Sensitization and translocation of TRPV1 by insulin and IGF-I

Oocytes were isolated from tricaine-anesthetized Xenopus laevis and separated from the follicular layer after incubation with collagenase (1–2 mg/ml). 3–7 days following cRNA injections (50–70 nl, 1 μg/μl), oocytes were stored at 18°C and used for experiments. Double electrode voltage clamp (-60 mV) was performed using a Warner amplifier (Warner Instruments, OC725C, Hamden, CT, USA) with 100% d.c. gain, digitized and stored on video tape. Experiments were performed at 21–23°C. Oocytes were placed in a Perspex chamber superfused (5–10 ml min^-1) with Ca²⁺-free Ringer solution containing (in mM): 100 NaCl, 2.5 KCl, 5 HEPES, pH 7.35. Within figures, current traces were shown as initial response to agonist juxtaposed on currents recorded following incubation with insulin, IGF-I and various inhibitors. Time course experiments consisted of current recording during capsaicin application in the presence of either control (first 20 min) or insulin (last 15 min) containing solutions. In all experiments, fold increase was calculated as agonist induced current amplitude in the presence of trophic factors and inhibitors divided by the agonist induced initial current amplitude under control conditions before their bath application. I-V relationships were measured using 500-ms voltage ramps from -80 mV to +80 mV.

Dorsal root ganglia were isolated from embryonic day 18 (E18) rats, triturated and cultured for 5–7 d in Neurobasal/B-27 (Life Technologies; Grand Island, NY) + 10% fetal bovine serum (FBS) on poly-D-lysine-coated glass coverslips. For perforated patch recording, the bath solution contained (in mM): 140 NaGlu, 2.5 KCl, 10 HEPES, 2 MgCl₂, 1 EGTA, pH 7.35 and the pipette solution contained (in mM): 130 NaGlu, 10 NaCl, 2.5 KCl, 10 HEPES, 1 MgCl₂, 0.2 EGTA, and amphotericin B (240 μg/ml). For cell-attached recording, the bath solution contained (in mM): 140 Kglu, 10 NaCl, 10 HEPES, 1 EGTA, 2 MgCl₂, pH 7.35 and the pipette contained (in mM): 140 NaGlu, 2.5 KCl, 10 HEPES, 2 MgCl₂, pH 7.35. Currents were recorded using a WPC 100 patch clamp amplifier (E. S. F. Electronic, Goettingan, Germany) or Axopatch 200B (Axon Instruments, Union City, CA). Data were digitized (VR-10B, Instrutech Corp.; Great Neck, NY) and stored on video tape.

For analysis, data were filtered at 2.5 kHz (-3db frequency with an 8-pole low-pass Bessel filter, Warner Instruments, LPF-8) and digitized at 5 kHz. Data analyses were performed on continuous stretches greater than 20 s from patches that contained one or two channels. Single channel current amplitude and P_o were calculated from all point amplitude histograms fitted with Gaussian functions (Microcal Origin; Northampton, MA).

Cells were grown on glass coverslips, then incubated with 5 μM Fluo-4AM (Molecular Probes; Eugene, OR) for 20 min at 37°C, washed with physiological buffer [(in mM): 140 NaCl, 5 HEPES, 2 CaCl₂, 1 MgCl₂, 2.5 KCl, pH 7.35], and treated with 500 nM capsaicin +/- 1 μM insulin immediately prior to analysis by confocal microscopy (Fluoview, Olympus; Mellville, NY). Data for each cell was quantified as the fluorescence after treatment (F) divided by initial fluorescence (F_o) at t = 0 (Fluoview software).

HEK 293 cells, which endogenously express IGF-I receptors [29] were cultured in Dulbecco's modified Eagle's medium (high glucose) supplemented with 10% fetal bovine serum, 50 units/ml penicillin, and 50 μg/ml streptomycin (Invitrogen; Carlsbad, CA) and maintained under 95% air/ 5% CO₂ at 37°C. Cells were transiently transfected with wild type TRPV1, TRPV1eGFP, TRPV1 S502/S800A mutant (gifted by D. Julius, N. Kadei, M. Tominaga, respectively), or TRPV1-V5-His tagged plasmid using Lipofectamine2000 reagent (Invitrogen) according to manufacturer's protocol. TRPV1-V5-His tagged plasmid was constructed in pcDNA3.1 vector using Topo Cloning kit (Invitrogen). To determine the membrane translocation of TRPV1, confocal images (1 μm sections) of GFP-tagged TRPV1 fluorescence was obtained. The intensity of the brightest membrane fluorescence was selected and quantified using MCID imaging software (Imaging Research Inc; St. Catherines, ONT)

