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Originally published In Press as doi:10.1074/jbc.M703551200 on August 28, 2007

J. Biol. Chem., Vol. 282, Issue 47, 34219-34228, November 23, 2007
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Succination of Protein Thiols during Adipocyte Maturation

A BIOMARKER OF MITOCHONDRIAL STRESS*

Ryoji Nagai{ddagger}1, Jonathan W. Brock{ddagger}, Matthew Blatnik{ddagger}, John E. Baatz§, Jennifer Bethard§, Michael D. Walla{ddagger}, Suzanne R. Thorpe{ddagger}, John W. Baynes{ddagger}2, and Norma Frizzell{ddagger}1

From the {ddagger}Department of Chemistry and Biochemistry, University of South Carolina, Columbia, South Carolina 29208 and §Children's Research Hospital and Department of Pharmacology, Medical University of South Carolina, Charleston, South Carolina 29243

Received for publication, April 27, 2007 , and in revised form, July 26, 2007.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Although obesity is a risk factor for development of type 2 diabetes and chemical modification of proteins by advanced glycoxidation and lipoxidation end products is implicated in the development of diabetic complications, little is known about the chemical modification of proteins in adipocytes or adipose tissue. In this study we show that S-(2-succinyl)cysteine (2SC), the product of chemical modification of proteins by the Krebs cycle intermediate, fumarate, is significantly increased during maturation of 3T3-L1 fibroblasts to adipocytes. Fumarate concentration increased ≥5-fold during adipogenesis in medium containing 30 mM glucose, producing a ≥10-fold increase in 2SC-proteins in adipocytes compared with undifferentiated fibroblasts grown in the same high glucose medium. The elevated glucose concentration in the medium during adipocyte maturation correlated with the increase in 2SC, whereas the concentration of the advanced glycoxidation and lipoxidation end products, N{epsilon}-(carboxymethyl)lysine and N{epsilon}-(carboxyethyl)lysine, was unchanged under these conditions. Adipocyte proteins were separated by one- and two-dimensional electrophoresis and ~60 2SC-proteins were detected using an anti-2SC polyclonal antibody. Several of the prominent and well resolved proteins were identified by matrix-assisted laser desorption ionization time-of-flight/time-of-flight mass spectrometry. These include cytoskeletal proteins, enzymes, heat shock and chaperone proteins, regulatory proteins, and a fatty acid-binding protein. We propose that the increase in fumarate and 2SC is the result of mitochondrial stress in the adipocyte during adipogenesis and that 2SC may be a useful biomarker of mitochondrial stress in obesity, insulin resistance, and diabetes.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The adipocyte is increasingly recognized as a dynamic cell that readily adapts to the changing nutritional status of the body. In a state of over-nutrition, the adipocyte responds by synthesizing and storing triglycerides, a process that may eventually lead to obesity and insulin resistance and then to diabetes. Chemical modification of proteins by advanced glycoxidation and lipoxidation end products (AGE/ALEs)3 is increased in diabetes and is strongly implicated in the development of diabetic complications (1–3). However, relatively little is known about the chemical modification of proteins in adipocytes or adipose tissue or the possible role of chemical modifications of proteins in the regulation of adipocyte metabolism during adipogenesis or diabetogenesis.

In general, AGE/ALEs are derivatives of lysine and arginine residues, formed by reaction of the amino or guanidino groups on protein with electrophilic intermediates in carbohydrate and lipid autoxidation or metabolism (4). In contrast to extracellular proteins, the lower pKa sulfhydryl group of cysteine residues is a more likely target for modification by electrophiles on intracellular proteins. We recently described S-(2-succinyl)cysteine (2SC) as a novel chemical modification of cysteine residues in proteins, formed by a Michael addition reaction between the Krebs cycle intermediate (KCI) fumarate and cysteine residues in protein (5). 2SC was detected in human serum albumin and was increased in the skeletal muscle protein and urine of diabetic rats. 2SC was also present at trace levels and increased with age in human skin collagen. Levels of 2SC correlated strongly with the concentration of the AGE/ALE, N{epsilon}-(carboxymethyl)lysine (CML) in both human skin collagen and skeletal muscle of diabetic rats, suggesting that 2SC might be a useful marker of oxidative stress and tissue damage in diabetes. In other work, we have determined that the increase in 2SC in muscle proteins in diabetes is caused by a comparable increase in intracellular fumarate concentration, suggesting that KCIs are elevated in muscle in diabetes as a result of mitochondrial stress or dysfunction. Based on these observations, we have begun to study the formation of AGE/ALEs and 2SC in other biological systems or conditions in which fumarate concentration is increased.

The 3T3-L1 fibroblast is a well studied model of cellular differentiation and adipogenesis. In response to insulin, glucose, dexamethasone, and isobutylmethylxanthine, it matures into a fat-laden adipocyte in vitro (6), a process that is characterized by an ≥10-fold increase in fumarate secretion into the medium (7). Adipocyte differentiation and maturation is associated with a dramatic shift in metabolic processes; cells rapidly metabolize glucose as they switch to triglyceride synthesis and storage. The maturation process depends on increased glucose uptake and utilization since lipid deposition increases with increasing glucose concentration in the medium (8). Like adipose tissue in diabetic animals, adipocytes become insulin-resistant during maturation in high glucose medium (8). Maturation at high glucose concentration is also accompanied by a significant increase in oxidative stress and mitochondrial superoxide production, apparently as a result of hyperpolarization of the mitochondrial inner membrane (IMM) (8). Thus, production of reactive oxygen species (ROS) can be decreased by treatment with uncouplers or by overexpression of uncoupling protein-1 (8). Alterations in mitochondrial function are also apparent in adipocytes from diabetic (db/db) mice (9), consistent with the role of mitochondrial stress or dysfunction in the development of diabetes and its complications.

