Changes in Expression of Skeletal Muscle Proteins between Obesity

ARTICLE pubs.acs.org/jpr

Changes in Expression of Skeletal Muscle Proteins between Obesity-Prone and Obesity-Resistant Rats Induced by a High-Fat Diet Dong Hyun Kim, Jung-Won Choi, Jeong In Joo, Xia Wang, Duk Kwon Choi, Tae Seok Oh, and Jong Won Yun* Department of Biotechnology, Daegu University, Kynungsan, Kyungbuk, Republic of Korea ABSTRACT: A primary goal in obesity research is to determine why some people become obese (obesity-prone, OP) and others do not (obesity-resistant, OR) when exposed to high-calorie diets. The metabolic changes that cause reduced adiposity and resistance to obesity development have yet to be determined. We thus performed proteomic analysis on muscular proteins from OP and OR rats in order to determine whether other novel molecules are involved in this response. To this end, rats were fed a low- or high-fat diet for 8 weeks and were then classified into OP and OR rats by body weight gain. OP rats gained about 25% more body weight than OR rats, even though food intake did not differ significantly between the two groups. Proteomic analysis using 2-DE demonstrated differential expression of 26 spots from a total of 658 matched spots, of which 23 spots were identified as skeletal muscle proteins altered between OP and OR rats by peptide mass fingerprinting. Muscle proteome data enabled us to draw the conclusion that enhanced regulation of proteins involved in lipid metabolism and muscle contraction, as well as increased expression of marker proteins for oxidative muscle type (type I), contributed to obesity-resistance; however, antioxidative proteins did not. KEYWORDS: high-fat diet, obesity-prone and -resistant rats, skeletal muscle proteome

D

iet-induced obesity (DIO) in rodents has been used as an animal model to investigate the interaction between the environment and genetic background. The model of DIO in the Sprague-Dawley (SD) rat is of special interest with regard to regulation of energy homeostasis. When out-bred SD rats are placed on an energy-dense high-fat diet (HFD), there is a wide distribution in body weight gain: a subset of animals becomes very obese (OP), whereas others remain as lean as animals fed even a HFD (OR).1,2 The DIO model better reproduces human obesity than genetic models of obesity, as there are only a few patients reported in the literature in which mutations of leptin or leptin receptor genes account for obesity.3 DIO develops only when rats are exposed to diets that are moderately high in fat and energy. Thus, it is analogous to many types of human obesity and permits the study of factors that predispose to and perpetuate obesity.4 Once exposed to a high energy diet, OR and OP rats differ markedly in their regulation of food intake and metabolic efficiency. A large number of factors have been shown to be associated with phenotypic expression of OR and OP traits, including strain (genetic differences), age, diet, physiological signaling, and responsiveness, as well as metabolic processes.5-12 However, factors that make some rats susceptible and others resistant to DIO are unclear. Several lines of evidence indicate that many factors are associated with differences in obesity susceptibility, including composition, fat oxidation and oxidative capacity, glucose uptake and disposal, thermogenic capacity, and mitochondrial activity in skeletal r 2010 American Chemical Society

muscle.6,13-16 Nevertheless, the metabolic changes that cause reduced adiposity and resistance to obesity development have not yet been determined. We therefore hypothesized that other factors are likely to contribute to this response. We thus performed proteomic analysis on muscular proteins from OP and OR rats to determine whether other novel molecules are involved in this response. Skeletal muscles are involved not only in contraction but also heat homeostasis and metabolic integration. Accumulating evidence shows that inefficient skeletal muscle lipid utilization may be associated with development of obesity.17 For this reason, muscle proteomics is an important tool for understanding whole-body physiology and pathogenesis of obesity.18-20 Therefore, in the present study, we tested this hypothesis using a proteomic approach in order to determine whether SD rats fed a HFD are able to avoid obesity development through an increase in body energy expenditure and lipid oxidation in the gastrocnemius muscle. A line of studies on differential proteome analysis were undertaken in order to profile muscular proteins in mammals under various physiological conditions, including aging,21-23 cold stress,24 chronic hypoxia,25 highintensity exercise,26 and burn injury.27 However, data on proteomic study in skeletal muscle in response to DIO are scarcely available.24,28 In the present study, we have used 2-DE, mass spectrometry, and immunoblotting to determine differential expression of Received: October 19, 2010 Published: December 10, 2010 1281

