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Carbonylation of Adipose Proteins in Obesity and Insulin Resistance

Identification of Adipocyte Fatty Acid-binding Protein as a Cellular Target of 4-Hydroxynonenal*
  • Paul A. Grimsrud
    Affiliations
    Department of Biochemistry, Molecular Biology and Biophysics, The University of Minnesota, Minneapolis, Minnesota 55455
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  • Matthew J. Picklo Sr.
    Affiliations
    Department of Pharmacology, Physiology, and Therapeutics, University of North Dakota School of Medicine and Health Science, Grand Forks, North Dakota 58203
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  • Timothy J. Griffin
    Affiliations
    Department of Biochemistry, Molecular Biology and Biophysics, The University of Minnesota, Minneapolis, Minnesota 55455
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  • David A. Bernlohr
    Correspondence
    To whom correspondence should be addressed: Dept. of Biochemistry, Molecular Biology and Biophysics, University of Minnesota-Twin Cities, 321 Church St. S E., Minneapolis, MN 55455. Tel.: 612-624-2712; Fax: 612-625-2163
    Affiliations
    Department of Biochemistry, Molecular Biology and Biophysics, The University of Minnesota, Minneapolis, Minnesota 55455
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  • Author Footnotes
    * This work was supported by National Institutes of Health Grant DK053189, the Minnesota Agricultural Experiment Station, and the Minnesota Obesity Center (to D A B.); by NHLBI, National Institutes of Health Grant T32 HL07741 (to P A G.); by National Institutes of Health Grant AG025371 (to T J G.); and by National Center for Research Resources Grant P20 RR17699-01 from the Center of Biomedical Research Excellence (to M J P.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
    The on-line version of this article (available at http://www.mcponline.org) contains supplemental material.
Open AccessPublished:January 06, 2007DOI:https://doi.org/10.1074/mcp.M600120-MCP200
      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 aldehyde-modified 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 ∼2–3-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 ∼3–4-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 1α-1 (eEF1α1), 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 ∼10-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.
      Reactive oxygen species (ROS)
      The abbreviations used are: ROS, reactive oxygen species; A-FABP, adipocyte fatty acid-binding protein (also referred to as aP2); E-FABP, epithelial fatty acid-binding protein (also referred to as KLBP or Mal-1); 4-HNE, trans-4-hydroxy-2-nonenal (also referred to as 4-hydroxynonenal); 4-ONE, trans-4-oxo-2-nonenal; 1,8-ANS, 1-anilinonaphthalene 8-sulfonic acid; PBST, PBS containing 0.05% Tween 20; ER, endoplasmic reticulum; HRP, horseradish peroxidase; GSTA4, glutathione S-transferase A4 (also referred to as GSTA4-4 or GSTa4); βME, β-mercaptoethanol; JNK, c-Jun N-terminal kinase; eEF1α1, eukaryotic elongation factor 1α-1.
      1The abbreviations used are: ROS, reactive oxygen species; A-FABP, adipocyte fatty acid-binding protein (also referred to as aP2); E-FABP, epithelial fatty acid-binding protein (also referred to as KLBP or Mal-1); 4-HNE, trans-4-hydroxy-2-nonenal (also referred to as 4-hydroxynonenal); 4-ONE, trans-4-oxo-2-nonenal; 1,8-ANS, 1-anilinonaphthalene 8-sulfonic acid; PBST, PBS containing 0.05% Tween 20; ER, endoplasmic reticulum; HRP, horseradish peroxidase; GSTA4, glutathione S-transferase A4 (also referred to as GSTA4-4 or GSTa4); βME, β-mercaptoethanol; JNK, c-Jun N-terminal kinase; eEF1α1, eukaryotic elongation factor 1α-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 (
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      ). 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 ROS-initiated peroxidation of polyunsaturated acyl chains of membrane phospholipids can result in a Hock cleavage and the subsequent formation of lipid-derived reactive aldehydes (
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      ).
      Of the variety of reactive aldehydes formed from lipid peroxidation, trans-4-hydroxy-2-nonenal (4-HNE) and trans-4-oxo-2-nonenal (4-ONE) have significant contributions to oxidative disease due to their high abundance and strong reactivity (
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      ). 4-HNE specifically is metabolized (detoxified) by being reduced to an alcohol (aldehyde reductase), oxidized to a carboxylic acid (aldehyde dehydrogenase), or conjugated to glutathione (glutathione S-transferase) (
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      Antioxidant role of glutathione S-transferases: protection against oxidant toxicity and regulation of stress-mediated apoptosis.
      ). However, some fraction of 4-HNE escapes metabolism and forms adducts with other nucleophiles including both protein and DNA. In the case of protein, 4-HNE reacts avidly with the side chains of cysteine and histidine residues as well as lysine albeit to a far lesser extent (
      • Carini M.
      • Aldini G.
      • Facino R.M.
      Mass spectrometry for detection of 4-hydroxy-trans-2-nonenal (HNE) adducts with peptides and proteins.
      ). Although the reactive lipid is formed in membranes, it can diffuse into the cytoplasm and nucleus due to its relatively high solubility, allowing it to react with proteins localized far away from the initial site of oxidative formation (
      • Oberley T.D.
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      Localization of hydroxynonenal protein adducts in normal human kidney and selected human kidney cancers.
      ).
      4-HNE modification of protein has been shown to have a variety of effects on its targets. Alkylation by 4-HNE frequently inhibits the activity of enzymes; examples include the Na+-K+-ATPase and NADP+-dependent isocitrate dehydrogenase (
      • Miyake H.
      • Kadoya A.
      • Ohyashiki T.
      Increase in molecular rigidity of the protein conformation of brain Na +-K+-ATPase by modification with 4-hydroxy-2-nonenal.
      ,
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      Inactivation of NADP+-dependent isocitrate dehydrogenase by lipid peroxidation products.
      ). In some cases, however, 4-HNE modification has been suggested to increase the activity of key regulatory proteins such as the dimerization and ligand-independent activation of the epidermal growth factor receptor (
      • Liu W.
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      • Kato M.
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      4-Hydroxynonenal triggers an epidermal growth factor receptor-linked signal pathway for growth inhibition.
      ) or the activation of the Nrf2 (nuclear factor erythroid 2-related factor 2) transcription factor, leading to increased expression of genes implicated in the antioxidant response (
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      γ-Glutamyl transpeptidase is induced by 4-hydroxynonenal via EpRE/Nrf2 signaling in rat epithelial type II cells.
      ,
      • West J.D.
      • Marnett L.J.