After 36 h of TRPV1 transfection, the cells (grown on 100 mm tissue culture plate) were insulin-treated and biotinylated with membrane impermeable NHS-LC-biotin (1.5 mg/ml in PBS; Pierce, Rockford, IL) as per manufacturer's protocol. Labeled cells were lysed in RIPA buffer and immunoprecipitated overnight with 10 μl of goat anti-VR1 polyclonal antibody/ Protein A/G Plus-agarose (Santa Cruz Biotechnology, Santa Cruz, CA) following manufacturer's protocol. Immunoprecipitates were eluted with 2X SDS sample buffer, separated on 7.5% SDS-PAGE, and transferred to PVDF membrane. To detect total TRPV1, blots were probed with rabbit anti-VR1 polyclonal antibody (1:1000; Affinity Bioreagents, Golden, CO). Specific antibody binding was detected using HRP-conjugated anti-rabbit secondary antibody (1:20,000; Jackson Immunoresearch, West Grove, PA) and Super Signal reagent (Pierce). To detect surface TRPV1, blots were stripped and probed with neutravidin-HRP (1:20,000; Pierce). Chemiluminescence was captured in Hitachi CCD Bio Genetic Systems after exposing the blot to Super Signal reagent (Pierce). Data were quantitated using LabWorks analysis software (UVP Inc., Upland, CA) and surface TRPV1 was normalized using total TRPV1. TRPV1-V5-His transfected cells were biotinylated as above and purified using Ni-NTA agarose (Qiagen, Valencia, CA) as per manufacturer's protocol.

Cells were plated on glass coverslips coated with 40 μg ml^-1 poly-L-lysine, treated with insulin/IGF/PDBu after 36 h of transfection, and fixed with 4% paraformaldehyde for 1 h. The cells were probed with rabbit anti-VR1 antibody (1:10,000; Affinity Bioreagents) followed by rhodamine red-X conjugated anti-rabbit secondary antibody (1:50; Jackson Immuno Research) following method described by Santa Cruz Biotechnology. Confocal images (1 μm sections) were captured using 570 nm laser. The brightest membrane fluorescence was selected from control and treated cells and quantified using MCID imaging software (Imaging Research Inc.).

First, we tested whether insulin and/or IGFs influence TRPV1-mediated membrane currents. The effect of insulin and IGF-I on cloned TRPV1 was characterized through dual-electrode voltage clamp (V_m = -60 mV) of Xenopus oocytes injected with TRPV1 cRNA. Insulin (1 μM) significantly potentiated the response to capsaicin (500 nM) greater than 2 fold (Fig. 1a). The time course of insulin-induced potentiation of capsaicin response shows a significant increase in current amplitude for 5, 10 and 15 minute incubation periods compared to initial levels (Fig. 1b). Dose response curves of capsaicin show that both the sensitivity (EC₅₀ shifted from 0.9 to 0.6 μM) and the maximal response (normalized to 1 μM capsaicin induced current before insulin application) increased (from 1.8 to 2.4) after incubation of oocytes with insulin (circles) for 5 min (Fig. 1c). A representative current-voltage relationship (I-V curve) confirms the outward rectification characteristic of TRPV1 current and shows that, in the presence of insulin, amplitude is increased at both positive and negative potentials (Fig. 1d). In addition, current potentiation was also seen with heat evoked responses (Fig. 1e) and protons (Fig. 1f), an endogenous TRPV1 agonist. These experiments show that insulin is increasing TRPV1-mediated currents and making the receptor more responsive to exogenous and endogenous activators.

Oocytes endogenously express insulin-like growth factor receptors (IGFR) rather than insulin receptors (IR) [30]. Insulin binds to IGFR with 100–1000 times lower affinity than IR. On the other hand, IGF-I binds to IGFR with much higher affinity, so we tested whether lower concentrations of IGF-I could potentiate TRPV1. Indeed, 20 nM IGF-I potentiated pH currents to a similar degree as 1 μM insulin (Fig. 1g). Potentiation was dramatically reduced by the addition of tyrphostin A47 (100 μM; 10 min), a selective inhibitor for the insulin family of receptor tyrosine kinases (Fig. 1h). Together these data suggest that in oocytes, insulin and IGF-I are acting through IGFR to potentiate TRPV1 (Fig. 1i).

Since insulin and IGF-I functions vary with cell type, it was important to establish that current potentiation also occurred in sensory neurons that express native TRPV1 [15, 21]. Therefore, we determined the effect of insulin and IGF-I on capsaicin-induced TRPV1 responses using cultured DRG neurons. For whole cell recordings, the perforated patch technique was used to prevent desensitization and tachyphylaxis, minimize intracellular disruption, and maintain intact signaling cascades. In this setting, insulin (1 μM) induced a potentiation of the capsaicin (100 nM) response (Fig. 2a), which returned to control levels 20 minutes after its removal (Fig. 2b, inset). Like oocytes, similar results were seen with IGF-I, signifying that insulin was binding IGFR in DRG neurons (Fig. 2c).

To confirm electrophysiological experiments, measurements of Ca²⁺-uptake were performed using confocal microscopy (Fig. 2d). In DRG neurons, capsaicin (500 nM) produced a Ca²⁺ influx that was increased in the presence of insulin. This shows that insulin elevated TRPV1-regulated Ca²⁺ mobilization. Together this data, confirm that the potentiation seen with cloned TRPV1 in oocytes is present in native TRPV1 expressing peripheral sensory neurons as well.