We show here that both the fumarate concentration in adiocytes and the 2SC content of adipocyte proteins increase dramatically as a function of both time and glucose concentration during maturation of 3T3-L1 fibroblasts to adipocytes. We also identify several target proteins modified by 2SC in the adipocyte, but not in undifferentiated fibroblasts grown in high glucose medium, and show that in contrast to 2SC, the AGE/ALEs, CML and CEL, do not increase in response to oxidative stress during adipocyte differentiation and maturation. Our results suggest that irreversible, nonenzymatic modification of thiol proteins by fumarate may contribute to alterations in metabolism in response to mitochondrial stress during the transitions from obesity to insulin resistance and diabetes.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Chemicals—Unless otherwise noted, all chemicals were purchased from Sigma/Aldrich. [U-13C3,15N]Cysteine and d8-lysine were from Cambridge Isotope Laboratories (Woburn, MA). 2SC and [U-13C3,15N]2SC were synthesized as previously described (5). CML, d4-CML, CEL, and d8-CEL were also synthesized and analyzed as previously described (10, 11).

Polyacrylamide gels, polyvinylidene difluoride membrane, ECL chemiluminescent substrate, and BioSafe Coomassie Blue stain were purchased from Bio-Rad, and large 27.6 x 21.2-cm pre-cast gels were obtained from Jule Biotechnologies (Milford, CT). Immobiline IPG strips and ampholytes for isoelectric focusing were purchased from GE Healthcare, and Sypro Ruby gel stain and Sypro Ruby protein blot stain were from Molecular Probes (Eugene, OR). Bovine pancreatic DNase I was obtained from Pierce.

Synthesis of 2-Succinylcysteamine (2SCEA) and Preparation of Anti-2SC Antibody—2SCEA was prepared by reacting cysteamine (500 mM) with a 10% molar excess of N-ethylmaleimide in phosphate buffer (200 mM, pH 7.4) for 1 h at room temperature in the dark. 2SCEA was recovered by hydrolysis in 6 M HCl for 6 h at 110 °C followed by desalting on a Dowex-50 cation exchange resin and lyophilization. 2SCEA was detected by amino acid analysis using cation exchange chromatography on a 250 x 3-mm polydivinylbenzene sulfonate column and the sodium buffer system from Pickering Laboratories (Mountain View, CA) with post-column derivatization using o-phthaldialdehyde and fluorescence detection at excitation = 375 nm, and emission = 425 nm. 2SCEA was then collected by chromatography without post-column reaction, desalted on Dowex 50 cation exchange resin, and lyophilized. The chemical purity and identity of 2SCEA was confirmed by proton NMR, and its concentration was determined by amino acid analysis using a valine standard.

Purified rabbit polyclonal antibody against 2SC was prepared by Eurogentec (San Diego, CA). Briefly, purified 2SCEA was cross-linked to keyhole limpet hemocyanin with glutaraldehyde. A New Zealand White rabbit was inoculated with 2SCEA-keyhole limpet hemocyanin (KLH) and Freund's complete adjuvant followed by three booster inoculations with 2SCEA-KLH and Freund's incomplete adjuvant. Antiserum was collected, and the polyclonal antibody was purified by affinity chromatography on a 2SCEA-glutaraldehyde resin. By direct enzyme-linked immunosorbent assay, the purified anti-2SCEA antibody showed reactivity to 2SC-RNase and 2SC-ovalbumin but not with unmodified RNase, ovalbumin, or keyhole limpet hemocyanin. The antibody did not cross-react with N-carboxymethylalbumin, prepared by reductive alkylation with glyoxylic acid using NaCNBH3 (10) nor with proteins reduced with dithiothreitol in denaturing buffer (below) and carboxymethylated with iodoacetic acid.

Fibroblast and Adipocyte Studies—Murine fibroblasts (3T3-L1) were obtained from the American Type Culture Collection (Manassas, VA) and cultured on 10-mm plates in basal Dulbecco's modified Eagle's medium (Cellgro, Herndon, VA) containing 5 mM D-glucose supplemented with 10% fetal bovine serum (Atlanta Biologicals, Atlanta, GA). Medium was replaced at 48-h intervals throughout. Differentiation of confluent fibroblasts into adipocytes was induced by the addition of dexamethasone (0.3 µM), 3-isobutyl-1-methylxanthine (0.5 mM), insulin (10 µg/ml), and glucose (30 mM) in complete culture medium for 2.5 days (6). This mixture was then replaced with basal medium containing a range of glucose concentrations (5–30 mM) and 5 µg/ml insulin for a further 2–8 days (maturation period) during which time lipid droplets accumulated in the cytoplasm. Undifferentiated fibroblasts incubated in basal growth medium alone (5 mM glucose) or with high glucose (30 mM) served as controls. Cells were harvested in 500 µl of lysis buffer (phosphate-buffered saline containing 1% Triton X-100) and were sheared using a 21-gauge needle attached to a 1-ml syringe. The cell lysate was pulse-sonicated at 2 watts using a Model 100 sonic dismembrator (Fisher) for 1 min before DNase treatment (200 units) for 15 min in lysis buffer. Protein content was determined by the Lowry assay (11).

Triglyceride Analysis—Triglyceride concentration was measured in 5-µl aliquots of cell lysates using the Thermo DMA triglyceride assay kit and standard (Thermo Electron Corp., Waltham, MA) according to the manufacturer's instructions.

