| Evidence for the presence of insulin and its signaling has been demonstrated in thecentral nervous system (CNS) and the peripheral nervous system (PNS). Although it hasbeen demonstrated that insulin receptor (IR) is abundant in the brain and spinal cord, thedistribution pattern is irregularly and distinctly. In rodents, IR is selectively distributed incerebral cortex, hippocampus and spinal cord. Moreover, rather than glia cells, themajority of the IRs locate on neurons, suggesting a direct role of insulin signaling inneuronal activity.Aberrant insulin signaling has been recognized in neuronal dysfunction, particularly in neurodegenerative disease, such as Alzheimer’s disease (AD), Parkinson’s disease (PD).However, compared with brain, the rate of insulin uptake demonstrates that spinal cordtakes up insulin more rapidly. However, there are fewer reports about the specific role ofinsulin signaling in spinal cord with attached dorsal root ganglion (DRG). Our previousreport indicate that in spinal dorsal horn, insulin signaling impairment is associated withpainful diabetic neuropathy in type2diabetes. Except for diabetes, otherpathophysiological conditions such as injury-induced pain also contribute to thedevelopment of impaired insulin sensitivity. Earlier observation in humans suggests thatacute pain leads to insulin resistance, indicating an underlying relationship between painand insulin signaling, but the mechanisms remain poorly understood.Based on these considerations, we sought to detect the changes of IR and insulinsignaling in pain.1Distribution of IR in the dorsal root ganglion and spinal cordTo visualize the distinct localization of IR in the spinal cord, we performed in situhybridization and immunohistochemistry.(1) To detect the distribution of IR in DRG and spinal cord, we performeddouble-labeled immunohistochemistry. Results showed that in DRG, the IR-positiveneurons were mainly found to be small-(diameter≤20μm) and medium-sized (20μm <diameter≤35μm)(Fig.1A). Moreover, IR-positive neurons colocalized with isolectin B4(IB4, the marker for nonpeptidergic unmyelinated fibers and their cell bodies) andcalcitonin gene related peptide (CGRP, the marker for peptidergic fibers and their cellbodies) in dorsal root ganglion (DRG).(2) In the lumbar spinal cord, IR-positive elements were densely distributed in spinaldorsal horn (Fig.1B). Our results showed that IR mRNA was widely expressed in both thegrey matter and the white matter of the lumbar spinal cord. However, the highest densityof IR mRNA signals was revealed in the superficial layer of spinal dorsal horn, laminae Iand II, where are considered as the fundamental area in nociceptive transmission in theCNS. Consistent with in situ hybridization, fluorescent immunostainings with IR antibody showed that the IR-positive cells were evident in the grey matter of the dorsal horn. Todefine the cell type of IR-positive cells, we conducted double-immunolabellings with celltype-specific markers (NeuN for neuron, GFAP for astrocyte), respectively. In thesuperficial layer of the spinal cord,93.9%of IR-positive products co-expressed withNeuN.2Down-regulation of insulin signaling is involved in painful diabetic neuropathy(1) Streptozotocin (STZ)-induced type1diabetic rats exhibited hyperglycemia, bodyweight loss and mechanical allodynia. In both DRG and spinal dorsal horn, IR-labeledelements decreased significantly. Moreover, NeuN-immunopositive decreased in diabeticrats. However, the activity of JAK2-STAT3pathway didn’t changed significantly.Treatment through intrathecal injection of insulin at the early stage of the painful diabeticneuropathy (PDN) could alleviate mechanical allodynia.(2) In type2diabetic animal model, the expressions of IR and phosphorylated insulinreceptor substrate-1(pIRS-1) decreased and the JAK2/STAT3pathway activated in thespinal dorsal horn in ob/ob mice with mechanical allodynia. Treatment through intrathecalinjection of AG490, an inhibitor of the JAK2/STAT3pathway, alleviated mechanicalallodynia in ob/ob mice and prevented impaired insulin signaling in spinal cord.3The underlying mechanism of insulin signaling in inflammatory pain(1) In DRG, the majority of TRPV1-postive cells were small-and medium-sizedneurons (Fig.1A), which is in accordance with previous reports. In DRG,85.6%ofTRPV1-positive neurons were colocalized with IRs. In the superficial layer of spinaldorsal horn, the TRPV1-positive terminals colocalized with IRs. The morphologicalevidence suggests that there might be an interaction between insulin signaling and TRPV1in DRG and spinal dorsal horn.(2) In complete Freund’s adjuvant (CFA)-induced inflammatory pain animals, theexpression of TRPV1increased in spinal dorsal horn with the development inflammatorypain. However, in the superficial layer of spinal dorsal horn, IR-positive elements decreased significantly. More importantly, the decreased IR expressions were mainlyderived from membrane rather than intracellular. The expression of insulin receptorsubstrate-1(IRS-1) decreased from6h to48h after CFA injection to the left hind paw.At24h after CFA injection, pIRS-1decreased significantly. Activity of the PI3K-Aktpathway decreased at24h after CFA injection as well. Intrathecal injection of insulincould attenuate reduced heat stimulated paw withdrawal latency and relieved thedown-regulation of insulin signaling and inhibited the up-regulation of TRPV1.(3) The results from immunohistochemistry indicate the majority of TRPV1-positiveneurons express IRs in DRG, so we used the calcium imaging to observe the response toTRPV1agonist capsaicin in the presence of standard bath solution with or without insulinin acute dissociated small DRG neurons. Perfusion of DRG neurons with500nMcapsaicin for30s elicited a marked calcium influx. After incubated DRG cells with bathsolution containing500nM capsaicin and1M insulin for1min30s,11.56%positivecells respond to capsaicin in first30s turned into negative, indicating that insulin couldeffectively decrease the intracellular calcium.(4) To know whether intrathecal insulin itself could affect nociceptive behaviors(flinching/licking), we examined the behaviors in mice after insulin application for25min.There was no significance between saline-treated and0.05IU insulin-treated groups in10min to25min, demonstrating that insulin itself could not induce nociceptive behaviors inmice.(5) We further examined whether insulin influences basal heat sensitivity. Intrathecalinjected with saline or0.05IU insulin for10min and25min respectively, and then hotplate test was performed on mice. Our results showed that compared with control,0.05IUinsulin pretreatment for10min significantly increased the latency time to52.5°C in mice.Similarly, extended the time for insulin pretreatment to25min also resulted in theprolonged latency time of the hot plate response in insulin group. These behavioral resultsshow that intrathecal injection of insulin produces analgesic effects in acute thermalnociception.(6) We further assessed the effect of insulin on capsaicin-induced nociceptive behaviors in mice. Intraplantar injection of capsaicin is known to elicit acute pain in mice,exhibiting marked spontaneous pain behaviors, such as flinching or licking the hind paw.Compared with intrathecal saline-treated mice,0.05IU insulin significantly reducednociceptive behaviors induced by the injection of capsaicin (1.6g/20l) into the hindpaw in10min. Then the ipsilateral injected hind paw was applied with same amount ofcapsaicin again and tested for another10min, the pain behaviors by the2ndintraplantarinjection of capsaicin were significantly decreased in insulin-treated animals.Conclusions:(1) IRs express in neurons in the DRG and spinal dorsal horn, which have beendemonstrated to be involved in nociceptive transmission.(2) Insulin signaling impairment in spinal dorsal horn is associated with theprogressive PDN in both type1and type2diabetes.(3) Our findings suggest a role of insulin signaling in inflammatory pain, whichmight be associated with TRPV1activity. |
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