      Alterations in gene expression induced by the lipid peroxidation product, 4-hydroxy-2-nonenal.
      ). In addition to altering the activity of enzymes, 4-HNE alkylation has been shown to alter the rate of degradation of some proteins (alcohol dehydrogenase and αB-crystallin) (
      • Carbone D.L.
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      • Petersen D.R.
      4-Hydroxynonenal regulates 26S proteasomal degradation of alcohol dehydrogenase.
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      • Shang F.
      Ubiquitin-dependent lysosomal degradation of the HNE-modified proteins in lens epithelial cells.
      ).
      Obesity-linked insulin resistance has recently been causally linked to the development of ER stress and an increase in ROS (
      • Furukawa S.
      • Fujita T.
      • Shimabukuro M.
      • Iwaki M.
      • Yamada Y.
      • Nakajima Y.
      • Nakayama O.
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      • Matsuda M.
      • Shimomura I.
      Increased oxidative stress in obesity and its impact on metabolic syndrome.
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      ,
      • Houstis N.
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      Reactive oxygen species have a causal role in multiple forms of insulin resistance.
      ). Adipocytes isolated from C57Bl/6J mice with diet-induced obesity have increased reactive oxygen species (
      • Talior I.
      • Yarkoni M.
      • Bashan N.
      • Eldar-Finkelman H.
      Increased glucose uptake promotes oxidative stress and PKC-δ activation in adipocytes of obese, insulin-resistant mice.
      ). Oxidative stress in human and murine adipose tissue as well as in cultured 3T3-L1 adipocytes affects the secretion of adipokines such as adiponectin and tumor necrosis factor α (
      • Furukawa S.
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      • Shimabukuro M.
      • Iwaki M.
      • Yamada Y.
      • Nakajima Y.
      • Nakayama O.
      • Makishima M.
      • Matsuda M.
      • Shimomura I.
      Increased oxidative stress in obesity and its impact on metabolic syndrome.
      ,
      • Lin Y.
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      ). As adipokines secreted by adipose tissue have dramatic effects on insulin sensitivity in liver and muscle, determining the effects of oxidative stress in adipose tissue is an important step in understanding the molecular mechanisms of type 2 diabetes and systemic energy metabolism (
      • Bays H.
      • Mandarino L.
      • DeFronzo R.A.
      Role of the adipocyte, free fatty acids, and ectopic fat in pathogenesis of type 2 diabetes mellitus: peroxisomal proliferator-activated receptor agonists provide a rational therapeutic approach.
      ). The modification of adipose proteins with lipid peroxidation products could be one contributing factor linking oxidative stress to insulin resistance.
      In this study we investigated the modification of adipose proteins by 4-HNE and other aldehydes in lean and obese C57Bl/6J mice. To accomplish this, hydrazide chemistry was used for coupling with aldehyde adducts on proteins (
      • Mirzaei H.
      • Regnier F.
      Affinity chromatographic selection of carbonylated proteins followed by identification of oxidation sites using tandem mass spectrometry.
      ). Biotin hydrazide tagging allowed evaluation of the relative extent of carbonylation as well as the identification of novel targets of such modifications, including the adipocyte fatty acid-binding protein (A-FABP).

      RESULTS

      To assess the relative extent of carbonylation on adipose tissue proteins in lean and obese mice, biotin hydrazide was used as a chemical tag to couple proteins with free carbonyl groups. As an initial means of verifying the ability of biotin hydrazide to detect carbonylated proteins, purified E-FABP, a known target of 4-HNE modification, was evaluated (
      • Bennaars-Eiden A.
      • Higgins L.
      • Hertzel A.V.
      • Kapphahn R.J.
      • Ferrington D.A.
      • Bernlohr D.A.
      Covalent modification of epithelial fatty acid-binding protein by 4-hydroxynonenal in vitro and in vivo. Evidence for a role in antioxidant biology.
      ). 4-HNE modification was confirmed by the presence of a mass shift of 156 Da when the protein was analyzed by MALDI-TOF MS (Fig. 1). The peak corresponding to a mass of 156 Da larger than that of E-FABP was not detected in the absence of incubation with 4-HNE (Supplemental Fig. 1). After 4-HNE-modified and unmodified E-FABP were incubated with biotin hydrazide, samples were resolved by SDS-PAGE, transferred to PVDF membrane, and incubated with FITC-streptavidin. As shown in Fig. 1, inset, biotin hydrazide modified only 4-HNE-modified E-FABP and not the native form.
      Figure thumbnail gr1
      Fig. 1Detection of 4-HNE modification of E-FABP. Purified recombinant mouse E-FABP was incubated with or without 0.5 mm 4-HNE in vitro, and the extent of modification was evaluated using MALDI-TOF MS. Inset, 10 μg of 4-HNE-modified and unmodified E-FABP were coupled with biotin hydrazide, resolved by SDS-PAGE, transferred to a PVDF membrane, and blotted with FITC-streptavidin.
      Using the methods initially developed for detecting 4-HNE modification of E-FABP, soluble adipose protein extracts were coupled with biotin hydrazide, resolved by SDS-PAGE, transferred to membranes, and blotted with HRP-streptavidin. Reaction variables such as biotin hydrazide concentration, temperature, protein amount, and reaction time were altered to optimize the detection of endogenous carbonylation (Supplemental Fig. 2). The reaction was relatively insensitive to temperature or biotin hydrazide concentration but was dependent upon the amount of protein in the reaction. Standard conditions of 0.5 mm biotin hydrazide, 50 μg of protein, 2 h, and 22 °C were chosen for all subsequent biotin hydrazide/HRP-streptavidin blotting experiments. To confirm that biotin hydrazide is highly specific for aldehydes, as has been demonstrated previously for dinitrophenylhydrazine (
      • Yan L.J.
      • Orr W.C.
      • Sohal R.S.
      Identification of oxidized proteins based on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, immunochemical detection, isoelectric focusing, and microsequencing.
      ), soluble adipose protein was incubated with NaBH4 to reduce aldehydes to primary alcohols. After dialysis, the protein was coupled with biotin hydrazide and blotted with HRP-streptavidin (Supplemental Fig. 3). The signal intensity for the reduced protein was decreased significantly, confirming that the signal intensity from the unreduced protein coupled with biotin hydrazide was primarily from aldehyde groups on the proteins.
