A number of clinical and biochemical studies demonstrate that obesity and insulin resistance are associated with increases in oxidative stress and inflammation. Paradoxically, insulin sensitivity can be enhanced by oxidative inactivation of cysteine residues of phosphatases, and inflammation can be reduced by S-glutathionylation with formation of protein-glutathione mixed disulfides (PSSG). Although oxidation of protein-bound thiols (PSH) is increased in multiple diseases, it is not known whether there are changes in PSH oxidation species in obesity.
In this work, the hypothesis that obesity is associated with decreased levels of proteins containing oxidized protein thiols was tested.
Design and Methods:
The tissue levels of protein sulfenic acids (PSOH) and PSSG in liver, visceral adipose tissue, and skeletal muscle derived from glucose intolerant, obese-prone Sprague-Dawley rats were examined.
The data in this study indicate that decreases in PSSG content occurred in liver (44%) and adipose (26%) but not skeletal muscle in obese rats that were fed a 45% fat-calorie diet versus lean rats that were fed a 10% fat-calorie diet. PSOH content did not change in the tissue between the two groups. The activity of the enzyme glutaredoxin (GLRX) responsible for reversal of PSSG formation did not change in muscle and liver between the two groups. However, levels of GLRX1 were elevated 70% in the adipose tissue of the obese, 45% fat calorie-fed rats.
These are the first data to link changes in S-glutathionylation and GLRX1 to adipose tissue in the obese and demonstrate that redox changes in thiol status occur in adipose tissue as a result of obesity.
Obesity and its ensuing comorbidities such as insulin resistance are of great national and international concern. Several studies indicate that obesity leads to an elevation in oxidative stress (1-4). While obesity is linked to oxidative stress, several lines of evidence indicate that generation of reactive oxygen species, for example, hydrogen peroxide, and protein oxidation are necessary for the enhancement of insulin signaling. Studies by Wu and colleagues indicate that insulin treatment leads to reversible, thiol-mediated decreases in protein tyrosine phosphatase (PTP) activity in adipocytes and that generation of hydrogen peroxide as a result of NADPH-oxidase 4 activity enhances insulin sensitivity (5, 6). Kobayashi et al. demonstrate that insulin resistance adipocytes have elevated glutathione (GSH) and that elevated GSH impairs insulin signaling (7). Transgenic mice that are deficient in the peroxide clearance enzyme glutathione peroxidase-1 (GPX1; EC 188.8.131.52) have elevated insulin sensitivity; whereas GPX1-overexpressing mice demonstrate insulin resistance (8-10). Other data indicate that inflammation, a sequalae of obesity, is downregulated by the S-glutathionylation of NF-κB pathway proteins (11-13). However, scant data exist regarding protein thiol modifications oxidation in the obese state.
The regulation of enzyme activity and protein–protein interaction by the oxidation state of protein-bound thiols (PSHs) is a rapidly growing field of study. In particular, interest has gathered owing to thiol oxidation as a redox switch that reversibly regulates protein function. In a simplified scheme (Figure 1), the thiolate form of the PSH reacts with hydrogen peroxide to yield a sulfenic acid (14). The sulfenic acid (PSOH) may be oxidized further to a sulfinic or sulfonic acid (PSO2H and PSO3H, respectively) (15). PSOH is a divergent point such that PSOH may react with another PSH to yield intramolecular/intermolecular disulfides (PSSP) or GSH (i.e., S-glutathionylation) to yield a mixed protein-GSH (PSSG) (16). In the case of PTP1B, the active Cys215 oxidizes to PSOH and then reacts with the amide nitrogen of Ser216 to form a sulfenyl amide (17). While direct enzymatic reversal of PSOH to PSH is not documented, the GLRX (EC 184.108.40.206) and thioredoxin (TXN; EC 220.127.116.11) family of enzymes are able to reduce the PSSG and PSSP to PSH, respectively (18).
