The transcriptional response to oxidative stress is part of, but not sufficient for, insulin resistance in adipocytes

Abstract

Insulin resistance is a major risk factor for metabolic diseases such as Type 2 diabetes. Although the underlying mechanisms of insulin resistance remain elusive, oxidative stress is a unifying driver by which numerous extrinsic signals and cellular stresses trigger insulin resistance. Consequently, we sought to understand the cellular response to oxidative stress and its role in insulin resistance. Using cultured 3T3-L1 adipocytes, we established a model of physiologically-derived oxidative stress by inhibiting the cycling of glutathione and thioredoxin, which induced insulin resistance as measured by impaired insulin-stimulated 2-deoxyglucose uptake. Using time-resolved transcriptomics, we found > 2000 genes differentially-expressed over 24 hours, with specific metabolic and signalling pathways enriched at different times. We explored this coordination using a knowledge-based hierarchical-clustering approach to generate a temporal transcriptional cascade and identify key transcription factors responding to oxidative stress. This response shared many similarities with changes observed in distinct insulin resistance models. However, an anti-oxidant reversed insulin resistance phenotypically but not transcriptionally, implying that the transcriptional response to oxidative stress is insufficient for insulin resistance. This suggests that the primary site by which oxidative stress impairs insulin action occurs post-transcriptionally, warranting a multi-level 'trans-omic' approach when studying time-resolved responses to cellular perturbations.Insulin resistance is a major risk factor for various metabolic diseases, such as type 2 diabetes, cardiovascular disease, and some cancers. Although its underlying mechanisms are elusive, insulin-responsive tissues such as adipose tissue undergo oxidative stress during insulin resistance 1,2 . Indeed, we have previously shown that oxidative stress unifies numerous triggers of insulin resistance in adipocytes and myotubes 3 . These include hyperinsulinaemia, inflammation, and glucocorticoids in vitro, as well as nutrient oversupply in vivo 3 . Thus, oxidative stress is an etiological component of insulin resistance 4,5 , yet how it impairs insulin action remains elusive.Oxidative stress arises from the aberrant production or defective scavenging of reactive oxygen or nitrogen species. These species can react with a range of macromolecules -in particular, they can oxidise exposed cysteine residues within proteins 6 , altering signalling and cellular physiology. To protect the cell from oxidative stress, the cell has two major redox buffering pools, governed by glutathione and thioredoxin. Although both thiol antioxidants, they are not redundant, serving to regulate distinct cellular signalling and metabolic pathways 7 . Inhibiting

Oxidant-Induced Alterations in the Adipocyte Transcriptome: Role of the Na,K-ATPase Oxidant Amplification Loop

“…The major pathways that were altered in HFD fed mice were integrin signaling, nuclear factor erythroid 2 – related factor 2 (NRF2)-mediated oxidative stress response, B cell receptor signaling, IL8 signaling, PI3K/AKT signaling, mTOR signaling, nerve growth factor (NGF) signaling, platelet derived growth factor (PDGF) signaling and peroxisome proliferator-activated receptor (PPAR) signaling. The early and late obesity-induced inflammatory gene profile of adipose tissue in HFD were also reported recently [ 26 ]. The major pathways found to have significant alterations were ECM receptor interaction, focal adhesion pathways, pathways linked to inflammation, including NF-κβ and TNFα signaling, chemokine signaling pathways and leukocyte trans endothelial migration pathways.…”

“…In particular, marked changes in PPARγ signaling were noted in both in vitro and in vivo models as has been noted by Kim and coworkers [ 42 ]. Recent reports have demonstrated altered gene regulation of mediators involved in PPAR signaling in 3T3-L1 cells under oxidative stress conditions [ 26 , 43 , 44 ]. Similarly, large scale dysregulation of genes in response to oxidative stress in all three models were noted in pathways related to lipid metabolism, mitochondrial biogenesis and anti-oxidant defense as well as the JAK-STAT, TGFβ and cytokine signaling pathways.…”

“…Advancement in transcriptomics provides a remarkable opportunity to study functional implications of the genetic variability in adipose tissue under different pathological conditions. Oxidative stress triggers a coordinated transcriptional response in adipocytes that in turn regulate various molecular pathways associated with adipocyte function, inflammation and oxidative stress [ 26 , 27 ]. The trans-omic network analysis in adipocytes exposed to oxidative stress provides insight into the molecular level interactions, and thereby interpretation of the associated biochemical events and their regulation.…”

“…The major inflammatory genes like MCP-1, TNF-α, IL-6, IL-8 and IL-1B were found to be dysregulated under inflammatory condition in adipocytes. The cellular response to oxidative stress and its role in insulin resistance have been previously studied in 3T3-L1 adipocytes which demonstrate the role of major pathways including MAPK signaling, insulin signaling, TGF-β signaling, JAK/STAT signaling, adipocytokine signaling, Type II diabetes mellitus, cytokine receptor interaction and p53 signaling with significant alteration in genes [ 26 , 33 ]. Prior studies have demonstrated that under chronic renal failure, the retention of uremic toxins stimulate a systemic oxidative stress and inflammatory response in vivo [ 13 , 22 , 34 ].…”

Mitochondrial oxidants, but not respiration, are sensitive to glucose in adipocytes

“…Specifically, as mitoROS outweighs endogenous scavenging systems, the mitoROS likely reacts with a macromolecule required in the initial stages of insulin-stimulated glucose transport. Furthermore, since oxidative stress does not require long-term adaptive changes such as transcriptional modification to cause insulin resistance in adipocytes (29), it is likely to cause rapid changes in kinase signalling and/or GLUT4 trafficking. However, mitoROS did not affect Akt signalling (Fig 3), concurring with previous studies (11).…”