      To examine the profile of carbonylated proteins in various tissues as well as to confirm that the endogenous carbonylation detected by biotin hydrazide included 4-HNE-modified proteins, soluble protein extracts were prepared from mouse eye, lung, tongue, and adipose tissues. The protein samples were coupled with biotin hydrazide using the standard conditions, resolved by SDS-PAGE, transferred to PVDF membrane, and blotted with HRP-streptavidin. In addition, parallel samples were subjected to immunochemical analysis using an anti-4-HNE antibody. As shown in Fig. 2, different patterns of protein carbonylation were detected in the various tissues, and in general, the profiles of modified proteins were similar, although the biotin hydrazide method appeared to be more sensitive and detected more proteins than did the anti-4-HNE antibody. As hydrazide chemistry has been shown previously to be specific for carbonyl groups (
      • Yan L.J.
      • Orr W.C.
      • Sohal R.S.
      Identification of oxidized proteins based on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, immunochemical detection, isoelectric focusing, and microsequencing.
      ), the additional bands detected by biotin hydrazide may arise from other carbonyl modifications besides 4-HNE that are known to be prominent (
      • Uchida K.
      4-Hydroxy-2-nonenal: a product and mediator of oxidative stress.
      ,
      • Stadtman E.R.
      Protein oxidation and aging.
      ). Moreover some bands detected by the anti-4-HNE antibody were not observed with the biotin hydrazide method and may be the result of nonspecific reactivity often observed with antibody detection (dark smear at ∼55 kDa seen in all samples).
      Figure thumbnail gr2
      Fig. 2Detection of carbonylated proteins in different murine tissues. Soluble extracts (50 μg) from eye (E), lung (L), tongue (T), or adipose tissue (A) were either coupled with biotin hydrazide (using the standard conditions) and detected by blotting with HRP-streptavidin (left panel) or immunoblotted with anti-4-HNE antibodies (right panel) to detect carbonylated proteins. An additional sample that had not been coupled with biotin hydrazide (−BH) was subjected to HRP-streptavidin blotting as a control. Molecular mass (kDa) of protein standards is indicated on the left.
      To investigate the extent of carbonylation of proteins in obesity, equal amounts (50 μg) of soluble proteins from epididymal adipose tissue of lean and obese mice were coupled with biotin hydrazide, resolved by SDS-PAGE, transferred to PVDF membrane, and blotted with HRP-streptavidin (Fig. 3 A). Quantification of signal intensities by densitometry (normalized to total protein) indicated increased (p < 0.05) carbonylation of proteins (∼2–3-fold) in the samples from the obese mice relative to the lean counterparts (Fig. 3B). This finding agrees well with those reported by Talior et al. (
      • Talior I.
      • Yarkoni M.
      • Bashan N.
      • Eldar-Finkelman H.
      Increased glucose uptake promotes oxidative stress and PKC-δ activation in adipocytes of obese, insulin-resistant mice.
      ) who measured a 2-fold increase in ROS in adipocytes from high fat-fed mice compared with control chow-fed animals. Consistent with increased ROS in adipose tissue of high fat-fed mice, extracts from obese animals exhibited a 3–4-fold decrease (p < 0.05) in GSTA4 protein compared with lean animals (Fig. 4). GSTA4 catalyzes the Michael addition of 4-HNE to glutathione (with high specificity for 4-HNE as a substrate), and its decreased abundance may contribute to the increased reactive aldehydes in adipose tissue from high fat-fed mice (
      • Yang Y.
      • Trent M.B.
      • He N.
      • Lick S.D.
      • Zimniak P.
      • Awasthi Y.C.
      • Boor P.J.
      Glutathione-S-transferase A4–4 modulates oxidative stress in endothelium: possible role in human atherosclerosis.
      ,
      • Engle M.R.
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      • Gaddy D.
      • Montague D.C.
      • Ceci J.D.
      • Yang Y.
      • Awasthi S.
      • Awasthi Y.C.
      • Zimniak P.
      Physiological role of mGSTA4–4, a glutathione S-transferase metabolizing 4-hydroxynonenal: generation and analysis of mGsta4 null mouse.
      ).
      Figure thumbnail gr3
      Fig. 3Carbonylation of proteins in adipose tissue from lean and obese mice.A, soluble proteins (50 μg) from adipose tissue of lean (L) and obese (O) mice were coupled with biotin hydrazide, resolved by SDS-PAGE, transferred to PVDF, and blotted with HRP-streptavidin. Molecular masses (kDa) of the proteins are indicated on the left. B, relative carbonylation (indicated as percentage of the average value for obese mice) was determined from the blot in A by densitometry (normalized to total protein). Values are expressed as mean ± S.E. (n = 3; *, p < 0.05).
      Figure thumbnail gr4
      Fig. 4Protein levels of GSTA4 in lean and obese mice. Soluble protein (50 μg) from adipose tissue of lean and obese mice was resolved by SDS-PAGE, transferred to PVDF, and immunoblotted with anti-GSTA4 antibody. Relative abundance (indicated as percentage of the average value for lean mice) was determined by densitometry (normalized to total protein). Values are expressed as mean ± S.E. (n = 8; *, p < 0.05). Inset, representative lean (L) and obese (O) samples from anti-GSTA4 immunoblot.
      To identify proteins that are targets of aldehyde modification, soluble adipose proteins from obese mice were coupled with biotin hydrazide and enriched via affinity chromatography using immobilized monomeric avidin. Bound proteins were eluted with biotin, digested with trypsin, and subjected to LC-ESI MS/MS. Proteins were identified by analyzing parent and fragment ions (Supplemental Fig. 4) using SEQUEST. All data were filtered using the SEQUEST parameters described under “Experimental Procedures.” The experiment was performed multiple times and repeated in the absence of biotin hydrazide coupling to control for nonspecific enrichment by the avidin column. After removing all proteins that were enriched without biotin hydrazide coupling, a list of soluble proteins identified as targets of aldehyde modification in adipose tissue was generated (Table I). Peptides matched to keratins, serum proteins, trypsin, and hypothetical proteins were also removed from the list. The identified proteins sorted into the following clusters: carbohydrate and lipid metabolism, signal transduction, antioxidant enzymes/cell stress response, nucleic acid metabolism, protein synthesis/degradation, and structural/motor proteins. Of particular note was A-FABP/aP2, a cytoplasmic fatty acid carrier protein and a member of the multigene family of lipid-binding proteins that includes previously characterized E-FABP (
      • Bennaars-Eiden A.
      • Higgins L.
      • Hertzel A.V.
      • Kapphahn R.J.
      • Ferrington D.A.
      • Bernlohr D.A.
      Covalent modification of epithelial fatty acid-binding protein by 4-hydroxynonenal in vitro and in vivo. Evidence for a role in antioxidant biology.
      ).
