Role of Fyn-mediated NMDA receptor function in prediabetic neuropathy in mice

Abstract

Diabetic neuropathy is a common complication of diabetes. This study evaluated the role of Fyn kinase and N-methyl-d-aspartate receptors (NMDARs) in the spinal cord in diabetic neuropathy using an animal model of high-fat diet-induced prediabetes. We found that prediabetic wild-type mice exhibited tactile allodynia and thermal hypoalgesia after a 16-wk high-fat diet, relative to normal diet-fed wild-type mice. Furthermore, prediabetic wild-type mice exhibited increased tactile allodynia and thermal hypoalgesia at 24 wk relative to 16 wk. Such phenomena were correlated with increased expression and activation of NR2B subunit of NMDARs, as well as Fyn-NR2B interaction in the spinal cord. Fyn(-/-) mice developed prediabetes after 16-wk high-fat diet treatment and exhibited thermal hypoalgesia, without showing tactile allodynia or altered expression and activation of NR2B subunit, relative to normal diet-fed Fyn(-/-) mice. Finally, intrathecal administrations of Ro 25-6981 (selective NR2B subunit-containing NMDAR antagonist) dose-dependently alleviated tactile allodynia, but not thermal hypoalgesia, at 16 and 24 wk in prediabetic wild-type mice. Our results suggested that Fyn-mediated NR2B signaling plays a critical role in regulation of prediabetic neuropathy and that the increased expression/function of NR2B subunit-containing NMDARs may contribute to the progression of neuropathy in type 2 diabetes.

Keywords: Fyn kinase; NMDA; neuropathic pain; prediabetes; spinal cord.

Fig. 1.

Effects of high-fat diet treatment on prediabetic neuropathy in wild-type mice. Prediabetic mice exhibited tactile allodynia (A) and thermal hypoalgesia (B) (n = 10/group). Mice were fed with normal diet or high-fat diet for 16 wk and were behaviorally tested for tactile responses to flexible von Frey filaments (first day) and for thermal algesia by paw withdrawal test (second day) at 16 wk or 24 wk. *Significant difference relative to normal diet control (P < 0.05); ^#significant difference relative to 16 wk (P < 0.05).

Fig. 2.

Effects of high-fat diet treatment on Fyn-mediated NR2B activation (n = 10/group). To explore the putative mechanisms underlying the effects of high-fat diet treatment on prediabetic neuropathy, spinal cord tissues were collected from each mouse at 16 wk or 24 wk in wild-type mice. Western blot analysis was performed on total NR2B levels (A), pNR2B-to-NR2B ratio (B), and Fyn kinase levels (C). D: to study physical interaction between Fyn kinase and NR2B, coimmunoprecipitation (Co-IP) assays were conducted by immunoprecipitation of the spinal cord samples with mouse monoclonal anti-Fyn antibody, followed by probing the blots with the antibody against NR2B (WB). *Significant difference relative to normal diet control (P < 0.05); ^#significant difference relative to 16 wk (P < 0.05).

Fig. 3.

Effects of high-fat diet treatment on prediabetic neuropathy in Fyn^−/− mice (n = 10/group). Prediabetic Fyn^−/− mice did not exhibit tactile allodynia (A) but showed thermal hypoalgesia (B). Mice were fed with normal diet or high-fat diet for 16 wk and were behaviorally tested for tactile responses to flexible von Frey filaments (first day) and for thermal algesia by paw withdrawal test (second day) at 16 wk or 24 wk. *Significant difference relative to normal diet control (P < 0.05); ^#significant difference relative to 16 wk (P < 0.05).

Fig. 4.

Effects of high-fat diet treatment on expression and activation of NR2B in prediabetic Fyn^−/− mice (n = 10/group). Western blot analysis was performed on Fyn kinase levels (A), total NR2B levels (B), and pNR2B-to-NR2B ratio (C). *Significant difference relative to normal diet control (P < 0.05); ^#significant difference relative to 16 wk (P < 0.05).

Fig. 5.

Comparison of effects of high-fat diet treatment on expression and activation of NR2B subunit in the spinal cord of wild-type and Fyn^−/− mice. Western blot analysis was performed on total NR2B levels (A) and pNR2B-to-NR2B ratio (B). *Significant difference relative to normal diet control (P < 0.05); ^#significant difference relative to 16 wk (P < 0.05).

Fig. 6.

Effects of intrathecal administration of Ro 25-6981 on prediabetic neuropathy in wild-type mice. Intrathecal administrations of Ro 25-6981 were conducted through the intervertebral space in unanesthetized mice between L₅ and L₆ of the spinal cord. Mice were behaviorally tested for tactile responses to flexible von Frey filaments (first day) (A) and for thermal algesia by paw withdrawal test (second day) (B) at 16 wk or 24 wk. *Significant difference relative to vehicle (P < 0.05); ^#significant difference relative to 16 wk (P < 0.05).