Carbonylation of Adipose Proteins in Obesity and Insulin Resistance

Abstract

Obesity is a state of mild inflammation correlated with increased oxidative stress. In general, pro-oxidative conditions lead to production of reactive aldehydes such as trans-4-hydroxy-2-nonenal (4-HNE) and trans-4-oxo-2-nonenal implicated in the development of a variety of metabolic diseases. To investigate protein modification by 4-HNE as a consequence of obesity and its potential relationship to the development of insulin resistance, proteomics technologies were utilized to identify aldehydemodified proteins in adipose tissue. Adipose proteins from lean insulin-sensitive and obese insulin-resistant C57Bl/6J mice were incubated with biotin hydrazide and detected using horseradish peroxidase-conjugated streptavidin. High carbohydrate, high fat feeding of mice resulted in a ϳ2-3-fold increase in total adipose protein carbonylation. Consistent with an increase in oxidative stress in obesity, the abundance of glutathione S-transferase A4 (GSTA4), a key enzyme responsible for metabolizing 4-HNE, was decreased ϳ3-4-fold in adipose tissue of obese mice. To identify specific carbonylated proteins, biotin hydrazide-modified adipose proteins from obese mice were captured using avidin-Sepharose affinity chromatography, proteolytically digested, and subjected to LC-ESI MS/MS. Interestingly enzymes involved in cellular stress response, lipotoxicity, and insulin signaling such as glutathione S-transferase M1, peroxiredoxin 1, glutathione peroxidase 1, eukaryotic elongation factor 1␣-1 (eEF1␣1), and filamin A were identified. The adipocyte fatty acid-binding protein, a protein implicated in the regulation of insulin resistance, was found to be carbonylated in vivo with 4-HNE. In vitro modification of adipocyte fatty acid-binding protein with 4-HNE was mapped to Cys-117, occurred equivalently using either the R or S enantiomer of 4-HNE, and reduced the affinity of the protein for fatty acids ϳ10-fold. These results indicate that obesity is accompanied by an increase in the carbonylation of a number of adipose-regulatory proteins that may serve as a mechanistic link between increased oxidative stress and the development of insulin resistance. Molecular & Cellular Proteomics 6:624 -637, 2007. Reactive oxygen species (ROS)1 and reactive nitrogen species are generated as a result of normal metabolic processes in the cell, including the uncoupling of the electron transport chain in the mitochondria and the oxidation of excess NADPH by NADPH oxidase (1). Oxidative stress occurs due to increased pro-oxidative conditions or the decline of antioxidant systems and is highly correlated with a wide variety of inflammatory and metabolic disease states, including Alzheimer disease, macular degeneration, lung dysfunction, and obesity-linked insulin resistance (2-5). Although the precise role of oxidative stress in disease mechanisms is not completely understood, ROS are known to be highly reactive with proteins, DNA, carbohydrates, and lipids in the cell. The ROSinitiated peroxidation of polyunsaturated acyl chains of membrane phospholipid...

NAD(P)H:quinone oxidoreductase 1 activity reduces hypertrophy in 3T3-L1 adipocytes

“…Our results support data demonstrating a decrease in glutathione S-transferase A4 in adipocytes from obese mice and a decrease in methionine sulfoxide reductase activity in visceral adipose from obese animals [52,53]. The extent to which decreases in antioxidant proteins are occurring as a result of pathological processes in adipose tissue versus resulting from the natural differentiation of preadipocytes to adipocytes still needs more clarification.…”

Low Aerobic Capacity and High-Fat Diet Contribute to Oxidative Stress and IRS-1 Degradation in the Kidney

“…Measurement of reactive aldehydes was performed as previously described [28]. Briefly, kidney tissue samples were homogenized in coupling buffer (100 m M sodium acetate, 20 m M NaCl, 0.1 m M EDTA, pH 5.5) supplemented with protease inhibitors (Complete; Roche Diagnostics).…”

Oxidative Stress and Covalent Modification of Protein with Bioactive Aldehydes

“…Proteins susceptible to reactive carbonyl modification in rat liver homogenates from nitropropane-treated animals have also been identified using this method (56). In work investigating the possible linkage between obesity and oxidative protein damage, such technology was utilized to identify aldehydemodified proteins from adipose tissue of lean insulin-sensitive and obese insulin-resistant C57BL/6J mice (24). Adipocyte FABP, a protein implicated in the regulation of insulin resistance, was found to be a target of 4-HNE modification in vivo.…”

“…The inactivation of several membrane transporters, including the Na ϩ -K ϩ -ATPase and glucose (GLUT3) transporters, by lipid-derived aldehydes in the brain has been linked to neurodegenerative disorders (27,28). The modification of the adipocyte FABP (as well as the epithelial isoform) by 4-HNE occurs on a conserved cysteine residue (Cys 117 ), decreases the protein's affinity for fatty acids, and may contribute to obesity-linked insulin resistance (24,29). Cytosolic and mitochondrial NADP ϩ -dependent isocitrate dehydrogenase isoforms are inactivated by lipid peroxidation products, which may lead to the dysregulation of NADPH levels (30).…”

“…Finally, cytoplasmic FABP can scavenge both the R-and S-stereoisomers via covalent protein adduction (24).…”