GC-MS Analysis of 2SC, CML, and CEL in Cell Lysates—Protein (2 mg) from whole cell lysate was used for analysis of AGEs and 2SC. After overnight reduction at 4 °C with 100 mM NaBH4 in 0.2 M borate buffer, pH 9.2, the protein was precipitated with an equal volume of 20% trichloroacetic acid. The protein pellet was re-suspended in 200 µl of 0.1 M phosphate buffer, pH 7.4, and lipids were removed by the addition of 1 ml of ice-cold acetone with vortexing, after which the samples were placed on ice for 15 min followed by centrifugation at 4 °C at 2000 x g for 10 min. The pellets were dried under nitrogen before the addition of isotopically labeled internal standards (d8-lysine, U-13C3, 15N-2SC, d4-CML, and d8-CEL), then hydrolyzed in 6 M hydrochloric acid at 110 °C for 24 h. The samples were dried in vacuo, and brown materials were removed by application to a C-18 Sep Pak column (1 ml; Waters, Milford, MA) followed by elution of polar amino acids with H2O. N,O-Trifluoroacetyl methyl ester derivatives were prepared (12) and then analyzed by multiple reaction monitoring GC-MS/MS on a TSQ 7000 (Thermo-Finnigan, Waltham, MA). The injection port was maintained at 200 °C, and the temperature program was 90 °C hold for 2 min, 10 °C/min from 90 to 140 °C, 3 °C/min to 220 °C, 15 °C/min to 300 °C, then hold at 300 °C for 5 min. The parent and daughter ion pairs monitored were: lysine m/z 180 > 69, d8-lysine, m/z 188 > 69; 2SC, m/z 284 > 242, U-13C3, 15N-2SC m/z 288 > 246; CML m/z 392 > 191; d4-CML m/z 396 > 195; CEL m/z 379 > 206; d8-CEL m/z 387 > 214. Quantification of analytes in protein samples was performed by isotope dilution mass spectrometry based on standard curves constructed from mixtures of known amounts of heavy labeled and natural abundance standard. The amounts of all analytes were normalized to the lysine content of the sample.

Analysis of Fumarate Concentration—Fumarate was measured in whole cell lysates (1 mg protein) according to the method of Tanaka et al. (13). Briefly, the cell lysates were treated with 20 µl of hydroxylamine at room temperature for 30 min in glass tubes before the addition of 100 nmol of nonanoic acid as an internal standard. The samples were acidified with 500 µl of 3 M hydrochloric acid, then extracted twice with 2 ml ethyl acetate and dried under nitrogen. The trimethylsilyl derivatives were prepared by the addition of 100 µl of acetonitrile and 100 µl of N-methyltrimethylsilyltrifluoroacetamide (Pierce). After incubating at 60 °C for 15 min, the samples were analyzed by multiple reaction monitoring GC-MS/MS on the TSQ 7000. For analysis of nonanoic acid and fumarate, the injection port was maintained at 200 °C, and the temperature program was 80 °C hold for 3 min, 10 °C/min from 80 to 300 °C, then hold at 300 °C for 5 min. The parent and daughter ion pairs monitored were: fumarate, m/z 245 > 147, and nonanoic acid, m/z 215 > 74. Quantification of fumarate was based on standard curves constructed from known mixtures of fumarate and nonanoic acid standards.

One-dimensional PAGE and Western Blotting—Fibroblast or adipocyte protein (50 µg) was diluted to 30 µl in deionized water before adding fresh Laemmli loading buffer containing 5% beta-mercaptoethanol (14). The samples were boiled for 10 min at 95 °C and then loaded onto 12.5% Tris-HCl polyacrylamide gels; electrophoresis was carried out at 200 V for 55 min. The separated proteins were transferred to a polyvinylidene difluoride membrane at 20 V for 45 min, and the membranes were blocked overnight in 5% nonfat dry milk. The membrane was probed with 0.5 µg of polyclonal anti-2SC antibody dissolved in 1% nonfat dry milk for 1 h. Blots were washed in 20 mM Tris-HCl, pH 7.3, containing 0.05% Tween 20 followed by the addition of goat anti-rabbit horseradish peroxidase-labeled secondary antibody (Zymed Laboratories Inc., Carlsbad, CA). After a series of washes, 2SC immunoreactivity was detected using ECL chemiluminescent substrate exposed to CL-XPosure film (Pierce).

Two-dimensional PAGE—Isoelectric focusing was performed on an Ettan IPGPhor II (GE Healthcare). Both fibroblast and adipocyte samples (250 µg of protein for 17-cm gels at pH 4–7 and 700 µg for 24-cm gels at pH 4.5–5.5) were precipitated with acetone, then dissolved in re-hydration buffer (7 M urea, 2 M thiourea, 2% CHAPS, 1% immobilized pH gradient (IPG) buffer, and 50 mM dithiothreitol). The samples were loaded in the IPGphor strip holder before the addition of the Immobiline IPG strips and were allowed to rehydrate overnight for 18 h before isoelectric focusing at 21 °C on the IPGphor IEF system using the following step programs; linear at 500 V for 45 min, linear at 1000 V for 1 h, linear for 8000 V·h for a total of 27 kV·h for 17-cm strips or linear at 200 V for 1 h, linear at 500 V for 1 h, linear at 1000 V for 45 min, linear at 2000 V for 45 min, and gradient to 3500 V for 28 h for a total of 101 kV for 24-cm strips. After electrophoresis the strips were each equilibrated in 15 ml of equilibration buffer I (6 M urea, 0.4 M Tris-HCl, pH 8.8, containing 30% glycerol (v/v), 2%SDS (w/v), 1% dithiothreitol) then shaken gently for 15 min at 21 °C. The solution was decanted and replaced with 15 ml of equilibration buffer II (6 M urea, 0.4 M Tris-HCl, pH 8.8, containing 30% glycerol (v/v), 2%SDS (w/v), 1% iodoacetamide) and incubated with gentle shaking for an additional 15 min. After equilibration, the strips were loaded onto 12.5% precast gels; the 17-cm strips were electrophoresed on a Bio-Rad Protean II system, and the 24-cm strips were electrophoresed on an Ettan Dalt 6 system (GE Healthcare). The separated proteins were fixed with 7.5% acetic acid, 10% methanol for 1 h before overnight staining with Sypro Ruby gel stain. The stained gels were imaged on a Typhoon 9400 multilaser scanner (GE Healthcare). A duplicate gel was transferred onto polyvinylidene difluoride membrane and fixed as above before staining for 15 min with Sypro Ruby blot stain. The blot was washed in water and imaged in the same manner as the gel. The blot was then washed in 150 mM Tris-HCl, pH 6.8, for 10 min before blocking and immunostaining with anti-2SC antibody as described above. 2SC-positive spots on the immunoblots were overlaid with the Sypro-stained membrane and matched to the corresponding spots on the Sypro stained gel using DeCyder two-dimensional differential analysis software (GE Healthcare). The protein spots were then manually excised from the gel using the Ettan Spot Picker (GE Healthcare).

