Van Buren JJ, Bhat S, Rotello R, Pauza ME, Premkumar LS.

DK065742 NIDDK NIH HHS , NSO42296 PHS HHS , R01 DK065742 NIDDK NIH HHS , R56 DK065742 NIDDK NIH HHS

	Figure 1. Insulin and IGF-I potentiate TRPV1 currents in Xenopus Oocytes. a, A representative dual-electrode whole-oocyte experiment showing potentiation of capsaicin induced TRPV1 currents following 15 min incubation of insulin (see Methods). b, Time course for capsaicin induced current (■: 500 nM capsaicin, 30 sec application) before and after insulin exposure at 5 (P < .05), 10 (P < .01), and 15 min (P < .01) incubation times (n = 7). c, Dose response curve of capsaicin before (squares) and after 5 μM insulin (circles) application, the currents were normalized to 1 μM capsaicin before insulin application (n = 2 to 4, before and after insulin). Both the sensitivity (EC50 shifted from 0.9 to 0.6 μM) and maximal response increased (1.8 to 2.4). d, Representative I-V relationships (1 s, 1 mV step ramp from -80 to +80 mV) before and after insulin treatment that demonstrates the outward rectification typical of TRPV1 channels. e, Potentiation of heat-induced currents by insulin. f, Potentiation of pH induced currents by insulin. g, Potentiation of pH induced currents by IGF-I. h, Tyrphostin A47, an IR / IGFR antagonist, blocked insulin potentiation. i, Summary graph showing fold increase in TRPV1 currents following 10 min incubation with control, insulin (cap: n = 5, P < .01; pH: n = 4, P < .01), IGF-I (n = 3, P < .01), and insulin + tyrophostin A47 (n = 3, P < .01). Results are expressed as increase in current amplitude relative to initial capsaicin or pH response.
	Figure 2. Insulin and IGF-I potentiate native TRPV1 currents in DRG neurons. a, Capsaicin (100 nM, applied at 2 min intervals) response was enhanced by insulin (1 μM) under perforated patch conditions. b, Time course of insulin induced potentiation of capsaicin currents (■: 500 nM capsaicin, 20 sec application) (n = 4, P < .01 at 5, 10, and 15 min incubation times). Inset shows the time course for experiment in Fig. 2a. c, Summary graph showing fold increase in TRPV1 currents following 10 min treatment with control (n = 15), insulin (1 μM; n = 11, P < .01) and IGF-I (20 nM; n = 4, P < .01). d, Under confocal microscopy, insulin (1 μM, 2 min) potentiated intracellular Ca2+ rise in response to 500 nM capsaicin (n = 10, P < .05). e, Capsaicin induced single channel current activity recorded at +60 mV was increased by exposure of cell to insulin (1 μM). f, Mean capsaicin induced open probability (Po) in the absence and presence of insulin (n = 5, P < .05). g, Mean current amplitude observed from single channel recordings (n = 5).
	Figure 3. Singnaling cascades utilized by Insulin and IGF-I. a, Summary graph showing fold increase of current in the presence of IGF-I and the inhibitors: genistein (50 μM, n = 6, P < .05), lavendustin A (100 μM, n = 7, P < .05), wortmannin (100 nM, n = 5, P < .05) and BIM (200 nM, n = 5, P < .05). b, A PKC phosphorylation site TRPV1 mutant (S502A/S800A) (n = 6, P < .01, upper trace) and cytochalasin D (1 μM, n = 4, P < .01, lower trace) completely blocked insulin potentiation.
	Figure 4. Insulin and IGF-I translocate TRPV1 to the plasma membrane. a, Potentiation of saturating concentrations of capsaicin response by insulin and PDBu. Application of 5, 10 and 20 μM capsaicin shows saturation of current response (ai), exposure (2–5 min) of insulin (aii) or PDBu (aiii) caused a 50% increase in current amplitude induced by 20 μM capsaicin (aiv) (insulin n = 4 P < .01; PDBu n = 5 P > .01). b, Representative Western blot of surface protein and total TRPV1 from control and insulin-treated (10 μM, 15 min) HEK293 cells expressing TRPV1 (probed with anti-TRPV1 antibody). Surface represents fraction of biotinylated TRPV1 and total represents total amount of TRPV1 in immunoprecipitate. Quantitative analysis of insulin's effect on surface expression, with mean densities of surface bands normalized to control values for samples run on the same gel (n = 4, P < .01). c, Immunohistochemistry performed TRPV1 transfected HEK cells that were exposed to IGF-I (20 nM, 15 min), insulin (10 μM, 15 min) and PKC agonist, PDBu (10 μM, 15 min). Quantification of relative optical intensities (ROI, normalized as surface/cytosol for each cell): (control: n = 5; IGF-I: n = 7, P < .05; insulin: n = 3 P < .01; and PDBu: n = 7, P < .01). d, Confocal image showing a significant increase (3.17 ± 0.52 fold, n = 9; P < .01) in fluorescence intensity at the membrane 5 min after exposure to IGF-1 (50 nM) in HEK cells heterologously expressing GFP-TRPV1 fusion protein as compared to before IGF-1 application.

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