      Table IIdentification of carbonylated proteins in adipose tissue
      Because A-FABP was found to be an in vivo target of aldehyde modification in adipose tissue by LC-ESI MS/MS, adipose extracts from A-FABP-null mice were used to confirm that A-FABP was modified with 4-HNE (Fig. 5). When soluble adipose protein extracts from wild type mice were subjected to aldehyde detection via biotin hydrazide coupling/HRP-streptavidin blotting, a prominently modified protein was detected at ∼15 kDa that was absent in protein extracts from A-FABP-null mice. When these same samples were subjected to immunoblotting with an anti-4-HNE antibody a similar result was observed. To further verify that A-FABP is a target of 4-HNE modification in vivo, the protein was purified from mouse adipose tissue. Both biotin hydrazide/streptavidin blotting and immunoblotting with anti-4-HNE antibody confirmed that A-FABP was modified with 4-HNE in vivo (Fig. 5).
      Figure thumbnail gr5
      Fig. 5In vivo carbonylation and 4-HNE modification of A-FABP.Left, soluble protein (50 μg) from adipose tissue of A-FABP null (−/−) and wild type (Wt) mice was evaluated using either biotin hydrazide coupling or immunochemically using anti-4-HNE and anti-A-FABP antiserum. Right, A-FABP was purified from murine adipose tissue and subjected to a parallel analysis.
      To determine the stoichiometry of in vivo 4-HNE modification of A-FABP, quantitative immunoblotting using antibodies directed to 4-HNE and A-FABP was utilized (Supplemental Fig. 5). The fraction of A-FABP modified with 4-HNE in adipose tissue of obese mice was calculated by determining the concentrations of total A-FABP and 4-HNE-modified A-FABP in tissue extracts. Through immunodetection of either A-FABP or 4-HNE-modified A-FABP, it was estimated that 6–8% of total A-FABP is modified by 4-HNE in adipose tissue of obese mice. Given the total abundance of A-FABP is ∼250 μm in adipocytes (
      • Matarese V.
      • Buelt M.K.
      • Chinander L.L.
      • Bernlohr D.A.
      Purification of adipocyte lipid-binding protein from human and murine cells.
      ), the concentration of 4-HNE-modified protein was estimated to be 15–20 μm.
      To further characterize the modification of A-FABP with 4-HNE, purified protein was incubated with 0.5 mm (R)- or (S)-4-HNE or a 50:50 racemic mixture for various lengths of time, and the extent of modification was assessed using MALDI-TOF mass spectrometry (linear mode). As shown in Fig. 6, there was no statistical difference between the extent of modification of the protein by (R)- or (S)-4-HNE compared with the racemic mixture.
      Figure thumbnail gr6
      Fig. 6In vitro modification of A-FABP by 4-HNE enantiomers. Purified A-FABP was incubated with 0.5 mm (R)-4-HNE, (S)-4-HNE, or a 50:50 racemic mixture. After various time points, the protein was subjected to MALDI-TOF MS analysis. The relative extent of 4-HNE modification was estimated from spectral peak intensities. The experiment was done in triplicate with error bars shown. Squares represent (R)-4-HNE, circles represent (S)-4-HNE, and diamonds indicate 50:50 racemic mixture.
      To identify the site of 4-HNE modification of A-FABP in vitro, His6-tagged A-FABP was incubated with 4-HNE and enzymatically digested with either trypsin or Glu-C, and the resulting peptides were subjected to MALDI-TOF MS/MS analysis (
      • Jenkins-Kruchten A.E.
      • Bennaars-Eiden A.
      • Ross J.R.
      • Shen W.J.
      • Kraemer F.B.
      • Bernlohr D.A.
      Fatty acid-binding protein-hormone-sensitive lipase interaction. Fatty acid dependence on binding.
      ). The m/z values of abundant ions in the initial MS spectra demonstrated A-FABP sequence coverage of 47.7% for Glu-C and 73.5% for trypsin (data not shown). For both trypsin and Glu-C digestions, parent ions with m/z values corresponding to a peptide with Cys-117 modified by 4-HNE (1076.5 for trypsin and 1628.8 for Glu-C) were isolated and subjected to fragmentation to generate MS/MS spectra (Fig. 7). Many of the ions in the MS/MS spectra observed corresponded to those in the predicted fragmentation pattern for the peptides with Cys-117 modified by 4-HNE (Table II). The 4-HNE modification itself appeared to undergo significant neutral loss fragmentation during the collision-induced dissociation process (labeled as [M + H]+1 − 156 for parent ion and with * for fragment ions in Fig. 7), a commonly observed event during MS/MS analysis of 4-HNE-modified peptides (
      • Carbone D.L.
      • Doorn J.A.
      • Kiebler Z.
      • Sampey B.P.
      • Petersen D.R.
      Inhibition of Hsp72-mediated protein refolding by 4-hydroxy-2-nonenal.
      ,
      • Carbone D.L.
      • Doorn J.A.
      • Kiebler Z.
      • Ickes B.R.
      • Petersen D.R.
      Modification of heat shock protein 90 by 4-hydroxynonenal in a rat model of chronic alcoholic liver disease.
      ) resulting in parent and fragment ions with 4-HNE modification intact in some cases and absent in others. The presence of b ions at 104.0 and 260.1 m/z in the spectrum for the Glu-C-digested peptide (Fig. 7A, labeled as b1 and b1*) verify that the 4-HNE modification is on Cys-117 as these m/z values correspond to N-terminal b1 ions consisting only of cysteine with and without the addition of 4-HNE. The 104.0 m/z peak represents the unmodified Cys-117 with the 260.1 m/z peak representing the same cysteine with the 4-HNE modification intact, giving it an additional 156 m/z units. Furthermore the additional 156 m/z units was only seen on b ions for the tryptic peptide in which Cys-117 is part of the given fragment (ions indicative of 4-HNE modification at Cys-117 are shown in bold in Table II). No fragments besides those containing Cys-117 were found to have 4-HNE adducts, and no other 4-HNE-modified peptides were detected for either digest. A-FABP contains no histidine residues and only one additional cysteine, Cys-1, making it the only other strong candidate as a potential site of in vitro 4-HNE modification. Although ions with m/z values corresponding to peptides with Cys-1 in the unmodified (alkylated with iodoacetic acid) state were detected for both trypsin and Glu-C, the corresponding ion for a 4-HNE-modified peptide was absent in each case. The reactivity of lysine with 4-HNE is much weaker than that of cysteine or histidine, and we found no evidence for a lysine modification on A-FABP at a detectable level. Furthermore the MS/MS spectra in Fig. 7 were submitted to Mascot to search against the mouse database in an unbiased way using 4-HNE as a variable modification. For both the trypsin and Glu-C digests, the Cys-117 HNE-modified peptide derived from A-FABP was the top scoring sequence match with Molecular Weight Search scores of 14 for trypsin and 39 for Glu-C (where a Molecular Weight Search score of greater than 32 indicates identity or extensive homology at p < 0.05) (
      • Pappin D.J.