In this work, we tested the hypothesis that obesity induces selective decreases in the content of proteins containing oxidized protein thiols. We examined tissue levels of PSOH and PSSG in liver, visceral adipose tissue, and skeletal muscle derived from glucose-intolerant, obese-prone Sprague-Dawley rats. Our data indicate that PSSG content decreased in liver and adipose but not in skeletal muscle and that GLRX1 content was elevated in visceral adipose tissue. Additionally, we introduce the novel use of N-methyl-2-vinylpyridine (M2VP) as a thiol derivatization agent for use in thiol proteomic studies.
Methods and Procedures
N-Ethylmaleimide (NEM), 5,5′-dithio-bis-(2-nitro-benzoic acid) (DTNB), 5,5-dimethyl-1,3-cyclohexanedione (dimedone/DMD), iodoacetic acid (IAA), protease inhibitor cocktail, Triton® X-100, guanidine hydrochloride, sodium dodecyl sulfate (SDS), diethylenetriaminepentaacetic acid (DTPA), and 2,6-di-tert-butyl-4-methy phenol (BHT) were purchased from Sigma/Aldrich (St. Louis, MO). Dithiothreitol was purchased from Research Products International Corp. (Mount Prospect, IL).
Preparation of M2VP
Synthesis of M2VP was performed by the Core Synthesis Facility at North Dakota State University. Under nitrogen atmosphere, 0.95 equivalents of methyl trifluoromethanesulfonate was added slowly to the solution of 2-vinyl pyridine in dry hexane (3 ml/mmol) at −10°C. After stirring for another 3 h at −10°C, the formed white solid was collected by filtration and was washed with hexane. Then the solid was dried under vacuum for 2 h, and the product was obtained as white solid with a yield of 82%.
Mass spectral analysis of the resulting product by Ion Trap-Time of Flight hybrid MSn (Shimadzu SSI, USA) showed a primary ion in positive mode of 120 m/z, with the major fragment ion elicited at 92 m/z. The triflate counter ion was evident in negative mode at 149 m/z.
The 1H NMR spectrum of the resulting compound was recorded on a Bruker AVANCE 500 MHz NMR spectrometer. Chemical shifts, δ are reported in ppm relative to SiMe4 in D2O. 1H NMR (D2O, δ): 4.23 (s, 3H, NCH3), 6.07 (d, J = 10 Hz, 1H, CH), 6.30 (d, J = 17 Hz, 1H, CH), 7.09 (dd, J1 = 17 Hz, J2 = 10 Hz, 1H, CH), 7.81 (m, 1H, Harom), 8.13 (m, 1H, Harom), 8.39 (m, 1H, Harom), 8.62 (m, 1H, Harom).
All experiments were performed in accordance with the NIH guidelines for use of live animals and were approved by the Institutional Animal Care and Use Committee of the USDA/Agricultural Research Service, Grand Forks Human Nutrition Research Center. Obese-prone male rats (Crl:OP[CD] strain code: 463; Charles River Laboratories International Inc.) up to 4 weeks of age were placed on a low-fat (10% fat calorie) AIN93-based powdered diet for 4 weeks.
At the end of 4 weeks, animals were regrouped by in vivo fat/lean body mass ratio with half of the animals placed on a high-fat (45% fat calorie) AIN93-based diet (n = 5) and the remaining animals continuing on the 10% fat calorie diet (n = 5). Diet composition has been published previously (19). Animals remained on their respective diets for the remaining 12 weeks. Food consumption and weights per animal were recorded daily. Whole body composition analysis was accomplished using an EchoMRI-700 whole body composition analyzer (Echo Medical Systems, LLC, Houston, TX) (20). Animals were euthanized with a 1.37:1 mixture of ketamine (100 mg/ml):xylazine (100 mg/ml) at 1 ml/kg body weight, i.p. and exsanguinated by descending vena cava blood draw. Tissues were quickly removed, frozen in liquid nitrogen, and stored at −80°C until use.
