INOS as a Driver of Inflammation and Apoptosis in Mouse Skeletal Muscle after Burn Injury: Possible Involvement of Sirt1 S-Nitrosylation-Mediated Acetylation of p65 NF-κB and p53.

Harumasa Nakazawa,Masao Kaneki,Ronald G Tompkins,Kyungho Chang,Kazuhiro Ishimaru,Kentaro Shimokado,Shohei Shinozaki,Shingo Yasuhara,Takashi Yasukawa,J A Jeevendra Martyn,Yong-Ming Yu,Partha Mukhopadhyay

doi:10.1371/journal.pone.0170391

Abstract

Inflammation and apoptosis develop in skeletal muscle after major trauma, including burn injury, and play a pivotal role in insulin resistance and muscle wasting. We and others have shown that inducible nitric oxide synthase (iNOS), a major mediator of inflammation, plays an important role in stress (e.g., burn)-induced insulin resistance. However, it remains to be determined how iNOS induces insulin resistance. Moreover, the interrelation between inflammatory response and apoptosis is poorly understood, although they often develop simultaneously. Nuclear factor (NF)-κB and p53 are key regulators of inflammation and apoptosis, respectively. Sirt1 inhibits p65 NF-κB and p53 by deacetylating these transcription factors. Recently, we have shown that iNOS induces S-nitrosylation of Sirt1, which inactivates Sirt1 and thereby increases acetylation and activity of p65 NF-κB and p53 in various cell types, including skeletal muscle cells. Here, we show that iNOS enhances burn-induced inflammatory response and apoptotic change in mouse skeletal muscle along with S-nitrosylation of Sirt1. Burn injury induced robust expression of iNOS in skeletal muscle and gene disruption of iNOS significantly inhibited burn-induced increases in inflammatory gene expression and apoptotic change. In parallel, burn increased Sirt1 S-nitrosylation and acetylation and DNA-binding capacity of p65 NF-κB and p53, all of which were reversed or ameliorated by iNOS deficiency. These results indicate that iNOS functions not only as a downstream effector but also as an upstream enhancer of burn-induced inflammatory response, at least in part, by Sirt1 S-nitrosylation-dependent activation (acetylation) of p65 NF-κB. Our data suggest that Sirt1 S-nitrosylation may play a role in iNOS-mediated enhanced inflammatory response and apoptotic change, which, in turn, contribute to muscle wasting and supposedly to insulin resistance after burn injury.

Highlights

Metabolic dysfunction in skeletal muscle is a major complication after major trauma, including burn injury, and negatively affects the clinical outcome of patients with major trauma [1, 2]
We have previously shown that Inducible nitric oxide synthase (iNOS) deficiency inhibits burn-induced insulin resistance [13], but the role of iNOS in inflammation and apoptosis has not been studied in burn or major trauma
To investigate the role of iNOS in acetylation of p65 Nuclear factor (NF)-κB and p53, we studied the effects of iNOS deficiency at 3 days post-burn injury. iNOS expression was increased in wild-type mice, but not iNOS KO mice, at 3 days after burn injury (Fig 2A and 2B)

Summary

Introduction

Metabolic dysfunction in skeletal muscle is a major complication after major trauma, including burn injury, and negatively affects the clinical outcome of patients with major trauma [1, 2]. These metabolic alterations include insulin resistance, hyperglycemia, hypermetabolism, increased lactate production, catabolism and muscle wasting. Burn injury suppresses insulin-stimulated glucose uptake by skeletal muscle compared with sham-burn in wild-type mice at 3 days after burn, which parallels impaired insulin-stimulated phosphorylation of insulin signaling molecules, insulin receptor, insulin receptor substrate-1, Akt and glycogen synthase kinase-3β [13]. The maximum effects of burn injury on both iNOS expression and insulin resistance were observed at 3 days after burn injury in mice [13]. The precise mechanisms by which iNOS induces and/or exacerbates stress (e.g., burn)- and obesity-induced insulin resistance are not well understood

Methods

Results

Conclusion