YM201636, an inhibitor of retroviral budding and PIKfyve-catalyzed PtdIns(3,5)P2 synthesis, halts glucose entry by insulin in adipocytes - PubMed (original) (raw)
YM201636, an inhibitor of retroviral budding and PIKfyve-catalyzed PtdIns(3,5)P2 synthesis, halts glucose entry by insulin in adipocytes
Ognian C Ikonomov et al. Biochem Biophys Res Commun. 2009.
Abstract
Silencing of PIKfyve, the sole enzyme for PtdIns(3,5)P(2) biosynthesis that controls proper endosome dynamics, inhibits retroviral replication. A novel PIKfyve-specific inhibitor YM201636 disrupts retroviral budding at 800 nM, suggesting its potential use as an antiretroviral therapeutic. Because PIKfyve is also required for optimal insulin activation of GLUT4 surface translocation and glucose influx, we tested the outcome of YM201636 application on insulin responsiveness in 3T3L1 adipocytes. YM201636 almost completely inhibited basal and insulin-activated 2-deoxyglucose uptake at doses as low as 160 nM, with IC(50)=54+/-4 nM for the net insulin response. Insulin-induced GLUT4 translocation was partially inhibited at substantially higher doses, comparable to those required for inhibition of insulin-induced phosphorylation of Akt/PKB. In addition to PIKfyve, YM201636 also completely inhibited insulin-dependent activation of class IA PI 3-kinase. We suggest that apart from PIKfyve, there are at least two additional targets for YM201636 in the context of insulin signaling to GLUT4 and glucose uptake: the insulin-activated class IA PI 3-kinase and a here-unidentified high-affinity target responsible for the greater inhibition of glucose entry vs. GLUT4 translocation. The profound inhibition of the net insulin effect on glucose influx at YM201636 doses markedly lower than those required for efficient retroviral budding disruption warns of severe perturbations in glucose homeostasis associated with potential YM201636 use in antiretroviral therapy.
Figures
Fig. 1
Effect of YM201636 on basal and insulin-induced glucose transport. Serum-starved 3T3L1 adipocytes were treated with the indicated concentrations of YM201636 (30 min), then stimulated with or without insulin (100 nM; 30 min) followed by 2DG assay. (A) Data from three independent experiments in triplicates (mean ± SEM); (B) the net insulin effect above basal calculated for each dose of YM201636 and expressed as a percent of the net insulin effect in cells not treated with YM201636; *p < 0.001.
Fig. 2
Effect of YM201636 on basal and insulin-induced GLUT4 translocation. 3T3L1 adipocytes, electroporated with HA-GLUT4-eGFP cDNA were serum-starved, treated with YM201636 (30 min), then stimulated with insulin (100 nM; 30 min) as indicted. Cells were analyzed by immunofluorescence microscopy. Shown is quantitation of the ratio of cell surface HA (Cy3)-signal to total GFP fluorescence in the HA-GLUT4-eGFP-expressing cells from three independent experiments, in which 10–20 cells/condition/experiment were analyzed by quantitative fluorescence microscopy as described in Materials and methods (mean ± SEM; *different vs. insulin-stimulated control, p < 0.001; #different vs. insulin-stimulated control, p < 0.025).
Fig. 3
Effect of YM201636 on Akt phosphorylation by insulin. Serum-starved 3T3L1 adipocytes were treated with YM201636 (30 min), then stimulated with insulin (100 nM; 10 min) or left untreated as indicated. Aliquots (50 μg) of the cell lysates, collected with protease and phosphatase inhibitors, were analyzed by immunoblotting with anti-phosphoSer473-Akt and anti-Akt antibodies. (A) Chemiluminescence detections of a representative blot with stripping between the antibodies. No phosphoSer47-Akt is seen in the absence of insulin. (B) Quantitation of the band intensity of the insulin-stimulated condition under different YM201636 concentrations normalized for total Akt, and presented as a percentage of the insulin-stimulated control (no YM201936, only vehicle); mean ± SEM, n = 3; #different vs. insulin-stimulated control, p<0.025; *different vs. insulin-stimulated control, p < 0.001.
Fig. 4
YM201636 inhibits both PIKfyve and insulin-activated class IA PI 3-kinase. Serum-starved 3T3L1 adipocytes were stimulated with or without insulin (100 nM; 10 min) as indicated. Anti-PIKfyve immunoprecipitates were incubated with YM201636 (100 nM; 15 min) or with vehicle and subjected to lipid kinase assay in the presence [γ-32P]ATP. (A) A representative autoradiogram of a TLC plate from three independent experiments. (B) PtdInsP samples recovered from the TLC plate in (A) (arrows) were deacylated and separated on HPLC column. Positions of 32P-GroPIns3P (3P) or 32P-GroPIns5P (5P) were determined from parallel runs of the respective deacylated 32P-labeled PtdInsP standards. In the absence of YM201636 (–), insulin increased GroPins3P to 546 ± 39% in comparison to basal GroPins3P in panel (a) (p < 0.001; n = 3), as calculated by peak area integration. In the presence of YM201636 (+), there was an insignificant insulin-dependent increase (111 ± 16%) of GroPins3P vs. basal GroPins3P in panel (a), indicating severe inhibition of the insulin-dependent activation of PI 3-kinase by YM201636. YM201636 also inhibited the basal PI 3-kinase activity, evidenced by the 52 ± 6% decrease in basal GroPins3P in panel (c) vs. GroPins3P in panel (a) (p < 0.001; n = 3).
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