Glucose tolerance tests
Oral glucose tolerance testing (OGTT) was conducted following 10 weeks on the normal fat and high fat diet. OGTT was performed following the recommendations of the Vanderbilt-NIH Mouse Metabolic Phenotyping Center for glucose tolerance testing (21). Animals were fasted for 15 h and then given glucose (1.34 g/kg lean body mass) orally as a solution dissolved in deionized water. Blood was drawn from the tail artery at baseline (pre glucose challenge), 7, 14, 21, 30, 60, and 90 min post glucose challenge. Immediately after each draw, whole blood glucose was measured in duplicate with glucose test strips (OneTouch® Ulta® LifeScan, Milpitas, CA).
Determination of thiol content
A six-fold volume of ice-cold methanol was added to protein solutions on ice followed by centrifugation at 14,000×g for 15 min at 4°C. The resulting pellet was washed with 1 ml of cold methanol twice with centrifugation. The final pellet was resuspended by sonication in a 1 ml solution of 1% (w/v) of SDS in homogenizing buffer. A portion of the resuspended sample was used for determination of PSH content using DTNB. Another portion was used for determination of protein content. Protein content for all assays was determined using Protein Assay Dye Reagent (Bio-Rad Laboratories, Inc., Hercules, CA) with bovine serum albumin as the standard.
Processing of tissue for PSOH and PSSG contents
Sodium acetate buffer (25 mM, pH 5.0) containing 1 mM DTPA and 0.05 mM BHT was used in all experiments unless otherwise noted. Buffers were sparged with argon gas to reduce dissolved O2 content. O2 content was monitored using a Milwaukee 600 dissolved oxygen probe (Milwaukee Instruments Inc., Rocky Mount, NC). The dissolved O2 content of the buffers were reduced from 7.9 mg/l to at least 0.2 mg/l or less and is comparable to results from others (22). Following de-oxygenation of the buffer, Triton® X-100 (0.5% final concentration w/v) M2VP (100 mM final), DMD (25 mM final), and protease inhibitors (1.0% final concentration v/v) were added followed by further argon sparging. M2VP was used to alkylate thiol residues, and DMD was used to derivatize PSOH residues. Tissues were sonicated using a Model 150I sonic dismembrator (Thermo Fisher Scientific Inc., Waltham, MA) with amplitude power setting at 50%. Hepatic tissue samples were sonicated in homogenizing buffer (10× w/v) and 1:1 with 2× SDS-PAGE sample loading buffer was added.
Adipose tissue samples were sonicated in homogenizing buffer (1:2 w/v), centrifuged at 5,000×g for 10 min at 4°C. To concentrate adipose proteins, proteins were precipitated using cold acetone. Briefly, the liquid infranatant layer between the fat layer and pelleted material was removed, sonicated, and centrifuged at 10,000×g for 10 min at 4°C. A nine-fold volume of ice-cold acetone was added to the supernatant, which was then vortexed and incubated on ice for 15 min, centrifuged at 2,000×g for 10 min at 4°C. The supernatant was removed and the pellet was resuspended in 250 μl homogenizing buffer.
Muscle tissue samples were sonicated in homogenizing buffer containing 6 M guanidine hydrochloride (5× w/v) for 1 min on an ice water bath. Guanidine was included to allow for complete solubilization of muscle proteins. Samples were cooled in ice for 3 min and then sonicated again for 30 s, centrifuged 10,000×g for 15 min at 4°C. Supernatant was incubated at 37°C for 15 min with vortexing every 5 min. Fifty microliters of sample was added to 200 μl 2× SDS loading buffer.
GLRX activity was measured in liver homogenates using cysteinyl-glutathione disulfide as a substrate coupled to the formation of GSSG and the oxidation of reduced nicotinamide adenine dinucleotide phosphate (NADPH) (A340 nm, ε = 6,200 M−1) (23). In experiments studying the effects of NEM and M2VP on GLRX activity, liver homogenates were passed through a ten-fold volume G10 sephadex column to remove M2VP and NEM. In other experiments, tissue-level GLRX activities were measured in the intact homogenate. Tissue homogenization either mechanically by Dounce homogenizer or by brief sonication (50% amplitude for less than 30 s) provided similar GLRX activity in liver homogenate. In the experiments presented, tissue samples were mechanically homogenized in buffer (1:10 w/v) and centrifuged 10,000×g for 5 min at 4°C.