Protein Identification by Mass Spectrometry—The excised spots were processed for MS analysis using an automated gel-processing robot (PerkinElmer Life Sciences MultiProbe II). The spots were placed in wells of ZipPlates (Millipore Inc., Billerica, MA) and washed twice with 25 mM ammonium bicarbonate in 5% acetonitrile, then dehydrated with 100% acetonitrile. Trypsin digestion was carried out overnight with sequence grade-modified trypsin by adding 15 µl of a 100 ng/ml solution in 25 mM ammonium bicarbonate. Tryptic peptides were drawn through the bottom of the ZipPlate wells then washed with 5% acetonitrile in 0.2% trifluoroacetic acid and a final wash with 0.2% trifluoroacetic acid. Peptides were eluted from the ZipPlate with 1.5 µl of 5 mg/ml{alpha}-cyano-4-hydroxycinnaminic acid in 50% acetonitrile containing 0.1% trifluoroacetic acid directly onto a MALDI plate insert then analyzed on an Applied Biosystems 4700 MALDI TOF-TOF. The data were searched using GPS software (Applied Biosystems, Foster City, CA) and Mascot (Matrix Science, London, UK).

Statistical Analysis—Data are summarized throughout as the means ± S.E. and are plotted using Sigma Plot 8 software (Systat Software, Inc. (SSI), Point Richmond, CA). Statistical analyses were performed using InStat (GraphPad software, San Diego, CA). Differences between groups were analyzed using the unpaired two-tailed t test.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Triglycerides and 2SC Increase in Adipocytes as a Function of Time and Glucose Concentration—The continuous formation of intracellular triglyceride vesicles over time and with increasing glucose concentrations was clearly visible by light microscopic observation of maturing 3T3-L1 adipocytes (not shown). The triglyceride content increased 3–4-fold compared with undifferentiated fibroblasts during 8 days of maturation in 30 mM glucose (63 versus 16 µg/mg cell protein, Fig. 1A). The increase in triglyceride content was also dependent on the glucose concentration in the medium during fibroblast differentiation (Fig. 1B). Fibroblasts cultured in 5 mM glucose and harvested at day 0 had essentially the same triglyceride content (~17 µg/mg cell protein) as fibroblasts grown for 8 days in 5 mM glucose (Fig. 1B).

2SC also increased as a function of time and glucose concentration in adipocytes, with the maximal rate of 2SC accumulation occurring between 2 and 4 days in culture and the maximum 2SC levels observed after 6–8 days of maturation (Fig. 2A). The level of 2SC in adipocytes was ~20-fold greater than that in fibroblasts after 8 days in culture in 30 mM glucose (adipocytes, 0.46 mmol 2SC/mol of lysine; fibroblasts, 0.025 mmol 2SC/mol of lysine, p < 0.0001, Fig. 2A). In a separate experiment, adipocyte 2SC also increased as a function of glucose concentration in the medium over a concentration range from 5–30 mM, rising from an average of 0.22 mmol 2SC/mol of lysine in 5 mM glucose to 0. 44 mmol 2SC/mol of lysine in 30 mM glucose (p = 0.02, Fig. 2B). 2SC also increased about 2-fold in undifferentiated fibroblasts after 8 days in culture in 30 mM glucose, but in general the increase in 2SC was dependent on both differentiation and glucose concentration. Although the concentrations of CML and CEL were comparable with that of 2SC in fibroblasts (Fig. 2, C versus A), neither CML nor CEL (Fig. 2C) changed significantly in either fibroblasts or adipocytes as a function of time of maturation. CML (Fig. 2D) and CEL (not shown) were also unaffected by glucose concentration during maturation.