dx.doi.org/10.1021/pr101048q | J. Proteome Res. 2011, 10, 1281–1292

Journal of Proteome Research

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Table 1. Composition of High-Fat Diet Used in This Study composition by weight component

g/kg

casein

200

cornstarch

155

sucrose

50

dextrose

132

cellulose

50

soybean oil

25

corn oil

0

lard mineral mix

175 35

vitamin mix

10

stored at -80 °C for further use. Tissues were mixed with rehydration buffer solution containing 7 M urea, 2 M thiourea, 4% CHAPS, 20 mM DTT, 2% IPG buffer of pH 3-10, and a trace of bromophenol blue and then homogenized. Extracts were centrifuged at 14,000 g for 20 min; the supernatant was then transferred into new tubes. Protein from the supernatant was precipitated using 20% trichloroacetic acid (TCA) and acetone prior to electrophoretic separation, and the precipitate was resuspended in rehydration buffer. The protein concentration was determined by the Bradford method29 using Bradford reagent (Sigma-Aldrich, St. Louis, MO, USA). All samples were stored at -80 °C until analysis. 2-DE

TBHQ

0.014

DL-methionine

0

L-cystine

3

choline bitartrate

2.5 composition by calories

component

%

protein carbohydrate

20 35

fat

45

total: 4776 kcal/kg

muscular proteins in response to HFD feeding. In order to avoid potential artifactual enrichment or loss of muscle proteins during extensive subcellular fractionation procedures, this comparative study employed total muscle extract. To the best of our knowledge, this is the first proteomics study to identify marker proteins in rodent muscle for determination of phenotype of obesity susceptibility and resistance.

’ MATERIALS AND METHODS Animal Experiments

Animal experiments were performed on Sprague-Dawley (SD) male rats purchased from DBL (Daehan Biolink, Seoul, Korea) at 4 weeks of age and housed one per cage in a room maintained under constant temperature at 22-25 °C with ∼55% relative humidity and on a 12/12-h light-dark cycle with lights on at 08:00 a.m. All procedures were conducted in accordance with the “Guide for the Care and Use of Laboratory Animals” published by the National Institutes of Health. A total of 37 rats were randomly selected and fed with an HFD for 8 weeks, based on a previous formula, with minor modifications, while the remaining 8 rats were kept on standard chow over the experimental period. Diet compositions used in this study are presented in Table 1. Body weight of each animal was measured every 2 days. Animals with body weights in the upper quartile were labeled as OP (n = 6), and those in the lower quartile were labeled as OR (n = 6) in the HFD-fed group. Preparation of Muscle Samples

Rat gastrocnemius muscle was taken from individual groups. Muscle tissues were excised immediately after sacrifice and then washed with a cold saline solution. The resulting samples were pulverized to a powder with liquid nitrogen in a mortar and