Proteins from tissues were separated by SDS-PAGE using 10% or 12% polyacrylamide gels (Invitrogen Corporation, Carlsbad, CA). Protein samples were added 1:1 with 2× loading buffer or 3:1 with 4× loading buffer (v/v). Following electrophoresis, proteins were transferred to a 0.45-μm polyvinylidene fluoride (PVDF) membrane and the resulting membrane was blocked in 5% non-fat dry milk (NFDM) dissolved in Tris-buffered saline (TBS) for 2 h at 20°C. Antibodies were diluted in 5% NFDM in TBS containing 0.1% Tween 20 (TBST) and incubated overnight at 4°C. Following washing of the blots, they were incubated for 2 h at 20°C in the appropriate anti-mouse or anti-rabbit secondary antibody (Promega, Madison, WI) conjugated to horseradish peroxidase diluted in 5% NFDM in TBST (1:6,000). Blots were developed using Pierce® ECL western blotting substrate (Thermo Fisher Scientific Inc., Waltham, MA) imaged on a Biospectrum® 500 imaging system and quantified using Vision Works™LS Image Acquisition and Analysis Software (UVP, Upland, CA).
The following antibodies were used: mouse anti-GSH antibody (1:2,000 dilution; Chemicon/Millipore, Temecula, CA), rabbit anti-PS-DMD antibody (1:3,000 dilution; Chemicon/Millipore, Temecula, CA), rabbit anti-GLRX1 antibody (1:2,000 dilution; Sigma, St. Louis, MO), and rabbit anti-glutathione-S-transferase Pi (GSTP; 1:2,000 dilution; Enzo Life Sciences, Plymouth Meeting, PA).
Dot blot analysis of liver, gastrocnemius, and perirenal adipose tissue samples prepared with DMD and M2VP provided saturable binding curves with anti-GSH and anti-DMD (not shown). Protein concentrations of 0.30 μg (liver) or 0.50 μg (gastrocnemius, peri-renal adipose tissue) per well were utilized as these concentrations were in the middle of the linear portion of the saturation curves. Proteins were diluted in TBS and loaded onto a 0.45-μm PVDF membrane using a Bio-Dot™ Apparatus (Bio-Rad Laboratories Inc.). Membranes were washed with TBS on the blotting apparatus, removed, and blocked in 5% NFDM/TBS. Antibody dilutions and incubations were conducted as described above.
Determination of hepatic and adipose GSH contents
GSH and glutathione disulfide (GSSG) content were determined using a modification of the method reported by Dringen and colleagues (24). Tissue samples were sonicated in 5% sulfosalicylic acid (SSA) in a ratio of 1:5 (w/v) for liver and 1:4 (w/v) for peri-renal adipose tissue. Homogenates were centrifuged at 12,000×g for 10 min. The liver supernatant was removed and assayed for GSH and GSSG activity. Adipose samples sonicated in the presence of SSA did not yield a distinct protein pellet as the large majority of the protein appeared to be associated with the floating lipid layer. The SSA-containing liquid layer was carefully removed by pipette, leaving a lipid/protein pellet. For GSH determination, liver supernatants were diluted 1:800 with 0.1 M sodium phosphate dibasic pH 7.4 containing 1 mM EDTA. Peri-renal adipose supernatants were diluted 1:5. Diluted supernatant (50 μl) was combined with 100 μl of the assay reaction mixture (0.3 mM NADPH, 0.24 mM DTNB, 2.24 units glutathione reductase). Absorbance was read at 405 nm at 25°C over a 2-min period. GSSG content was determined by pipetting 10 μl of SSA sample into 10 μl of 6 mM M2VP then adding 80 μl of 100 mM sodium carbonate, followed by incubation for 2 min at room temperature. Fifty microliters of M2VP-treated liver sample (1:20 final dilution) or 50 μl M2VP-treated adipose (1:10 final dilution) were then assayed with the same reaction mix concentrations used in the GSH assay. Standard curves were performed with GSH or GSSG as appropriate. The liver pellet was frozen and later dissolved in 0.5 N NaOH for protein analysis by Bio-Rad protein assay with final results being expressed GSH and GSSG content per mg protein. We attempted to acetone extract the lipid/protein floating layer to recover adipose proteins. However, this method did not yield a distinct protein pellet for protein determination. Thus, GSH and GSSG content for adipose is normalized to gram wet weight of starting tissue.