Adipocytes Increase Production of Fumarate with Time and in Response to Increasing Glucose Concentration—Because fumarate is a known precursor of 2SC (5) and has been reported to increase during adipocyte maturation (7), we examined the relationship between fumarate concentration and 2SC formation in fibroblasts and adipocytes. Fumarate did not change in fibroblasts cultured for up to 8 days in 30 mM glucose but increased gradually in adipocytes as a function of time, yielding a 5–6-fold increase at 6 days (fibroblasts, 0.28 nmol fumarate/mg of protein; adipocytes, 1.6 nmol fumarate/mg of protein, p = 0.01, Fig. 3A). Mean adipocyte fumarate content also rose when cells were differentiated at higher glucose concentration compared with fibroblasts cultured under the same conditions (Fig. 3B). Notably, fumarate concentrations in adipocytes increased 3-fold when glucose in the medium was increased from 5 to 10 mM, rising from 0.66 nmol of fumarate/mg of protein to 1.9 nmol/mg but did not change further with increasing glucose concentration. In contrast, in fibroblasts the mean fumarate concentration over all glucose concentrations was 0.42 nmol/mg of protein (Fig. 3B). In general, these experiments demonstrate that exposure to high glucose both enhanced the production of fumarate and increased the formation of 2SC on protein during adipocyte maturation. The stepwise change in fumarate concentration between 5 and 10 mM glucose without further change at higher glucose concentration (Fig. 3B) suggests a threshold effect; above 10 mM glucose there is no further increase in fumarate concentration. In contrast, in Fig. 2B, there is a stepwise increase in 2SC in response to increasing extracellular glucose concentration, suggesting that fumarate rapidly reaches a steady state concentration, whereas 2SC accumulates irreversibly in protein as a function of time.


Figure 1
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  		FIGURE 1.
Intracellular triglycerides increase during adipocyte maturation as a function of both time and extracellular glucose concentration. Adipocytes (open circles, open bars) and fibroblasts (closed circles, closed bars) were incubated in 30 mM glucose for 0–8 days (A) or with increasing concentrations of glucose for 8 days (B) as described under "Experimental Procedures." Triglycerides were measured in 5 µl of cell lysate and then normalized to the protein content of the cells. Data are for a representative experiment and are the mean ± S.E., n = 3; p < 0.01 for adipocytes cultured in 5 mM versus 20 or 30 mM (B).

 


Figure 2
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  		FIGURE 2.
Intracellular 2SC, but not CML or CEL, increases during adipocyte maturation as a function of both time and extracellular glucose concentration. Cell lysates (2 mg protein) from the same samples described in Fig. 1 were analyzed for 2SC, CML, and CEL by GC-MS as described under "Experimental Procedures." Adipocytes (open circles, open bars) and fibroblasts (closed circles, closed bars) were incubated in 30 mM glucose for 0–8 days (A and C) or with increasing concentrations of glucose (5–30 mM) for 8 days (B and D). 2SC, CML (circles), and CEL (squares) were quantified by multiple reaction monitoring-GC-MS/MS. Data are for a representative experiment and are the mean ± S.E., n = 3. Note difference in scales for 2SC versus CML and CEL concentrations.

 
Detection of 2SC-Proteins in Adipocytes—To determine which proteins were being modified by 2SC during adipocyte maturation, a polyclonal anti-2SC antibody was prepared in rabbits. Using one-dimensional SDS-PAGE analysis, the antibody detected 2SC-proteins in whole cell lysates of both fibroblasts and adipocytes cultured in either 5 or 30 mM glucose for 8 days (Fig. 4). In contrast to fibroblasts, which appeared to show only one 2SC-protein at ~50 kDa, adipocytes matured in 5 mM glucose showed 3 protein bands, whereas adipocytes differentiated in 30 mM glucose yielded ~30 protein bands of varying intensity. In this and subsequent two-dimensional PAGE experiments there was no detectable staining in control experiments omitting either anti-2SC or second antibody.

Separation of 2SC-Proteins by Two-dimensional PAGE—Preliminary experiments using pH 3–10 IPG strips indicated that the majority of 2SC-modified proteins fell within the pH range of 4–7 (data not shown). Therefore, further analysis of fibroblast and adipocyte lysates was performed on narrower range, pH 4–7, 17-cm IPG strips (Fig. 5, A and B, respectively). The immunoblotting of duplicate gels (Fig. 5, C and D) showed 2SC-proteins in both fibroblasts and adipocytes, consistent with the results of one-dimensional analysis. The single band which appeared as the lone 2SC-protein in fibroblasts in Fig. 4 appeared as two distinct spots on the two-dimensional gel (Fig. 5C). Separation of adipocyte proteins by two-dimensional PAGE using a pH 4–7 gradient revealed ~60 adipocyte-specific 2SC-proteins (Fig. 5D). Despite improvements in the separation of the adipocyte proteins on the larger gel, it was difficult to match the immunoblot to the Sypro-stained gel. Therefore, a greater protein load was further separated on a narrower range (pH 4.5–5.5) 24-cm IPG strip (Fig. 6A). As in the above experiment the immunoreactive 2SC-proteins in adipocytes were detected on duplicate blots using the anti-2SC antibody (Fig. 6, B and C). Staining the blot with Sypro before treatment with anti-2SC antibody facilitated matching the 2SC-protein spots on the immunoblot with those on the Sypro-stained gel. Although 2SC immunostaining appeared very intense for some of these spots, it was still difficult to identify all of the corresponding proteins on the gel. In total, 29 spots were matched and excised from the gel and processed for identification by MALDI-TOF/TOF.


Figure 3
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  		FIGURE 3.
Fumarate accumulates in adipocytes during maturation as a function of time and extracellular glucose concentration. Cell lysates (1 mg of protein) from the same samples described in Fig. 1 were analyzed for fumarate content by GC-MS as described under "Experimental Procedures." Adipocytes (open circles, open bars) and fibroblasts (closed circles, closed bars) were incubated in 30 mM glucose for 0–8 days (A) or with increasing concentrations of glucose for 8 days (B). Data are for a representative experiment and are the mean ± S.E., n = 3.