      • Hojrup P.
      • Bleasby A.J.
      Rapid identification of proteins by peptide-mass fingerprinting.
      ). Together this evidence demonstrates that Cys-117 is the site of 4-HNE modification on A-FABP in vitro.
      Figure thumbnail gr7
      Fig. 7MS/MS spectra of 4-HNE-modified A-FABP peptides. A-FABP was modified with 0.5 mm 4-HNE in vitro and digested with either Glu-C protease (A) or trypsin (B). In each case, the ion with m/z corresponding to the predicted 4-HNE-modified peptide was isolated and subjected to fragmentation. Both the [M + H]+1 parent ion peak corresponding to the 4-HNE-modified peptide as well as the peak corresponding to the neutral loss of 4-HNE ([M + H]+1 − 156) are labeled. The b and y ions observed are labeled as well as several observed a ions, an immonium ion of an arginine residue (Imm. Arg.), and ions corresponding to the loss of water (−H2O). Fragment ions demonstrating neutral loss of 4-HNE are indicated with an asterisk (*).
      Table IIIons observed in MS/MS fragmentation of 4-HNE-modified A-FABP peptides
      To verify that Cys-117 is the in vivo site of modification (and in agreement with the reactivity of the protein with 4-HNE in vitro), A-FABP purified from mouse adipose tissue was digested with Glu-C and subjected to MALDI-TOF MS. The spectrum obtained had 67.9% sequence coverage for A-FABP. Peaks at 1641.8 and 1530.6 m/z were detected, corresponding to unmodified peptides containing Cys-1 (acetylated) and Cys-117 alkylated with iodoacetic acid. Although no peak was observed corresponding to a peptide with Cys-1 modified with 4-HNE or 4-ONE, a cluster of peaks at 1626.6, 1627.6, and 1628.6 m/z (signal:noise between 4 and 5 for each peak) was observed (Supplemental Fig. 6) and is in agreement with the theoretical monoisotopic m/z values of the predicted Glu-C peptide with Cys-117 modified with 4-ONE (1626.8) and 4-HNE (1628.8). Although it is unclear whether the 1628.6 m/z peak is part of the isotope envelope for a monoisotopic peak at 1626.6 m/z (as the 1627.6 peak likely is) or whether it is the result of an independent modification, these m/z values are in agreement with the modified Glu-C peptide observed in the in vitro studies (Table II). In addition, this peak cluster was absent in a control Glu-C digest of E. coli-expressed recombinant A-FABP, which had 74.0% sequence coverage for A-FABP and contained the peak corresponding to the peptide with Cys-117 alkylated with iodoacetic acid (data not shown).
      To investigate the effect of 4-HNE modification of A-FABP on its fatty acid binding activity, recombinant protein was modified with 4-HNE, and the binding of the surrogate fluorescent ligand 1,8-ANS was monitored (
      • Kane C.D.
      • Bernlohr D.A.
      A simple assay for intracellular lipid-binding proteins using displacement of 1-anilinonaphthalene 8-sulfonic acid.
      ). Fig. 8 shows the binding isotherm for the native and 4-HNE-modified protein with native A-FABP exhibiting a Kd of 2.05 ± 0.17 μm and the 4-HNE-modified A-FABP exhibiting a Kd of 23.20 ± 4.18 μm (p < 0.05).
      Figure thumbnail gr8
      Fig. 8Effect of 4-HNE modification of A-FABP on 1,8-ANS binding. After modification with or without 0.5 mm 4-HNE, A-FABP was titrated into 1,8-ANS, and the binding isotherm was calculated from monitoring changes in fluorescence. The experiment was done in triplicate, and the results shown are representative of the three trials. Squares represent untreated A-FABP, and triangles represent 4-HNE-modified A-FABP.

      DISCUSSION

      Obesity and type 2 diabetes are correlated with an increase in ER stress (
      • Furukawa S.
      • Fujita T.
      • Shimabukuro M.
      • Iwaki M.
      • Yamada Y.
      • Nakajima Y.
      • Nakayama O.
      • Makishima M.
      • Matsuda M.
      • Shimomura I.
      Increased oxidative stress in obesity and its impact on metabolic syndrome.
      ,
      • Ozcan U.
      • Cao Q.
      • Yilmaz E.
      • Lee A.H.
      • Iwakoshi N.N.
      • Ozdelen E.
      • Tuncman G.
      • Gorgun C.
      • Glimcher L.H.
      • Hotamisligil G.S.
      Endoplasmic reticulum stress links obesity, insulin action, and type 2 diabetes.
      ) and increased ROS (
      • Furukawa S.
      • Fujita T.
      • Shimabukuro M.
      • Iwaki M.
      • Yamada Y.
      • Nakajima Y.
      • Nakayama O.
      • Makishima M.
      • Matsuda M.
      • Shimomura I.
      Increased oxidative stress in obesity and its impact on metabolic syndrome.
      ,
      • Ozcan U.
      • Cao Q.
      • Yilmaz E.
      • Lee A.H.
      • Iwakoshi N.N.
      • Ozdelen E.
      • Tuncman G.
      • Gorgun C.
      • Glimcher L.H.
      • Hotamisligil G.S.
      Endoplasmic reticulum stress links obesity, insulin action, and type 2 diabetes.
      ). One possible contributing factor of obesity to insulin resistance is oxidative stress-induced lipid peroxidation and the modification of proteins by reactive aldehydes such as 4-HNE and 4-ONE. To gain insight into the extent of carbonylation of adipose proteins, biotin hydrazide was used as an affinity tag for proteins with free aldehyde groups (
      • Mirzaei H.
      • Regnier F.
      Affinity chromatographic selection of carbonylated proteins followed by identification of oxidation sites using tandem mass spectrometry.
      ). Whereas other studies have demonstrated an increase in oxidative stress markers such as malondialdehyde and ROS measurements (
      • Furukawa S.
      • Fujita T.
      • Shimabukuro M.
      • Iwaki M.
      • Yamada Y.
      • Nakajima Y.
      • Nakayama O.
      • Makishima M.
      • Matsuda M.
      • Shimomura I.