Data were analyzed using one-way ANOVA, two-way ANOVA, or Student's t-test as appropriate using GraphPad Prism version 5.00 for Windows (GraphPad Software, San Diego, CA, USA, www.graphpad.com). Statistical significance was taken as P ≤ 0.05.
The initial portion of this work was to develop a facile method for the preparation of multiple tissues for analysis of thiol oxidation (PSOH, PSSG) on proteins. In addition to limiting dissolved O2, alkylation of thiol groups by agents such as NEM and IAA is used to prevent artifactual oxidation of thiols. We compared the ability of NEM, IAA, and M2VP (Figure 2A) to cause thiol depletion at pH 5.0. We tested M2VP as an alternate thiol alkylating agent owing to studies indicating that 2-vinylpyridine derivatives have favorable thiol alkylation properties below neutral pH (25). Furthermore, the aromatic quaternary amine moiety of M2VP allowed for satisfactory solubility at concentrations ≥100 mM in aqueous solution. NEM and M2VP had similar thiol depletion characteristics on liver homogenate with approximately 50% of PSHs being alkylated. Conversely, IAA produced little thiol alkylation at pH 5.0. These results comparing NEM and IAA are similar to those obtained by Rogers and colleagues (26). Because of the poor alkylation results of IAA, we did not examine IAA further.
While NEM and M2VP were not able to completely alkylate all protein thiols, we compared the ability of NEM and M2VP to prevent the oxidation of thiols to PSOH (Figure 2B). PSOH is derivatized with DMD to form an immunoreactive adduct (PS-DMD) recognized by antibodies (14, 27). Our data indicated that formation of PSOH residues occurs on proteins likely as a consequence of tissue preparation as no exogenous oxidants were added to generate PSOH residues. DMD reactivity was blocked by sodium arsenite, confirming that SOH residues were present and were reacting with DMD to form the immunoreactive adduct (not shown) (14). Both M2VP and NEM potently prevented PSOH formation in homogenized liver samples (Figure 2B). However, some NEM-treated proteins had immunoreactivity toward the SOH/DMD antibody (Figure 2B). This cross reactivity may be the result of NEM and DMD both possessing cyclic diketone moieties.
S-Glutathionylation is recognized as a modulator of protein function. We hypothesized that similar to the increase in PSOH formation on tissue homogenization, levels of PSSG would increase and that increases in PSSG content would be blocked by the thiol alkylators NEM and M2VP. In contrast to our hypothesis, our data indicated that NEM and M2VP increased the levels on PSSG on selective proteins (Figure 2C,D). This finding may be the result of GLRX activity cleaving the GSH residue from the target protein. The active site thiol residue of GLRX responsible for the cleavage of the GSH residue has a pKa of approximately 3.5, and, thus, GLRX may be active even in our acidic buffer system (23). Our data show that NEM and M2VP both inhibited GLRX activity in liver homogenates (Figure 2E).
To determine the degree to which levels of proteins with PSOH and PSSG were changed in obese animals, we utilized the obese-prone Sprague-Dawley rats (28-30). These rats became glucose intolerant, gained more total body weight, gained additional adipose, and gained significantly, but slightly, more lean body mass when fed a high-fat (45% fat derived calories from lard) diet versus a 10% fat calorie diet (Figure 3A–D). Our data are comparable with those by Louis and colleagues (28) demonstrating that obese-prone Sprague-Dawley rats become hyperglycemic and insulinemic. Feeding data are provided in Supporting Information S1.
From these animals, we analyzed liver, visceral (peri-renal) adipose tissue, and the gastrocnemius muscle (as a model skeletal muscle) as these tissues are relevant to the development of obesity and insulin resistance. We analyzed tissues for content of PSSG and PSOH by use of dot blot for total signal and western blot for analysis of specific proteins. We chose both the methods as the dot blot would provide quantitative information and the western blot would provide information regarding protein masses.