 
Identification of 2SC-Proteins in Maturing Adipocytes—Table 1 provides a list of the 13 spots among 29 processed that were positively identified by MALDI-TOF/TOF; the top scoring peptide for each of these is indicated in Table 1. For proteins with only one positive peptide identification, the mass spectrum was examined to confirm the sequence of the peptide. The isoelectric points and molecular weights of these proteins were consistent with their location in the gel using the ExPasy ProtParam tool. Furthermore, each of the identified proteins contained at least one free cysteine residue. The data analysis procedure also included a search for any ions with an additional mass of 117, corresponding to the addition of fumarate to a cysteine residue, with formation of 2SC. In agreement with the theoretical monoisotopic masses for several cysteine-containing peptides, a mass corresponding to the presence of a 2SC residue was found for ~50% of the proteins (Table 1). Further mass spectrometric analysis of greater amounts of these proteins after immunoprecipitation with specific antibodies will be necessary to confirm the identity of the 2SC-proteins and quantify the site and extent of modification. Two other proteins were also identified, glyceraldehyde-3-phosphate dehydrogenase and adiponectin, and are the subjects of separate studies.


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  		TABLE 1
Summary of MS and MS/MS data used to identify 2SC-proteins The data obtained after processing the 2SC-immunoreactive protein spots by MALDI-TOF/TOF were analyzed using the MASCOT database-searching algorithm. The calculated isoelectric points and NCBI reference numbers for the identified proteins are listed. Column 5 indicates the percent total sequence covered by the observed masses of the peptide mass fingerprint (PMF) obtained from MS data. Additional data were obtained from MS/MS sequencing; peptides with a significant ion score were considered positive MS/MS identifications after sequence examination. The number of peptides identified by MS/MS for each protein is listed in column 6 along with the % confidence interval (% CI). The final column indicates the peptide mass fingerprint obtained for several proteins, which corresponds to possible succinated cysteines in each of these proteins. Further sequencing of these peptides will be required to confirm the actual sites of modification.

 


Figure 4
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  		FIGURE 4.
Immunoblot analysis of 2SC-proteins. Total cell protein (30 µg) from fibroblasts cultured in 30 M glucose or adipocytes cultured in either 5 or 30 mM glucose was immunoblotted with polyclonal anti-2SC antibody as described under "Experimental Procedures." A single band was detected in fibroblast (Fib) lysates. In contrast, adipocyte lysates cultured in 5 mM glucose had 3 bands (5 mM), and adipocytes in 30 mM glucose (30 mM) had up to 30 bands corresponding to 2SC-proteins. Equal protein loading was determined by the Lowry assay and confirmed by Coomassie Blue staining of the gel lanes. Numbers at the left are molecular weights of marker proteins.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
2SC as a Biomarker of Mitochondrial Stress—Maturation of adipocytes in vitro is characterized by a metabolic shift toward lipogenesis, which increases with increasing glucose concentration in the medium. Mitochondria are especially active under these circumstances, both for substrate flux and interconversion and for energy production. At high glucose concentration, there is evidence of mitochondrial stress, based on both hyperpolarization of the IMM and increased ROS production (8). The hyperpolarization and resulting backpressure on, or reduction of the electron transport chain is the likely source of increased production of ROS since treatment with uncouplers or overexpression of uncoupling proteins decreases ROS production in cells grown in high glucose media (8, 15). The hyperpolarization of the IMM probably also contributes to the increase in the intracellular concentrations and secretion of fumarate and other KCIs by adipocytes (7). Although these KCIs, including fumarate, may be produced in the cytoplasm from intermediates in lipogenesis, the hyperpolarization of the IMM suggests that the accumulation of KCIs is of mitochondrial origin, possibly because the supply of glucose exceeds the metabolic needs of the cell, either exceeding the requirements for lipogenesis or perhaps in response to a feedback mechanism for limiting excess intracellular lipid accumulation. In either case the hyperpolarization of the IMM, the increase in ROS production, and the accumulation of KCIs during adipogenesis at high glucose concentrations are consistent with mitochondrial stress. Similar increases in production of ROS and concentration of KCIs develop in muscle during strenuous exercise when increased reduction of the electron transport chain results from the lack of an adequate supply of the terminal electron acceptor, oxygen, during oxygen debt or hypoxia (16, 17). Under these circumstances KCIs would move from the mitochondrion to the cytosol by passive transport systems for dicarboxylic acids. The mitochondrial dicarboxylic transporter, mDIC, is expressed at high levels in mouse adipose tissue (18), and fumarate translocation into the cytosol is supported both by its appearance in the growth medium during differentiation of fibroblasts to adipocytes (7) and by the fact that a large number of the 2SC-proteins identified are located primarily in the cytoplasm or endoplasmic reticulum of adipocytes (Table 2). In other work, we have observed that the increase in 2SC in skeletal muscle protein of diabetic rats (5) is also the result of an increase in intracellular fumarate concentration, possibly because of pseudohypoxia (19) or even true hypoxia as a result of vascular dysfunction in diabetes. Overall, the increase in fumarate and other KCIs appears to develop during mitochondrial stress whether it occurs as a result of high rates of lipogenesis or under conditions of oxygen debt. The resulting increase in 2SC, detected in both diabetic animals (5) and in adipocytes in cell culture, may therefore serve as a useful biomarker of mitochondrial stress. McDevitt et al. (20) have noted a 2–3-fold increase in fumarate concentration in the urine of Zucker obese (non-diabetic) rats, suggesting that increases in 2SC may be characteristic of obesity and insulin resistance as well as diabetes.