      Increased oxidative stress in obesity and its impact on metabolic syndrome.
      ,
      • Talior I.
      • Yarkoni M.
      • Bashan N.
      • Eldar-Finkelman H.
      Increased glucose uptake promotes oxidative stress and PKC-δ activation in adipocytes of obese, insulin-resistant mice.
      ), to our knowledge this is the first report of direct protein modification in adipose tissue linked to obesity. The ∼2–3-fold increase in protein carbonylation accompanying high fat feeding agrees with other measures of oxidative stress (
      • Talior I.
      • Yarkoni M.
      • Bashan N.
      • Eldar-Finkelman H.
      Increased glucose uptake promotes oxidative stress and PKC-δ activation in adipocytes of obese, insulin-resistant mice.
      ). In addition, 37 targets of aldehyde modification were identified in adipose tissue of the obese mice.
      Several proteins we identified in this study have been reported previously to be carbonylated or 4-HNE-modified specifically in other systems. Glyceraldehyde-3-phosphate dehydrogenase and glutathione S-transferase M1 have been shown to be targets of 4-HNE (
      • Gelfi C.
      • De Palma S.
      • Ripamonti M.
      • Eberini I.
      • Wait R.
      • Bajracharya A.
      • Marconi C.
      • Schneider A.
      • Hoppeler H.
      • Cerretelli P.
      New aspects of altitude adaptation in Tibetans: a proteomic approach.
      ). In addition, dihydropyrimidinase-related protein 2 has been shown to be carbonylated in brain of Alzheimer disease patients, and triose-phosphate isomerase 1 has been found to be carbonylated in yeast upon treatment with hydrogen peroxide (
      • Castegna A.
      • Aksenov M.
      • Thongboonkerd V.
      • Klein J.B.
      • Pierce W.M.
      • Booze R.
      • Markesbery W.R.
      • Butterfield D.A.
      Proteomic identification of oxidatively modified proteins in Alzheimer's disease brain. Part II: dihydropyrimidinase-related protein 2, β-enolase and heat shock cognate 71.
      ). As we found similarities between the detection of proteins with biotin hydrazide and anti-4-HNE antibody in adipose and other tissues, we presume that many of the proteins identified by enrichment via biotin hydrazide coupling are 4-HNE-modified (Fig. 2). However, as amino acid side chain oxidation and other lipid peroxidation products can also result in aldehyde groups on proteins (
      • Uchida K.
      4-Hydroxy-2-nonenal: a product and mediator of oxidative stress.
      ,
      • Stadtman E.R.
      Protein oxidation and aging.
      ), additional studies need to be done to confirm the type of modification present on each specific protein of interest as we have done for A-FABP.
      Work from this laboratory has shown previously that E-FABP is a target of 4-HNE modification in retina and that the site of modification is Cys-120 (corresponding to Cys-117 in A-FABP) (
      • Bennaars-Eiden A.
      • Higgins L.
      • Hertzel A.V.
      • Kapphahn R.J.
      • Ferrington D.A.
      • Bernlohr D.A.
      Covalent modification of epithelial fatty acid-binding protein by 4-hydroxynonenal in vitro and in vivo. Evidence for a role in antioxidant biology.
      ). A-FABP and E-FABP are members of the intracellular fatty acid-binding protein multigene family, share ∼60% amino acid identity, and have essentially superimposable secondary and tertiary structures (
      • Hohoff C.
      • Borchers T.
      • Rustow B.
      • Spener F.
      • van Tilbeurgh H.
      Expression, purification, and crystal structure determination of recombinant human epidermal-type fatty acid binding protein.
      ,
      • Gutierrez-Gonzalez L.H.
      • Ludwig C.
      • Hohoff C.
      • Rademacher M.
      • Hanhoff T.
      • Ruterjans H.
      • Spener F.
      • Lucke C.
      Solution structure and backbone dynamics of human epidermal-type fatty acid-binding protein (E-FABP).
      ). The defining feature of the FABP multigene family is a large interior water-filled cavity that serves as the hydrophobic ligand-binding domain. Lipid binding to A-FABP occurs within a defined fatty acid binding site with the carboxyl group coordinated by Arg-106, Arg-126, and Tyr-128 (
      • Reese-Wagoner A.
      • Thompson J.
      • Banaszak L.
      Structural properties of the adipocyte lipid binding protein.
      ). The decreased binding affinity of 4-HNE-modified A-FABP for the hydrophobic probe 1,8-ANS (Fig. 8) agrees with the finding of Cys-117 as the site of modification (Fig. 7) and is consistent with a variety of crystallographic and NMR studies on the A-FABP apo- and holoprotein forms. X-ray crystallographic analysis of A-FABP in a variety of fatty acid-bound forms (palmitate, stearate, and arachidonate) have shown that the C3 to C6 region of a bound lipid lies in close proximity to Cys-117 (
      • LaLonde J.M.
      • Bernlohr D.A.
      • Banaszak L.J.
      X-ray crystallographic structures of adipocyte lipid-binding protein complexed with palmitate and hexadecanesulfonic acid. Properties of cavity binding sites.
      ,
      • Xu Z.
      • Bernlohr D.A.
      • Banaszak L.J.
      The adipocyte lipid-binding protein at 1.6-Å resolution. Crystal structures of the apoprotein and with bound saturated and unsaturated fatty acids.
      ,
      • LaLonde J.M.
      • Levenson M.A.
      • Roe J.J.
      • Bernlohr D.A.
      • Banaszak L.J.
      Adipocyte lipid-binding protein complexed with arachidonic acid. Titration calorimetry and X-ray crystallographic studies.
      ). Moreover chemical modification of Cys-117 in vitro with a battery of sulfhydryl reagents decreased the affinity of A-FABP for fatty acids, and fatty acid binding attenuated Cys-117 modification (
      • Buelt M.K.
      • Bernlohr D.A.
      Modification of the adipocyte lipid binding protein by sulfhydryl reagents and analysis of the fatty acid binding domain.
      ). Prior to this study, only long and very long chain fatty acids were believed to bind within the cavity (
      • Kane C.D.
      • Bernlohr D.A.
      A simple assay for intracellular lipid-binding proteins using displacement of 1-anilinonaphthalene 8-sulfonic acid.
      ). This report implies that the ligand binding specificity of FABPs is broader than previously considered and extends to medium chain aldehydes. It will be interesting in the future to determine whether testis FABP, which exhibits the same structurally conserved cysteine residue, is also 4-HNE-modified.