2-DE was performed by an optimized procedure, as described previously,30,31 using protein extracts from the gastrocnemius muscle of six rats per each experimental group, which consisted of normal controls who were fed a low fat diet and OP and OR rats fed an HFD. In addition, in order to minimize gel-to-gel variation, an internal standard for muscle generated by pooling an aliquot of protein extracts (100 μg) in each (n = 6) and three sheets of 2-DE gels per each group were prepared. For analytical gels, IPG strips of pH 3-10 nonlinear (NL) (18 cm; GE healthcare, Buckinghamshire, U.K.) were rehydrated for 20 h in rehydration buffer containing 100 μg protein. After rehydration, isoelectric focusing (IEF) was conducted using the PROTEIN IEF cell (BioRad, Hercules, CA, USA) at 20 °C, 15 min at 250 V, 3 h at 250-10,000 V, 6 h at 10,000 V, and then held at 500 V. For the second dimension, gel strips were equilibrated for 15 min in 6 M urea, 2% SDS, 30% glycerol, and 50 mM Tris (pH 8.8), followed by replacement of DTT with 2.5% iodoacetamide for 15 min. The strips were loaded on a 20 20 cm2 SDS-polyacrylamide gel at 25 °C, 15 mA/gel, and the gels were then stained using a silver staining method. Silver staining was performed as follows: Gels were fixed for 30 min in 50% ethanol (Duksan Pure Chemicals, Anshan, Korea) and 5% acetic acid (Duksan Pure Chemicals), followed by 10 min in 30% ethanol, and water-washed for 5 min three times. Gels were sensitized for 10 min in 0.02% sodium thiosulfate (Sigma-Aldrich), followed by 0.5 min water washes three times, and incubated for 25 min in 0.3% silver nitrate (AgNO3; purity 99.9%, Kojima Chemicals, Sayama, Japan). Following two 0.5 min water washes, proteins were visualized with developing solution containing 3% sodium carbonate, 0.02% sodium thiosulfate, and 0.05% formalin, and then stopped with 6% acetic acid. Image Analysis

Gels were imaged on a UMAX PowerLook 1120 (Maxium Technologies, Akron, USA), and the resulting 16-bit images were converted to TIF format prior to export and analysis. Differential protein expression pattern was performed using a modified version of Imagemaster 2D software V4.95 (GE healthcare). For each experiment, a reference gel was selected at random from gels of the control group, and protein spots were then automatically detected. Automatic spot detection was refined by adjustment of detection parameters (number of smoothing passes, saliency of the spot feature, and minimum area). Detection parameters were set as follows: 2 smooth passes, a 10 saliency value, and 15 minimum areas. If necessary, the spots were edited manually. Spots detected from the other gels in the control data set were matched to those in the selected reference gel. Relative optical density and relative volume were also calculated to correct for differences in gel staining. Each spot intensity volume was processed by background subtraction and total spot volume normalization; the resulting spot volume 1282

dx.doi.org/10.1021/pr101048q |J. Proteome Res. 2011, 10, 1281–1292


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Table 2. Proteins Showing Differential Expression in Rat Gastrocnemius Muscle in Normal, OP, and OR rats alterations (% volume)b spot no.

protein

acc. no.a

theoretical Mw (kDa)

theoretical pI

normal

OP

OR

coveragec

scored

1 2 3 4

rCG43676-like glycogen phosphorylase ATP synthase subunit R adenylosuccinate synthase like 1-like isoform 2 acyl-coenzyme A thioesterase 2 elongation factor 1-γ cytosolic aspartate aminotransferase protein DJ-1 electron transfer flavoprotein subunit R R-enolase rCG60221, isoform CRA_b unidentified creatine kinase unidentified succinate dehydrogenase flavoprotein subunit R B-crystallin desmin myoglobin unidentified pyruvate dehydrogenase E1 component subunit β triose phosphateisomerase 1 tropomyosin R NADH dehydrogenase iron-sulfur protein 3 pyruvate degydrogenase complex X vinculin 2-oxoglutarate dehydrogenase