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  		TABLE 2
Subcellular location, number of cysteines, and functions of 2SC-protein in adipocytes

 


Figure 5
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  		FIGURE 5.
Detection of 2SC-proteins by two-dimensional PAGE analysis of fibroblast and adipocyte lysates. Whole cell lysates (200 µg of protein) from either fibroblasts (A) or adipocytes (B) were separated on 17-cm pH 4–7 IPG strips and then electrophoresed on a 12.5% SDS-PAGE gel. Proteins were stained with Sypro Ruby and visualized at 450 nm. Duplicate gels were electrophoresed in the same manner followed by immunoblotting with anti-2SC to detect modified proteins in fibroblasts (C) and adipocytes (D). The fibroblast 2SC-protein identified at 50 kDa in Fig. 3 appears as two spots in C, whereas the adipocytes (D) have up to 60 2SC-proteins. Numbers at the right are molecular weights of marker proteins. The circle in D identifies proteins encircled on the higher resolution gel in Fig. 6B.

 


Figure 6
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  		FIGURE 6.
Identification and isolation of 2SC-proteins. Whole cell lysates (700 µg of protein) from adipocytes were separated on 24-cm pH 4.5–5.5 IPG strips and electrophoresed on a 12.5% SDS-PAGE gel. Separated proteins were stained with Sypro Ruby and visualized at 450 nm (A). A duplicate gel was transferred to polyvinylidene difluoride membrane and immunoblotted with anti-2SC antibody. A 2-min exposure of the immunoblotted membrane (B) revealed multiple intense spots corresponding to the 2SC-modified proteins; the circle represents the region circled in Fig. 5D. A longer exposure of the immunoblot (1 h) revealed a total of up to 60 protein spots (C). Protein spots that were positively identified by MALDI-TOF/TOF are identified by numbers in this figure and in Table 1.

 
In the present work we show that the accumulation of fumarate (Fig. 3, A and B) and formation of 2SC (Fig. 2, A and B) in differentiating adipocytes closely tracks the commitment to lipogenesis (Fig. 1, A and B) as a function of both glucose concentration and time of differentiation. The peak or plateau in fumarate concentration at 6 days in 30 mM glucose may result from a gradual decline in mitochondrial activity at the later stages of adipocyte maturation. Respiration, as measured by CO2 production and ATP levels, in 3T3-L1 cells cultured in 25 mM glucose also peaks at maturation days 6–8 (21). In all experiments fibroblasts contained substantially less fumarate and 2SC, even when grown in high glucose medium, indicating that it is not just the glucose concentration but the metabolic commitment to adipogenesis that leads to mitochondrial stress.

Although fumarate increased by 3–4-fold during adipogenesis (Fig. 3, A and B), succination4 of protein (2SC formation) was increased by about 10–20-fold (Fig. 2, A and B). The greater increase in succination of proteins could result from a decrease in glutathione (GSH) concentration in response to the increase in ROS production in adipocytes. The high pKa of the cysteine residue of GSH, ~9.0 (22), would limit its effectiveness in protecting nucleophilic cysteine residues in protein; the rate of succination of glyceraldehyde-3-phosphate dehydrogenase, for example, is ~250 faster than that of N-acetylcysteine at physiological pH (5). However, GSH is present at millimolar concentrations in cells, and a decrease in GSH concentration could explain the disproportionate increase in protein succination compared with fumarate concentration.

The 10–20-fold increase in 2SC in adipocytes compared with fibroblasts (Fig. 2, A and B) indicates that 2SC is exquisitely sensitive to changes in metabolism during maturation of the adipocyte. In contrast, there was no significant change in the AGE/ALEs, CML or CEL, with either time or glucose concentration. Levels of 2SC were also 10–20-fold higher than CML or CEL in adipocytes grown in 30 mM glucose (Fig. 2, A versus C), indicating that 2SC is a much more abundant modification formed in response to extracellular glucose and oxidative stress. Based on lower levels of cysteine compared with lysine in proteins, the fractional modification of cysteine residues by 2SC is even more significant compared with modification of lysine residues. The failure of CML and CEL to increase in response to oxidative stress during differentiation may result from slower rates of glycoxidation versus succination reactions. Changes in CML and CEL are more readily observed over longer time periods on long-lived, extracellular proteins.

Identification of 2SC-Proteins in the Adipocyte—There are only a few studies which identify intracellular proteins chemically modified by AGE/ALEs during growth of cells in high glucose medium (e.g. Refs. 23 and 24); in these studies only a single or a limited number of modified proteins was detected. In contrast, ~60 succinated proteins were readily detected in the adipocyte. Although only a few of these proteins have been identified thus far, these are clearly not bystander proteins. They have a diverse range of critical functions and include several cytoskeletal, cytosolic, and endoplasmic reticulum proteins (Fig. 6, Tables 1 and 2). Only one succinated protein, actin, was detected in fibroblasts (Fig. 4, lane 1); two isoforms of this protein, A-X and {gamma}-actin, were detected in both fibroblasts and adipocytes (Fig. 5, C and D). Actin is one of the most abundant structural proteins in all cell types and has a relatively long halflife, perhaps making it more susceptible to cumulative modification. Cysteine 374 has been identified as the primary site of modification of actin by lipid-derived electrophiles in vitro, and modification of this residue impairs actin polymerization (25). In addition to actin, two other cytoskeletal proteins, vimentin and tropomyosin 3, were succinated in adipocytes (Table 1). All three of these proteins were identified in a previous study on reaction of 15d-prostaglandin J2 with mesangial cells in vitro (26). Vimentin contains only one reactive cysteine that is readily modified by reversible processes such as oxidation and glutathionylation (27, 28); these modifications inhibit the formation of vimentin filaments (29). Mechanistically, succination of cytoskeletal proteins may weaken the integrity of the cytoskeleton as adipocytes differentiate and mature. Although we have not determined the quantitative extent of modification of any of these proteins, the chain is only as strong as its weakest link; even a small extent of modification of these cytoskeletal proteins may be significant in a biochemical context. Indeed, the weakening of actin and vimentin filaments may be necessary for the 3T3 fibroblast to accommodate to its evolving role during adipogenesis from a cell with negligible lipid stores to one with multiple small triglyceride vesicles and then with fewer, larger lipid vesicles.

