Insulin potentiates mechanical responses in small dorsal root ganglion neurons by increasing the sensitization of TRPV4 channels.

Abstract

We have previously reported that insulin potentiates the response to mechanical stimuli in small dorsal root ganglion (DRG) neurons. However, the mechanisms underlying the insulin-induced potentiated responsiveness to mechanical stimulation in sensory neurons remain unclear. Transient receptor potential vanilloid 4 (TRPV4) is expressed as a mechanosensitive channel in DRG neurons and is activated by mechanical stimuli. We therefore hypothesized that insulin augments the response to mechanical stimulation in small DRG neurons by enhancing sensitization of TRPV4 channels. Colocalization of TRPV4, insulin receptor (IR), and the C-fiber marker peripherin in small DRG neurons was evaluated by immunofluorescence, demonstrating that 53 ± 10% of TRPV4-positive small DRG neurons were colocalized with IR and peripherin. In in vitro whole cell patch clamp recordings from cultured DRG neurons, mechanically activated currents were significantly increased 5 min after the application of insulin (P = 0.0137) and such augmentation was suppressed by TRPV4 antagonist HC067047. We further examined the impact of insulin on the expression of the IR signaling pathway proteins in cultured DRG neurons using western blotting. Akt was significantly increased in cultured DRG neurons incubated with insulin (phospho-Akt: P = 0.0007, phospho/total Akt ratio: P = 0.0183). Furthermore, blocking IR signaling kinases, phosphoinositide 3-kinase (PI3K), and PKC suppressed the insulin-induced augmentation in TRPV4 agonist-induced currents (PI3K: P = 0.0074, PKC: P = 0.0028). Collectively, our results suggest that insulin-induced potentiation of mechanical response in small DRG neurons occurs through enhanced sensitization of TRPV4 channels.NEW & NOTEWORTHY We investigated whether insulin potentiates the sensitization of TRPV4 channels to mechanical stimulation in DRG neurons. Insulin-induced enhancement of mechanical response was suppressed by blocking TRPV4 channels. Furthermore, blockade of insulin receptor signaling pathways, PI3K and PKC, inhibited insulin-induced sensitization of TRPV4 channels. Our results provide evidence that insulin-induced potentiation of mechanical sensation in small DRG neurons is mediated through enhancing the sensitization of TRPV4 channels via the insulin receptor signaling pathway.