Downregulation of Adipose Glutathione S-Transferase A4 Leads to Increased Protein Carbonylation, Oxidative Stress, and Mitochondrial Dysfunction

Abstract

OBJECTIVEPeripheral insulin resistance is linked to an increase in reactive oxygen species (ROS), leading in part to the production of reactive lipid aldehydes that modify the side chains of protein amino acids in a reaction termed protein carbonylation. The primary enzymatic method for lipid aldehyde detoxification is via glutathione S-transferase A4 (GSTA4) dependent glutathionylation. The objective of this study was to evaluate the expression of GSTA4 and the role(s) of protein carbonylation in adipocyte function.RESEARCH DESIGN AND METHODSGSTA4-silenced 3T3-L1 adipocytes and GSTA4-null mice were evaluated for metabolic processes, mitochondrial function, and reactive oxygen species production. GSTA4 expression in human obesity was evaluated using microarray analysis.RESULTSGSTA4 expression is selectively downregulated in adipose tissue of obese insulin-resistant C57BL/6J mice and in human obesity-linked insulin resistance. Tumor necrosis factor-α treatment of 3T3-L1 adipocytes decreased GSTA4 expression, and silencing GSTA4 mRNA in cultured adipocytes resulted in increased protein carbonylation, increased mitochondrial ROS, dysfunctional state 3 respiration, and altered glucose transport and lipolysis. Mitochondrial function in adipocytes of lean or obese GSTA4-null mice was significantly compromised compared with wild-type controls and was accompanied by an increase in superoxide anion.CONCLUSIONSThese results indicate that downregulation of GSTA4 in adipose tissue leads to increased protein carbonylation, ROS production, and mitochondrial dysfunction and may contribute to the development of insulin resistance and type 2 diabetes.

Protein Carbonylation and Adipocyte Mitochondrial Function

“…GSTA4 is a cytoplasmic enzyme that translocates to the mitochondrion under conditions of oxidative stress (26). Previous results have demonstrated that selective loss of GSTA4 protein results in increased total protein carbonylation and mitochondrial dysfunction, yet the mitochondrial targets of such modification have not been identified (11). To address the role of protein carbonylation in adipocyte mitochondrial dysfunction, we utilized a strategy based on loss or gain-of-function of GSTA4.…”

“…Loss of GSTA4 in cultured 3T3-L1 adipocytes or in adipose tissue from high fat-fed C57Bl/6J mice leads to elevated mitochondrial carbonylation, diminished respiration, and altered metabolic flux through the tricarboxylic acid cycle and ␤-oxidation pathways (11). In addition, obesity-induced insulin resistance in humans positively correlates with protein carbonylation, indicating that increased oxidative stress leads to increased levels of carbonylated proteins (27).…”

“…Silencing GSTA4 in 3T3-L1 adipocytes leads to loss of ADPcoupled respiration in isolated mitochondria (11). However, because these cells have compensatory changes in substrate level phosphorylation and fatty acid uptake, it was important to assess their in situ respiratory capacity.…”

“…GSTA4-silenced (Kd) and scrambled (Scr) control adipocytes were generated as described previously (11). The extent of differentiation was identical between cell lines as evaluated by assessing lipid accumulation and the expression of adipocyte marker proteins including the adipocyte fatty acid binding protein.…”

“…The down-regulation of GSTA4 is specific for white fat (visceral and subcutaneous depots) and does not occur in brown fat, liver, or muscle (11,12). Our previous studies indicated that the level of protein carbonylation is elevated in the obese adipocyte and leads to the disruption of a number of metabolic pathways including ␤-oxidation, electron transport, and the citric acid cycle.…”