gi|293349643 gi|158138498 gi|40538742 gi|109479984

29.5 97.7 59.8 50.5

10.6 6.7 9.2 8.6

0.144 ( 0.019 0.194 ( 0.023 0.913 ( 0.07 0.067 ( 0.008

0.202 ( 0.001* 0.263 ( 0.059 1.401 ( 0.017* 0.029 ( 0.007*

0.146 ( 0.003** 0.206 ( 0.013 1.018 ( 0.123* 0.074 ( 0.011*

30 40 31 14

65 334 148 92

gi|6166586 gi|51948418 gi|220684

49.9 50.3 46.6

7.7 6.3 6.7

0.052 ( 0.011 0.078 ( 0.004 0.567 ( 0.046

0.095 ( 0.003* 0.094 ( 0.002* 0.721 ( 0.022

0.059 ( 0.01* 0.061 ( 0.006* 0.481 ( 0.028*

16 21 31

69 101 157

gi|16924002 gi|57527204

20.2 35.3

6.3 8.6

0.492 ( 0.026 0.262 ( 0.063

0.574 ( 0.034 0.35 ( 0.021

0.361 ( 0.039* 0.227 ( 0.037*

60 30

125 125

gi|158186649 gi|149066001

47.4 21.1

6.2 5.9 7.9

30

109

gi|18426858

72.6

6.8

0.19 ( 0.004* 0.055 ( 0.006* 0.039 ( 0.005* 0.382 ( 0.052 0.031 ( 0.002* 0.032 ( 0.002*

116 91

33.4

0.279 ( 0.021 0.102 ( 0.013 0.069 ( 0.006 0.363 ( 0.028* 0.044 ( 0.001 0.052 ( 0.005

30 25

gi|203480

0.221 ( 0.069 0.079 ( 0.012 0.049 ( 0.032 0.207 ( 0.097 0.026 ( 0.009 0.039 ( 0.012

12

86

gi|57580 gi|38197676 gi|11024650

19.9 53.4 17.2

6.8 5.2 7.8 6.2

0.283 ( 0.032* 0.343 ( 0.101 0.094 ( 0.02* 0.028 ( 0.027 0.029 ( 0.016

121 262 160

39.3

0.478 ( 0.007* 0.491 ( 0.013* 0.057 ( 0.001* 0.043 ( 0.006* 0.057 ( 0.001*

46 44 53

gi|56090293

0.612 ( 0.03 0.327 ( 0.126 0.161 ( 0.021 0.02 ( 0.005 0.069 ( 0.002

22

99

gi|38512111 gi|157787199 gi|157817227

27.2 32.7 30.3

7.1 4.7 7.1

0.021 ( 0.003 0.044 ( 0.002 0.113 ( 0.018

0.044 ( 0.005* 0.079 ( 0.019* 0.177 ( 0.003*

0.028 ( 0.021 0.051 ( 0.014 0.096 ( 0.023*

60 29 30

180 88 156

gi|60688224

41.0

8.8

0.039 ( 0.001

0.061 ( 0.001**

0.059 ( 0.013

24

106

gi|157822133 gi|62945278

117.1 117.4

5.8 6.3

0.008 ( 0.002 0.021 ( 0.003

0.019 ( 0.001* 0.038 ( 0.004*

0.011 ( 0.002* 0.027 ( 0.004

14 15

128 158

5 6 7 8 9 10 11 13 14 15 17 18 19 20 21 22 24 23 25 26 27 28

a NCBInr database accession number. b Statistical significance was determined by t-test, where p-value is *p < 0.05 and **p < 0.01; normal versus OP and OP versus OR group, respectively. c Percent of identified sequence to the complete sequence of the known protein. d Protein scores greater than 61 are significant (p < 0.05).

percentage (%Volume) was used for comparison. %Volume ratios of the differentially expressed protein spots were represented, and standard deviations were provided for elucidation of real changes, as shown in Table 2. For consideration as a real change in gastrocnemius muscle proteins in each group, spots were selected based on the following criteria: p < 0.05 and an average g1.2-fold or > -1.2-fold.32 Enzymatic Digestion of Protein in Gel

Protein spots were enzymatically digested in gel in a manner similar to that previously described by Shevchenko et al.33 using modified porcine trypsin. Gel pieces were washed with 50% ACN to remove SDS, salts, and stain. The gel was then dried to remove the solvent, rehydrated with trypsin (8-10 ng/μL), and incubated for 8-10 h at 37 °C. The proteolytic reaction was terminated by addition of 5 μL of 0.5% TFA. Tryptic peptides were recovered by combining the aqueous phase from repeated extractions of gel pieces with 50% ACN. After concentration, the peptide mixture was redissolved in buffer and desalted using C18ZipTips (Millipore, Watford, U.K.), and peptides were eluted with 1-5 μL of ACN. An aliquot of this solution was mixed with an equal volume of saturated solution of CHCA in 50% ACN, and 1 μL of the mixture was spotted onto a target plate.