The group of succinated proteins in adipocytes also included a number of enzymes and signaling molecules. Cathepsin B is a papain family protease with an active site cysteine residue; it has multiple roles in protein turnover and remodeling of the extracellular matrix under physiological and pathological conditions. It is also inactivated by reaction with electrophiles, including glyoxal and methylglyoxal (30). Inactivation of cathepsin B (and other thiol proteases) may inhibit degradation of cellular proteins, contributing to the greater increase in 2SC, compared with fumarate, during adipogenesis (Fig. 2, A and B, versus Fig. 3, A and B). Heat shock proteins are molecular chaperones that are highly sensitive to changes in the intracellular redox environment. One of the most important chaperone activities of Hsp 90beta is its "cradle-to-grave" regulation of the glucocorticoid receptor (31, 32). Hsp 90 co-chaperones this receptor to the nucleus where it regulates a large number of proteins associated with the progression of diabetes. Hsp 70 protein 5 (BiP) and protein disulfide isomerase are important "quality control" proteins in the endoplasmic reticulum. Large quantities of BiP are found at the endoplasmic reticulum pore where it interacts with nascent polypeptide chains to guide their proper folding (33). Fumarate modification of these proteins could interfere with protein transport and folding. Because the adipocyte is responsible for the secretion of a wide range of hormones (adipokines), including disulfide-dependent adiponectin and resistin, it is possible that modification of BiP and protein disulfide isomerase may affect hormone action in obesity, insulin resistance, and diabetes. Protein disulfide isomerase has an additional function as prolyl-4-hydroxylase. Among its activities, this enzyme catalyzes the hydroxylation of a proline residue in the transcription factor, hypoxia-inducible factor-1{alpha} (HIF), which is a rate-limiting step in proteasomal degradation of HIF. Fumarate (and succinate) are reversible inhibitors of this enzyme (34), leading to stabilization of hypoxia-inducible factor. Irreversible modification of protein disulfide isomerase/prolyl-4-hydroxylase by fumarate may provide a more permanent posttranslational mechanism for stabilization of hypoxia-inducible factor during hypoxia or pseudohypoxia in diabetes. Another 2SC-protein, 14-3-3{gamma}, is an abundant cytoplasmic adaptor molecule involved in modulating a wide range of intracellular signaling processes. Isoforms of 14-3-3 proteins are involved in insulin receptor substrate 1 intracellular trafficking in 3T3-L1 adipocytes in response to insulin stimulation (35, 36). The binding of 14-3-3 to insulin receptor substrate 1 attenuates its ability to recruit and activate phosphatidylinositol 3-kinase. As a result, insulin resistance develops, and insulin-stimulated glucose transport is affected. By targeting a key protein in the insulin signaling cascade, fumarate-dependent formation of 2SC may be intimately involved in the development of insulin resistance in the adipocyte and in other cells in diabetes. Overall, the various functions of the modified proteins identified in this study imply that succination may impact on a wide range of intracellular processes that are altered in obesity, insulin resistance, and diabetes. Further study of adipocyte proteins, the extent of their modification, and the effect of modification on function together with studies of adipose and other tissues in vivo are certain to provide fresh insights into the regulation and/or dysregulation of metabolism in obesity, insulin resistance, and diabetes. In this context we have observed 2–3-fold increases in fumarate and a greater than 10-fold increase in 2SC-proteins in epididymal fat of the db/db mouse model of obesity-dependent diabetes as early as 10–15 weeks of age and hope to translate the present studies on the adipocyte in vitro to an understanding of altered adipose tissue metabolism in diabetes.


   FOOTNOTES
 
* This work was supported by NIDDK, National Institutes of Health Research Grant DK-19971. 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. Back

1 Both authors contributed equally to this work. Back

2 To whom correspondence should be addressed: Dept. of Chemistry and Biochemistry, 631 Sumter St. (GSRC), University of South Carolina, Columbia, SC 29208. Tel.:/Fax: 803-777-7272; E-mail: john.baynes{at}sc.edu.

3 The abbreviations used are: ALE, advanced lipoxidation end-product; 2SC, S-(2-succinyl)cysteine; AGE, advanced glycoxidation end-product; CEL, N{epsilon}-(carboxyethyl)lysine; CML, N{epsilon}-(carboxymethyl)lysine; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; IMM, inner mitochondrial membrane; IPG, immobilized pH gradient; KCI, Krebs cycle intermediate; GC/MS, gas chromatography-mass spectrometry; 2SCEA, S-(2-succinyl)cysteamine; MALDI-TOF, matrix-assisted laser desorption ionization time-of-flight; ROS, reactive oxygen species; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; ROS, reactive oxygen species. Back

4 We propose the term "succination" to describe the modification of protein cysteine residues by fumarate, yielding 2SC. This distinguishes succination from succinylation, in which an ester, thioester, or amide bond would be formed. Back



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 

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M. Blatnik, N. Frizzell, S. R. Thorpe, and J. W. Baynes
Inactivation of Glyceraldehyde-3-Phosphate Dehydrogenase by Fumarate in Diabetes: Formation of S-(2-Succinyl)Cysteine, a Novel Chemical Modification of Protein and Possible Biomarker of Mitochondrial Stress
Diabetes, January 1, 2008; 57(1): 41 - 49.
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