First, we analyzed PSSG content owing to data indicating that PSSG is regulated through formation and reversal. Content of PSSG was significantly reduced in the peri-renal adipose (26%) and liver (44%) of animals fed the 45% fat calorie diet compared to the 10% fat-fed animals (Figure 4). Western blot analyses of the samples demonstrated decreases in the intensity of GSH-modified proteins rather than changes protein targets. No changes in PSSG content or protein targets were observed in gastrocnemius muscle. Easily visualized proteins were abundant high molecular weight species; however, we recognize that changes in less-abundant proteins may have occurred.
PSOH is a precursor to PSSG, and, thus, PSOH content and PSOH-modified proteins were studied (Figure 5) (16, 31, 32). No changes in total PSOH content by dot blot were observed in the tissues studied from 10% fat and 45% fat animals. There were no apparent changes in PSOH-modified targets in liver and muscle. There were differences in PSOH target proteins in adipose; however, these differences were not consistent. Unlike PSSG, multiple proteins in all the tissues contained PSOH residues.
A decrease in the content of PSSG proteins may be the result of a decrease in GSTP or an increase in GLRX activity. Recent data indicate that GSTP is able to catalyze the formation of PSSG with PSOH and GSH as substrates (16, 32). We tested the extent to which GSTP protein was altered by obesity. However, there was no difference between lean and obese animals in liver, peri-renal adipose, or gastrocnemius muscle (data not shown).
The decreases in PSSG may also be the result of elevated GLRX. We examined tissues for GLRX1 content owing to newer data implicating GLRX1 in inflammation and vascular dysfunction (11-13, 33-36) (Figure 6). A 70% increase in GLRX1 protein was observed in the peri-renal adipose tissue of obese rats when compared with the lean controls (Figure 6A). No changes in GLRX1 were observed in liver from lean versus obese rats (Figure 6B). A single, 25-kD GLRX1 immunoreactive protein was observed in gastrocnemius muscle (not shown). While this immunoreactive protein did not change with diet, we are unsure as to its identity as GLRX1. Analysis of total GLRX activity in liver or gastrocnemius muscle demonstrated no obesity-induced changes (Figure 6C). GLRX activity was below the level of detection in peri-renal adipose under our assay conditions.
We tested whether the decreases in PSSG content in liver and visceral adipose would be reflected by changes in GSH and GSSG content. However, there were no significant differences in GSH and GSSG content or GSH:GSSG ratios in liver or adipose between lean and obese animals (Table 1). However, there was a remarkable difference (approximately ten-fold) in the GSH:GSSG ratios when comparing liver to visceral adipose in either lean or obese animals.
|10% Fat||45% Fat||10% Fat||45% Fat|
Obesity in humans is associated with increases in oxidative stress markers such as isoprostanes and protein-bound carbonyls in adipose tissue (1-4). Conversely, data indicate adipocytic insulin resistance is the result of elevated GSH content in the adipocytes (7). Whole animal studies indicate that depletion of GSH prevents obesity-induced insulin resistance (37). Thus, alterations in the redox state of protein thiols may play a role in adipose dysfunction. In this work, we tested the hypothesis that obesity induces tissue selective decreases in the content of proteins containing oxidized protein thiols, specifically those containing PSOH and PSSG, and residues. Our major findings are that decreases in S-glutathionylation of hepatic and adipose proteins occurred in obese versus lean rats and that elevated levels of GLRX1, which reverses S-glutathionylation, were found in the visceral adipose of obese rats.
