Elsevier

Behavioural Brain Research

Volume 326, 30 May 2017, Pages 69-76
Behavioural Brain Research

Soluble epoxide hydrolase inhibition alleviates neuropathy in Akita (Ins2 Akita) mice

https://doi.org/10.1016/j.bbr.2017.02.048Get rights and content

Highlights

  • Inhibiting the soluble epoxide hydrolase effectively blocks diabetic neuropathic pain.

  • SEH activity correlates with the disease pathogenesis in the Akita model.

  • Inhibiting sEH has a potential role in mitigating the progression of this pathology.

Abstract

The soluble epoxide hydrolase (sEH) is a regulatory enzyme responsible for the metabolism of bioactive lipid epoxides of both omega-6 and omega-3 long chain polyunsaturated fatty acids. These natural epoxides mediate cell signaling in several physiological functions including blocking inflammation, high blood pressure and both inflammatory and neuropathic pain. Inhibition of the sEH maintains the level of endogenous bioactive epoxy-fatty acids (EpFA) and allows them to exert their generally beneficial effects. The Akita (Ins2Akita or Ins2C96Y) mice represent a maturity-onset of diabetes of the young (MODY) model in lean, functionally unimpaired animals, with a sexually dimorphic disease phenotype. This allowed for a test of male and female mice in a battery of functional and nociceptive assays to probe the role of sEH in this system. The results demonstrate that inhibiting the sEH is analgesic in diabetic neuropathy and this occurs in a sexually dimorphic manner. Interestingly, sEH activity is also sexually dimorphic in the Akita model, and moreover correlates with disease status particularly in the hearts of male mice. In addition, in vivo levels of oxidized lipid metabolites also correlate with increased sEH expression and the pathogenesis of disease in this model. Thus, sEH is a target to effectively block diabetic neuropathic pain but also demonstrates a potential role in mitigating the progression of this disease.

Introduction

Diabetes is a growing worldwide epidemic and results in many comorbidities including hypertension, nephropathy, retinopathy and neuropathy [1]. Diabetic neuropathy has a profound negative impact on patients and available therapies have severe side effects and are often counter indicated for other comorbid conditions [2]. There is a great need for new therapeutics as well as relevant models to test them that have a potential for translation to man.

The Akita mouse model was discovered in the late 1990s and has been developed as a non-obese, maturity-onset of diabetes of the young (MODY) mouse model for Type I diabetes (Yoshioka 1997). It has important advantages compared to other models of diabetes used to study neuropathy, among other disorders. First, it is an autosomal dominant murine Ins2C96Y mutation with a progressive disease into adulthood which has a syntenic chromosomal conversion to human, and thus, is similar to the development of disease in humans. This obviates much of the argumentation around using streptozocin to chemically induce diabetes since streptozocin has been at times suggested to be neurotoxic [3]. Second, it is a non-obese model, and therefore is not subject to the complications of impaired motor function for behavioral tasks as observed with ob/ob or db/db mouse models. Third, it allows for an investigation of a dimorphic phenotype. The heterozygous Akita mice, particularly the males, progress to pernicious hyperglycemia and resulting secondary pathologies of diabetes providing a unique opportunity to study the physiopathologies of the disease and therapeutic interventions to alleviate them. Homozygous Ins2 Akita males are subject to high mortality at a young age [4] while the homozygous wildtype littermates are used as asymptomatic controls. Here we employ this mouse model to study the effects of enzyme inhibition on the developed diabetic neuropathy and probe differences in the sexually dimorphic expression of the phenotype to answer basic questions about the pathology in these animals.

The experiments here use the naturally occurring Akita mouse model to test the hypothesis that inhibiting the soluble epoxide hydrolase enzyme (sEH) mediates analgesia against the chronic pain of diabetic neuropathy. The sEH is a master regulatory enzyme in the arachidonic acid (ARA) cascade downstream of the cytochrome P450 enzymes that act on long chain polyunsaturated fatty acids (PUFA) as substrates. The sEH transforms epoxy-fatty acids (EpFA) into the vicinal diols of several classes of lipids including both omega-3 and 6 derived metabolites. The EpFA are stabilized in vivo by the inhibition of the sEH and have demonstrated anti-inflammatory, antihypertensive, and analgesic properties [5], [6], [7]. The EpFA have also demonstrated efficacy against chemically induced diabetic neuropathy [8]. Here we use the Akita mouse model to inquire if sEH inhibition is analgesic in this naturally progressing disease, and importantly, if there are sexual dimorphisms in the pain responses. We used the conditioned place preference assay to investigate if sEHI block neuropathic pain. We then tested both male and female Akita mice for sEH enzyme activity and examined the oxylipin profile in the same groups.

Section snippets

Animals

All procedures and animal care adhered the National Institutes of Health guide for the care and use of Laboratory animals (NIH Publications 8th Edition 2011) and were performed in accordance with the protocols approved by the Animal Use and Care Committee (IACUC) of the University of California, Davis. Great care was taken to minimize suffering of the animals and to reduce the number of animals used. Experiments on heterozygous Akita and wildtype littermate controls used mice bred from wild

Inhibiting sEH induces a CPP response in Akita mice with developed neuropathy

For all the behavioral tests the Akita mice were cohoused with their littermate controls for the duration of the experiments and randomly assigned to treatment or vehicle groups and tested intermingled for the studies. Only at sacrifice were the individual mice identified as Akita or littermate control and added to the appropriate groups. The Akita mouse model was used for these experiments because the Akita diabetic mice are lean, motor competent mice that can be assessed in these assays as a

Discussion

The sEH inhibitors (sEHI) are analgesic in several models of pain including inflammatory lipopolysaccharide and carrageenan induced pain which were some of the first models tested with sEHI [16], [17], [18]. Later the investigation broadened into testing neuropathic pain such as streptozocin induced diabetic neuropathy [7], [19]. However, there is debate whether this chemically induced model compares to the human disease [3]. Thus, we report here the naturally progressing Akita MODY model which

Conflict of interest

The University of California holds patents on the sEH inhibitors used in this study as well as their use to treat inflammation, inflammatory pain, and neuropathic pain. BD Hammock is a co-founder and K Wagner is an employee of EicOsis L.L.C., a startup company advancing sEH inhibitors into the clinic.

Acknowledgements

This work was supported by the National Institute of Environmental Health Sciences (NIEHS) Grant R01 ES002710, NIEHS Superfund Research Program P42 ES004699, National Institute of Neurological Disorders and Stroke (NINDS) U54 NS079202-01 and Grants NIEHST32ES007059, NIH 5T32DC008072-05 and 5T32HL086350-08 (to K.W.). This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors. A.V.G supported by a Hellman Fellowship. The content is

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