To understand the underlying molecular mechanism responsible for whole-cell potentiation, single channel currents were recorded in cell-attached patches from DRG neurons. Under this configuration, extracellular insulin would require receptor mediated signal transduction to facilitate changes in TRPV1 channel function recorded within the patch area (Fig. 2e). Channel open probability (P_o), a measure of the time the channel spends in the open state, induced by capsaicin (10 nM) increased following bath-application of insulin (1 μM), outside the patch area (Fig. 2e, lower graphs). P_o significantly changed from 0.15 ± 0.05 in control conditions to 0.43 ± 0.14 after insulin (Fig. 2f), without altering the single channel amplitude (Fig. 2g). Since insulin application was outside the patch, these data support intracellular signaling, as opposed to direct binding to TRPV1 as a mechanism for insulin-mediated potentiation.

IR and IGFR produce their effects via an overlapping set of downstream enzymes [29], so we sought to identify signaling pathways involved in TRPV1 modulation. First, receptor tyrosine kinase (RTK) involvement was assessed by pretreating oocytes with membrane permeable inhibitors before and during insulin application (Fig. 3a). The nonspecific RTK blockers genistein (50 μM, 60 min) and lavendustin A (100 μM, 60 min) significantly reduced IGF-I (20 nM, 10 min) potentiation. Second, wortmannin (100 nM, 15 min), a specific phosphatidylinositol 3-kinase (PI(3)K) inhibitor, reduced potentiation as well. Thus, we reasoned that insulin and IGF-I utilize receptor tyrosine kinases to activate PI(3)K and prompt a signaling cascade that leads to TRPV1 current potentiation.

PI(3)K mediates some of its effects through various isoforms of protein kinase C (PKC) [29]. Therefore, we tested in oocytes expressing TRPV1 whether insulin and IGF-I could activate PKC, an enzyme known to potentiate TRPV1 through channel phosphorylation [8, 31]. Bisindoylmaleimide (BIM; 200 nM, 60 min), a nonspecific PKC inhibitor, significantly decreased IGF-I potentiation (Fig. 3a). The importance of PKC was further demonstrated using a mutant TRPV1 (S502A/S800A), which lacks residues acting as substrates for PKC phosphorylation (31) (Fig. 3b, upper trace). Two consequences of these mutations were apparent. First, insulin/IGF-I potentiation was abolished. Second, current amplitude was smaller in oocytes injected with mutant TRPV1 compared to wild type, suggesting that phosphorylation might have an intrinsic effect on basal channel function. Thus, it appears that RTK, PI(3)K and PKC activation are required for current potentiation by insulin.

In a number of systems, insulin and/or IGF-I can increase surface content of effector molecules [32‐34]. We have used five different approaches to examine whether TRPV1 translocation occurs in response to insulin or IGF-I. First, we used cytochalasin D (1 μM, 60 min) to inhibit actin polymerization and decrease vesicular fusion to the plasma membrane in oocytes (Fig. 3b, lower traces). Cytochalasin D almost completely blocked TRPV1 current potentiation by insulin, implicating involvement of protein trafficking affecting translocation of both TRPV1 and PKC. Second, insulin and PDBu not only increased the potency but also the efficacy of capsaicin induced currents. In DRG neurons, at saturating concentrations of capsaicin (20 μM) (Fig. 4a _i ) the current amplitude increased (>50%) following exposure (2–5 min) to insulin (1 μM) (Fig. 4a _ii ) or PDBu (1 μM) (Fig. 4a _iii ) suggesting recruitment of new channels into the plasma membrane or activation of previously silent channels (see also Fig. 1c). Third, Western blot analysis of cell-surface biotinylated TRPV1 expressed in HEK cells was carried out to test whether TRPV1 itself was being translocated to the plasma membrane (Fig. 4b). Analysis of band densities indicates that insulin (10 μM, 15 min) doubled surface TRPV1 expression levels relative to controls. Fourth, relative surface to cytosol optical intensities were quantified by immunofluorescence microscopy (Fig. 4c). These results, obtained with antibodies specific for TRPV1, show that IGF-I (20 nM, 15 min), insulin (10 μM, 15 min) and PDBu (10 μM, 15 min), all significantly increased surface TRPV1 expressed in HEK cells (Fig. 4c). Fifth, similar results were seen in HEK cells transiently transfected with green fluorescence protein (GFP)-tagged TRPV1. Exposure of IGF-I (50 nM) significantly increased the fluorescence intensity of the membrane within five minutes, indicating the accumulation of TRPV1 on the membrane (Fig. 4d). Together, these data suggest the involvement of receptor translocation in TRPV1 current potentiation and show that, in the presence of insulin, TRPV1 is mobilized to the plasma membrane from the cytosol.