Our current data show that levels of PSSG are reduced in the adipose and hepatic tissues from the obese, glucose-intolerant animals. These findings agree with several lines of evidence indicating that levels of oxidative modification of macromolecules are necessary for optimum insulin signaling. Mice overexpressing GPX1 (EC 18.104.22.168) have insulin resistance (10). Conversely, GPX1-null animals have elevated insulin sensitivity in part owing to oxidative inactivation of the dual specificity phosphatase, phosphatase and tensin homolog (PTEN) (9). Insulin signaling is compromised in 3T3-LI adipocytes when treated with GSH or its analogs, but insulin signaling is enhanced following GSH depletion with buthionine sulfoximine (7). While GSH depletion conceivably also reduces the formation of PSSG, it would not limit modification of protein cysteine residues to PSOH, precursors of PSSG (Figure 1) that also would reduce protein activity. GSH depletion through limitation of GPX1 activity would lead to elevated peroxides that catalyze PSOH formation.
Our observation that GLRX1 protein is elevated in adipose tissue of obese rats is novel. GLRX1 is a low molecular weight protein (approximately 11 kDa) of the glutaredoxin superfamily, of which many members are conserved from yeast to mammals and have different functions (16, 18). GLRX1 is present in the cytosol and the intermembrane space of mitochondria and is highly selective toward reducing PSSG versus intramolecular/intermolecular protein disulfides. While the factors regulating the tissue-specific expression of GLRX1 are unclear, it is evident that signaling pathways are activated in the obese adipose tissue that increase GLRX1 levels.
Recent data indicate that GLRX1 activity can promote an inflammatory response. In this case, GLRX1 activity relieves the inhibition of nuclear factor kappa-B (NF-κB) activity via activation of inhibitor of NF-κB kinase subunit β (IKKβ; EC 22.214.171.124). IKKβ is inhibited through the formation of a PSSG residue at amino acid Cys 179 (34). Removal of GSH activates IKKβ that then subsequently phosphorylates NF-kappa-B inhibitor subunit beta (IK). Phosphorylation of IK causes detachment of IK from the NF-κB transcription factor complex thus allowing the NF-κB to enter the nucleus and stimulate tumor necrosis factor alpha (TNFα) and Interleukin-6 (IL6) (34). Other data indicate that NF-κB activation increases GLRX1 expression is a positive feed-forward manner (12). The extent to which GLRX1 is expressed in the various cell types constituting adipose tissues needs further study.
Mechanisms leading to the formation of PSSG were studied. Recent data indicate that GSTP can catalyze the formation of PSSG by adding GSH onto PSOH (32). However, there were no obesity-related changes in GSTP content in any of the tissues examined. The content of PSOH was not altered between lean and obese animals in the tissues examined, although there was at least one elevated PSOH in the lean adipose tissue compared to the obese tissue. This result is in contrast to elevations in protein carbonyls in adipose tissue as a result of obesity (2).
The extent to which a decrease in PSSG may be beneficial or detrimental to cellular function is nuanced. S-Glutathionylation of the mitochondrial respiratory proteins and uncoupling protein 2 (UCP2) inhibits the activities of these respective enzymes (38-40). Thus a decrease in S-glutathionylation in obesity may be viewed as an adaptive response to increase cellular energy metabolism. Conversely, as described above, decreased S-glutathionylation of the NF-κB pathways components can lead to elevated inflammation.
Our data from liver indicate the GLRX1 is not the only determinant of PSSG tissue content as the PSSG content was decreased without changes in GLRX1. We speculate that decreases in PSSG content could be the result of the presence of other GLRX family members or increases in thiol modifications by other species such as nitrosylation or carbonylation that are targeting the same protein bound thiols.
In summary, our data indicate that S-glutathionylation is reduced in the adipose and liver of obese animals with concomitant increase of GLRX1 in adipose. We are cognizant, however, that we have not performed a complete analysis of the oxidized thiol proteome. The extent to which S-glutathionylation regulates signaling pathways (e.g., insulin and inflammation) in adipose tissue needs further examination.
This publication and the use of the Core Synthesis Facility at North Dakota State University was made possible by NIH Grant Number P20 RR015566 from the National Center for Research Resources. Its contents are solely the responsibility of the authors and do not necessarily represent the official views of the NIH. We thank Dr. Irina Smoliakova, Department of Chemistry, University of North Dakota, for performing 1H NMR analysis of M2VP. We thank Dr. John Mieyal for his advice on measurement of GLRX activity and providing recombinant human GLRX1.