Acute augmentation of epoxygenated fatty acid levels rapidly reduces pain-related behavior in a rat model of type I diabetes

Abstract

The nerve damage occurring as a consequence of glucose toxicity in diabetes leads to neuropathic pain, among other problems. This pain dramatically reduces the quality of life in afflicted patients. The progressive damage to the peripheral nervous system is irreversible although strict control of hyperglycemia may prevent further damage. Current treatments include tricyclic antidepressants, anticonvulsants, and opioids, depending on the severity of the pain state. However, available therapeutics have drawbacks, arguing for the need to better understand the pathophysiology of neuropathic pain and develop novel treatments. Here we demonstrate that stabilization of a class of bioactive lipids, epoxygenated fatty acids (EpFAs), greatly reduces allodynia in rats caused by streptozocin-induced type I diabetes. Inhibitors of the soluble epoxide hydrolase (sEHI) elevated and stabilized the levels of plasma and spinal EpFAs, respectively, and generated dose-dependent antiallodynic effects more potently and efficaciously than gabapentin. In acute experiments, positive modulation of EpFAs did not display differences in insulin sensitivity, glucose tolerance, or insulin secretion, indicating the efficacy of sEHIs are not related to the glycemic status. Quantitative metabolomic analysis of a panel of 26 bioactive lipids demonstrated that sEHI-mediated antiallodynic effects coincided with a selective elevation of the levels of EpFAs in the plasma, and a decrease in degradation products coincided with the dihydroxy fatty acids in the spinal cord. Overall, these results argue that further efforts in understanding the spectrum of effects of EpFAs will yield novel opportunities in treating neuropathic pain.

Fig. 1.

Stabilization of EpFAs is antinociceptive in the type I diabetes-induced neuropathic pain model. (A) The sEHI, AUDA (n = 6 per group, 10 mg/kg s.c.), reduced mechanical allodynia and mechanical hyperalgesia but not the transient heat hyperalgesia elicited by STZ-induced type I diabetes (paired t test *P = 0.04, **P = 0.002). Heat hyperalgesia quantified by Hargreaves’ assay, allodynia by the von Frey assay, and mechanical hyperalgesia by the Randall–Selitto assay. Data are reported as grams of force or time in seconds required to elicit a withdrawal. In all subsequent panels and figures, data are reported as “percent of baseline” threshold. (B) Dose and time dependency of effect of AUDA (n = 6 per group, one-way ANOVA followed by Student Newman–Keuls test: ‡, P = 0.001, ♠, P = 0.03 vs. after STZ baseline, subcutaneous administration). (C) Dose- and time-dependent efficacy of a structurally different sEHI, TPAU compared with vehicle control (PEG400, subcutaneous administration). (D) Chemical structures of different classes of small molecule inhibitors of sEH used in this study. Although these molecules share the same urea pharmacophore, each probe has different chemical properties and in vitro potency values (IC₅₀) on recombinant rat sEH; 11, 79, and 9 nM for AUDA, TPAU, and TUPS, respectively. Potencies on rat sEH are determined using the fluorescent substrate, CMNPC, α-cyanocarbonate. All data in this and following figures are presented as mean ± SE of mean.

Fig. 2.

Positive modulation of EpFAs is highly efficacious against diabetic neuropathic pain without sensorimotor impairment. (A) Mean antiallodynic effect of TUPS on diabetic rats over the course of 4 d. TUPS (10 mg/kg i.p.) at a 10-fold lower dose is comparable to Gabapentin (100 mg/kg i.p.), which requires repeated administration to maintain pain relief (n = 6 per group, one-way ANOVA followed by Student Newman–Keuls test, drugs vs. vehicle P < 0.001 for all pair-wise comparisons). TUPS remained efficacious across all time points. Error bars in some groups are smaller than the point shown. (B) Inhibition of sEH does not affect the time rats maintain position on a constant-speed rotating rod (TUPS, 10 mg/kg i.p., n = 5, paired t test vs. baseline, P = 0.76). Diazepam was used as a positive control (5 mg/kg i.p., n = 6, paired t test vs. baseline, *P < 0.05). Compounds were administered 60 min before testing and a cutoff time of 180 s was imposed.

Fig. 3.

Inhibition of sEH modulates levels of EpFAs and dihydroxy fatty acids in plasma and spinal cord tissue. (A) Type I diabetes does not change the plasma levels of substrates and products of sEH (pre-STZ n = 6, post-STZ n = 18 and post-STZ+TPAU n = 6, one-way ANOVA, P = 0.1 for LA and AA metabolites, P = 0.07 for EPA metabolites and P = 0.09 for DHA metabolites). TPAU significantly alters the mean ratio of summed epoxy to dihydroxy fatty acids in diabetic rats, with the exception of EPA metabolites (Kruskal–Wallis one-way ANOVA followed by Dunn’s all pair-wise comparison, *P < 0.02). Table S1 displays the identity and detected quantity of the metabolites analyzed from plasma. (B) Plasma levels of two major prostanoids show no significant change between groups (one-way ANOVA, P = 0.62). (C) Inhibition of sEH leads to selective changes in the levels of spinal EpFAs and their degradation products, dihydroxy fatty acids expressed as ratios (pre-STZ n = 3, post-STZ n = 4, and post-STZ+TUPS n = 6, one-way ANOVA followed by Student Newman–Keuls test; ◆, P = 0.03 for post-STZ vs. post-STZ+TUPS for LA metabolites and *, P = 0.02 for DHA metabolites). (D) Spinal prostanoids are elevated by diabetes but not reduced by inhibition of sEH (one-way ANOVA followed by Student Newman–Keuls test, ◆, P = 0.03 for vehicle vs. post-STZ PGE₂ and *, P = 0.006 for vehicle vs. post-STZ PGD₂).