      The presence of a cluster of peaks containing ions with m/z 1626.6 and 1628.6 in the MALDI-TOF MS spectrum of in vivo purified A-FABP digested with Glu-C (Supplemental Fig. 6) is consistent with the finding of Cys-117 as the in vitro site of modification (Fig. 7). As further support for in vivo modification at Cys-117, digestion of recombinant E. coli-derived A-FABP (E. coli lacks large amounts of polyunsaturated fatty acid substrates for 4-ONE and 4-HNE synthesis) revealed no peak cluster at these m/z values. It is not surprising that a significant portion of the modified species of A-FABP in vivo may be alkylated with 4-ONE (consistent with 1626.6 peak) at Cys-117 in addition to 4-HNE (consistent with 1628.6 peak). Because 4-ONE contains a more electronegative keto function relative to the hydroxyl group of 4-HNE it has been measured to be significantly more reactive than 4-HNE (
      • Doorn J.A.
      • Petersen D.R.
      Covalent modification of amino acid nucleophiles by the lipid peroxidation products 4-hydroxy-2-nonenal and 4-oxo-2-nonenal.
      ).
      It has been shown previously that A-FABP has a role in insulin resistance in mice (
      • Hertzel A.V.
      • Smith L.A.
      • Berg A.H.
      • Cline G.W.
      • Shulman G.I.
      • Scherer P.E.
      • Bernlohr D.A.
      Lipid metabolism and adipokine levels in fatty acid binding protein null and transgenic mice.
      ), and A-FABP-null mice are relatively insulin-sensitive compared with wild type C57Bl/6J littermates. In light of this, covalent binding of 4-HNE may promote the holoprotein structure, mimicking the fatty acid-bound state. As the interaction of A-FABP and hormone-sensitive lipase has been shown previously to be fatty acid-dependent (
      • Jenkins-Kruchten A.E.
      • Bennaars-Eiden A.
      • Ross J.R.
      • Shen W.J.
      • Kraemer F.B.
      • Bernlohr D.A.
      Fatty acid-binding protein-hormone-sensitive lipase interaction. Fatty acid dependence on binding.
      ), the level at which A-FABP is modified with 4-HNE could have significant effects on lipolysis in the adipocyte. In this study it was estimated that 6–8% of A-FABP is modified with 4-HNE in adipose tissue of obese mice. Because of the high level of expression of A-FABP in adipose tissue (250 μm), this seemingly modest fraction that is modified would accumulate to relatively high concentration (
      • Miyake H.
      • Kadoya A.
      • Ohyashiki T.
      Increase in molecular rigidity of the protein conformation of brain Na +-K+-ATPase by modification with 4-hydroxy-2-nonenal.
      ,
      • Yang J.H.
      • Yang E.S.
      • Park J.W.
      Inactivation of NADP+-dependent isocitrate dehydrogenase by lipid peroxidation products.
      ,
      • Liu W.
      • Akhand A.A.
      • Kato M.
      • Yokoyama I.
      • Miyata T.
      • Kurokawa K.
      • Uchida K.
      • Nakashima I.
      4-Hydroxynonenal triggers an epidermal growth factor receptor-linked signal pathway for growth inhibition.
      ,
      • Levonen A.L.
      • Landar A.
      • Ramachandran A.
      • Ceaser E.K.
      • Dickinson D.A.
      • Zanoni G.
      • Morrow J.D.
      • Darley-Usmar V.M.
      Cellular mechanisms of redox cell signalling: role of cysteine modification in controlling antioxidant defences in response to electrophilic lipid oxidation products.
      ,
      • Zhang H.
      • Liu H.
      • Dickinson D.A.
      • Liu R.M.
      • Postlethwait E.M.
      • Laperche Y.
      • Forman H.J.
      γ-Glutamyl transpeptidase is induced by 4-hydroxynonenal via EpRE/Nrf2 signaling in rat epithelial type II cells.
      ,
      • West J.D.
      • Marnett L.J.
      Alterations in gene expression induced by the lipid peroxidation product, 4-hydroxy-2-nonenal.
      μm HNE-modified A-FABP) in obese mice. The relative extents of modification of A-FABP in lean and obese mice was consistent with the ∼2–3-fold increase in total protein carbonylation (Fig. 3), and increased modification of A-FABP may in part contribute to insulin resistance in obesity through altering hormone-sensitive lipase activity.
      A potential role for A-FABP is as an antioxidant protein, scavenging 4-HNE in the cell (
      • Bennaars-Eiden A.
      • Higgins L.
      • Hertzel A.V.
      • Kapphahn R.J.
      • Ferrington D.A.
      • Bernlohr D.A.
      Covalent modification of epithelial fatty acid-binding protein by 4-hydroxynonenal in vitro and in vivo. Evidence for a role in antioxidant biology.
      ). Glutathione, a well characterized antioxidant against 4-HNE, has been shown to be enantioselective toward (S)-4-HNE in its GST-catalyzed Michael adduct formation (
      • Hiratsuka A.
      • Tobita K.
      • Saito H.
      • Sakamoto Y.
      • Nakano H.
      • Ogura K.
      • Nishiyama T.
      • Watabe T.
      (S)-Preferential detoxification of 4-hydroxy-2(E)-nonenal enantiomers by hepatic glutathione S-transferase isoforms in guinea-pigs and rats.
      ). The fact that A-FABP forms adducts with both enantiomers of 4-HNE suggests that such a 4-HNE-scavenging role would not be completely overlapping with that of glutathione. As both stereoisomers of 4-HNE are formed in the cell, the ability of FABP to form adducts with (R)-4-HNE could be an important way of regulating concentrations of free 4-HNE.
      Several other proteins identified in this study as targets of carbonylation may contribute to insulin resistance and inflammation. The well characterized roles of peroxiredoxin 1 and glutathione peroxidase 1 are in reducing hydroperoxides in the cell, including lipid hydroperoxides (
      • Wood Z.A.
      • Schroder E.
      • Robin Harris J.
      • Poole L.B.
      Structure, mechanism and regulation of peroxiredoxins.
      ,
      • Drevet J.R.
      The antioxidant glutathione peroxidase family and spermatozoa: a complex story.
      ). However, peroxiredoxin and glutathione peroxidase enzymes have also been shown to be important for the regulation of signal transduction pathways through controlling the redox state of proteins and lipids. Specifically the nuclear localization of NFκB, a stress-activated transcription factor known to contribute to insulin resistance in adipose tissue through the control of adipokine expression, has been shown to be negatively regulated in part by peroxiredoxin and glutathione peroxidase proteins (
      • Kang S.W.
      • Chae H.Z.
      • Seo M.S.
      • Kim K.
