocytes with either PIA or PGE1 (but not nicotinic acid) rendered the cells completely resistant to the antilipolytic effect of insulin. The ability of insulin to stimulate autophosphorylation of the beta-subunit of the insulin receptor was decreased by approximately 30% in PIA-treated cells, and the dose-response curve was shifted to the right. Similarly, the ability of the receptor to phosphorylate poly(Glu4-Tyr1) was decreased by approximately 35%. This decrease in tyrosine kinase activity of the receptor may account for the decrease in insulin sensitivity of glucose transport but cannot account for the complete loss of antilipolysis. The findings suggest both a direct and indirect involvement of Gi proteins in insulin action.
Adenosine, and other A1 adenosine receptor agonists such as N6-phenylisopropyl adenosine (PIA) are potent inhibitors of adenylate cyclase and lipolysis in rat adipocytes [l]. A1 adenosine receptors are coupled to inhibition of adenylate cyclase by a pertussis toxin sensitive G-protein, G . Three subtypes of Gi have been identifed, Gil, Gi2 and Gi3, all of whicf; are present in adipocytes [2]. Recent evidence suggests that Gi2 is responsible for inhibition of adenylate cyclase [3,4].We have reported that rat adipocyte A adenosine receptors can be down-regulated by prolonged incubation oflthe cells with PIA IS]. Furthermore, we found by Western blot analysis using specific antipeptide antisera, that all three subtypes of Gi are down-regulated along with the adenosine receptor [6]. To determine whether other inhibitors of adenylate cyclase will down-regulate G;, and whether G . down-regulation can form a basis for heterologous desensitization, we ha& investigated the effects of prostaglandin El (PGE1) and of nicotinic acid.Adipocytes were isolated by collagenase digestion, and maintained in primary culture [A. Lipolysis was measured by following the rate of glycerol release, as described previously [5]. Relative levels of G-protein subunits were determined by Western blot analysis [6]. Briefly, cells were homogenized, and a crude membrane fraction was isolated by differential centrifugation. The membranes were separated on SDS-polyacrylamide gels (12.5% acrylamide, 0.06% bis-acrylamide), and then transferred electrophoretically to nitrocellulose. The blots were incubated with antisera SG1, which binds to a.1 and ai2 equally, I3B, which binds to a.3, BN2, which binds to 8-subunits or CS1, which binds to forms of as. (+he production and characterization of these antisera have been described [2,6]). The blots were visualized using alkaline phosphatase labelled goat anti-rabbit antibody, and then the bands were quantified using an LKB laser densitometer. PIA, PGE and nicotinic acid were each potent inhibitors of lipolysis in freshly isolateh adipocytes (Fig. 1). From these dose-response curves, maximally effective concentrations of each compound were selected for investigation of chronic effects on levels of G-proteins. Since PIA was I -" I -0 ZI o L I -c3 I O -'~ 10-0 1 0 4 10-7 1 0 4 1 0 4 10-4 10-3 Concentration (M) Fig. 1. Dose-resoonse curves for inhibition of lioolvsis. Adipocytes were incubated with the indicated concentrations of PIA (0-o), PGEl (A-A) or nicotinic acid (0-0). and the rate of glycerol release was measured over lh.maximally effective for Gi down-regulation at 300 nM [6], PGE1 was used at 3 pM, and nicotinic acid at a concentration of 100 pM. Adipocytes were incubated with these concentrations of the three antilipolytic compounds for 4 days, and then relative levels of Gi and Gs a-subunits, and of p-subunits were determined as described above. As in our previous report [6] PIA down-regulated all three forms of a;, to varying degrees. The effect of PGE1 was almost identical to that of PIA. Thus, ail...
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.