In the setting of obesity, endoplasmic reticulum (ER) stress has been identified as a prominent feature in metabolic tissues in both animal models and in humans. To cope with ER stress, cells activate the unfolded protein response (UPR) to mitigate stress. However, failure of the UPR results in chronic unresolved stress, contributing to the development of obesity-induced insulin resistance. Nitric oxide (NO) is a key mediator of obesity-associated inflammation. We recently demonstrated that NO-mediated protein modification (S- nitrosylation) impairs the RNase activity of inositol-requiring enzyme-? (IRE1?), resulting in unresolved ER stress and insulin resistance. Although nitric oxide synthase (NOS) provides the general intracellular NO pools for protein S-nitrosylation, the rate of these modifications is also affected by the targeted removal of NO groups by protein denitrosylation. Our strong preliminary data demonstrate that obesity impairs the activity of S- nitrosoglutathione reductase (GSNOR, a major denitrosylase), leading to elevated nitrosative stress in the liver. Furthermore, deletion or overexpression of GSNOR directly regulates the S-nitrosylation state of IRE1? and ER function in mice with diet-induced obesity (DIO). Notably, liver-specific GSNOR overexpression ameliorated obesity-associated insulin resistance. However, the molecular mechanism that underlies the regulation of ER homeostasis by GSNOR-mediated denitrosylation signaling is unknown. We propose to test the central hypothesis that obesity attenuates GSNOR-dependent protein denitrosylation, resulting in elevated nitrosative stress in the ER that contributes to obesity-associated hepatic insulin resistance. To test this hypothesis, we will undertake 2 specific aims.
In Aim 1, we will: 1) determine whether liver-specific GSNOR deletion impacts hepatic insulin sensitivity and whole body glucose homeostasis using DIO mouse model; 2) establish whether GSNOR regulates hepatic insulin action directly via modulation of UPR; and 3) assess the therapeutic potential of enhancing hepatic GSNOR activity in obese mice by glutathione supplementation and enhancing cellular nicotinamide adenine dinucleotide (NAD) metabolism.
In Aim 2, we will: 1) profile the ER S-nitrosylation proteome and characterize the endogenous S-nitrosylation sites on IRE1?; 2) address how GSNOR-mediated denitrosylation signaling modulates the IRE1? RNase activity; and 3) establish how GSNOR-mediated denitrosylation signaling affects IRE1? interactome formation. The approaches used here are innovative, combining the use of cellular and molecular biological analysis, biochemical analysis for S-nitrosylated proteins, elucidating the ER S-nitrosylation proteome, protein-RNA interaction analysis, and in vivo mouse metabolic profiling. The mechanisms elucidated in this project will provide further understanding of how inflammatory and ER stress pathways are integrated in the context of obesity-associated hepatic insulin resistance, dyslipidemia and ultimately type 2 diabetes. Insight into the mechanisms by which maladaptive organelle stress responses drive these pathologies should speed development of novel therapeutic targets for metabolic diseases.
The proposed project is relevant to public health in that it is expected to reveal a novel molecular mechanism that underlies the association between inflammation and endoplasmic reticulum homeostasis in the context of obesity, and to thereby advance knowledge in the fields of obesity and diabetes. Thus, the proposed research is relevant to the NIH mission of developing fundamental knowledge that will help to reduce the burdens of human disability.
|Wang, Jie-Mei; Qiu, Yining; Yang, Zhao et al. (2018) IRE1? prevents hepatic steatosis by processing and promoting the degradation of select microRNAs. Sci Signal 11:|