      • Baines I.C.
      • Rhee S.G.
      Mammalian peroxiredoxin isoforms can reduce hydrogen peroxide generated in response to growth factors and tumor necrosis factor-α.
      ,
      • Jin D.Y.
      • Chae H.Z.
      • Rhee S.G.
      • Jeang K.T.
      Regulatory role for a novel human thioredoxin peroxidase in NF-κB activation.
      ,
      • Kretz-Remy C.
      • Mehlen P.
      • Mirault M.E.
      • Arrigo A.P.
      Inhibition of IκB-α phosphorylation and degradation and subsequent NF-κB activation by glutathione peroxidase overexpression.
      ). Furthermore the presence of active site cysteine (peroxiredoxin 1) and selenocysteine (glutathione peroxidase) residues makes these enzymes strong candidates for being inactivated by alkylation with 4-HNE, which could play a role in activating the NFκB stress response in obesity. In addition, glutathione S-transferase and aldehyde dehydrogenase family members are important antioxidants against 4-HNE and other electrophilic lipids (
      • Uchida K.
      4-Hydroxy-2-nonenal: a product and mediator of oxidative stress.
      ). Their potential inactivation through modification could contribute to cellular stress by amplifying oxidative stress in obesity.
      The actin filament-binding protein filamin A has been shown to interact with the insulin receptor and inhibit insulin signal transduction (
      • He H.J.
      • Kole S.
      • Kwon Y.K.
      • Crow M.T.
      • Bernier M.
      Interaction of filamin A with the insulin receptor alters insulin-dependent activation of the mitogen-activated protein kinase pathway.
      ). The modification of filamin A with a lipid such as 4-HNE possibly could aid in its localization to the plasma membrane, increasing interactions with the insulin receptor and inhibiting insulin action. Calumenin, a protein involved in the regulation of calcium flux out of the ER, is also an intriguing target of carbonylation as it has been shown that ER stress is activated in obesity (
      • Ozcan U.
      • Cao Q.
      • Yilmaz E.
      • Lee A.H.
      • Iwakoshi N.N.
      • Ozdelen E.
      • Tuncman G.
      • Gorgun C.
      • Glimcher L.H.
      • Hotamisligil G.S.
      Endoplasmic reticulum stress links obesity, insulin action, and type 2 diabetes.
      ,
      • Jung D.H.
      • Mo S.H.
      • Kim D.H.
      Calumenin, a multiple EF-hands Ca2+-binding protein, interacts with ryanodine receptor-1 in rabbit skeletal sarcoplasmic reticulum.
      ). During conditions of ER stress, misfolded proteins accumulate in the ER lumen and initiate the unfolded protein response and the activation of JNK, which is known to increase serine phosphorylation of insulin receptor substrate-1 (
      • Ozcan U.
      • Cao Q.
      • Yilmaz E.
      • Lee A.H.
      • Iwakoshi N.N.
      • Ozdelen E.
      • Tuncman G.
      • Gorgun C.
      • Glimcher L.H.
      • Hotamisligil G.S.
      Endoplasmic reticulum stress links obesity, insulin action, and type 2 diabetes.
      ). As ER stress can be initiated by calcium release from the ER, the potential inactivation of calumenin by 4-HNE could contribute to the unfolded protein response and JNK activation in an indirect manor (
      • DuRose J.B.
      • Tam A.B.
      • Niwa M.
      Intrinsic capacities of molecular sensors of the unfolded protein response to sense alternate forms of endoplasmic reticulum stress.
      ). In addition, the modification of eukaryotic elongation factor 1α-1 (eEF1α1) could contribute to insulin resistance through the activation of a similar stress response. This protein has been shown to be involved in activating apoptosis under lipotoxic conditions in non-adipose cells (
      • Borradaile N.M.
      • Buhman K.K.
      • Listenberger L.L.
      • Magee C.J.
      • Morimoto E.T.
      • Ory D.S.
      • Schaffer J.E.
      A critical role for eukaryotic elongation factor 1A-1 in lipotoxic cell death.
      ). Although adipocytes do not undergo apoptosis in response to high amounts of lipid, it is possible that eEF1α1 is involved in activating other stress responses in adipocytes in obesity. Although these proteins are intriguing candidates for being targets of modification by 4-HNE or other lipid peroxidation products, additional studies are necessary to determine the specific modifications and potential effects in each case.
      The decreased amount of GSTA4 protein (∼3–4-fold) in adipose tissue of obese mice seen in this study (Fig. 4) is consistent with published microarray data demonstrating decreased GSTA4 mRNA expression in adipose tissue of obese C57Bl/6J mice (
      • Moraes R.C.
      • Blondet A.
      • Birkenkamp-Demtroeder K.
      • Tirard J.
      • Orntoft T.F.
      • Gertler A.
      • Durand P.
      • Naville D.
      • Begeot M.
      Study of the alteration of gene expression in adipose tissue of diet-induced obese mice by microarray and reverse transcription-polymerase chain reaction analyses.
      ). GSTA4 has been shown previously to be the primary GST isozyme responsible for metabolizing 4-HNE (
      • Yang Y.
      • Trent M.B.
      • He N.
      • Lick S.D.
      • Zimniak P.
      • Awasthi Y.C.
      • Boor P.J.
      Glutathione-S-transferase A4–4 modulates oxidative stress in endothelium: possible role in human atherosclerosis.
      ,
      • Engle M.R.
      • Singh S.P.
      • Czernik P.J.
      • Gaddy D.
      • Montague D.C.
      • Ceci J.D.
      • Yang Y.
      • Awasthi S.
      • Awasthi Y.C.
      • Zimniak P.
      Physiological role of mGSTA4–4, a glutathione S-transferase metabolizing 4-hydroxynonenal: generation and analysis of mGsta4 null mouse.
      ). As such, a 3–4-fold decrease in GSTA4 in adipose cells would presumably contribute to increased accumulation of 4-HNE and other lipid peroxidation products, resulting in elevated protein carbonylation as observed in this study. Further investigation is needed to determine whether protein carbonylation and GSTA4 abundance are components linking obesity to insulin resistance.

      Acknowledgments

      We thank all members of the Bernlohr and Griffin research groups who provided helpful suggestions regarding this work and specifically Lisa Smith and Wendy Wright for maintenance and care of the animals and Mikel Roe for assistance with instrument methods. We also thank Dr. LeeAnn Higgins and the staff of the University of Minnesota Center for Mass Spectrometry and Proteomics for instruction on instrument operation and the Minnesota Supercomputing Institute for maintenance of the SEQUEST cluster.

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