Protein Identification

Protein analysis was performed using an Ettan MALDI-TOF (Etthan MALDI-TOF Pro Evaluation module Version 2.0.16, GE healthcare). Peptides were evaporated with a N2 laser at 337 nm using a delayed extraction mode. They were accelerated with a 20 kV injection pulse for TOF analysis. Each spectrum was the cumulative average of 300 laser shots. Spectra were calibrated with trypsin autodigestion ion peak at m/z 842.510, 2211.1046 as internal standards. Keratin contamination peaks and trypsin ion peak at m/z 1045 were excluded from the list if at least two different keratins were identified from the same sample. Peptide masses were matched with the theoretical peptides of all proteins in the NCBI database using the Mascot search program (http://www.matrixscience.com). Protein score is -10(log P), where P is the probability that the observed match is a random event. Protein scores greater than 61 indicated significance (p < 0.05). The following parameters were used for the database search: trypsin as a cleaving enzyme; a maximum of one missed cleavage; 0.01% of maximum as peak threshold; iodoacetamide (Cys) as a complete modification; methionine as a partial modification; monoisotopic masses; and a mass tolerance of (0.1 Da. 1283



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Western Blot Analysis

Tissue lysates were prepared with RIPA buffer (Sigma-Aldrich), homogenized, and centrifuged at 14000 g for 20 min. Extracts were diluted in sample buffer (50 mM Tris of pH 6.8, 2% SDS, 10% glycerol, 0.1% bromophenol blue, and 5% β-mercaptoethanol) and heated for 5 min in a boiling bath. The samples were then subjected to SDS-PAGE for electrophoresis and transferred to PVDF membranes (NEN, Boston, MA, USA). The membranes were subsequently blocked with 5% nonfat dry milk in Tris-buffered saline (TBS-T, 10 mM Tris-HCl, 150 mM NaCl, pH 7.5) containing 0.1% Tween-20. After washing with TBS-T, the membranes were probed with primary antibody. The following antibodies were used in this study: anti-rabbit AMPK, p-AMPK, CPT-1, GLUT4, and troponin I (Santa Cruz Biotechnology, Santa Cruz, CA, USA); anti-goat UCP1, UCP2, UCP3, Cyt C, Gpx, and SOD2 (Santa Cruz Biotechnology); anti-mouse glycogen phosphorylase and β-actin (Santa Cruz Biotechnology); anti-rabbit ACC, p-ACC, and FAS (Cell Signaling Technology, Beverly, MA, USA),; anti-rabbit myoglobin (Young In Frontier, Seoul, Korea); and anti-rabbit tropomyosin R (Abcam, Cambridge, U.K.). After washing with TBS-T, the membrane was incubated for 2 h with horseradish peroxidase-conjugated anti-goat IgG, antimouse IgG, and anti-rabbit IgG secondary antibody (1:1000; Young In Frontier) and developed using Enhanced Chemiluminescence (Intron, Seoul, Korea). Western blot analysis was conducted by scanning with a UMAX PowerLook 1120 (Maxium Technologies) and digitalized using image analysis software (KODAK 1D, Eastman Kodak, Rochester, NY, USA).

Figure 1. Phenotype of OP and OR rats fed a HFD. Body weight gain of normal rats fed a LFD, as well as OP and OR rats fed a HFD. They were weighed every other day for the 56-day duration of the study. Data are presented as mean ( SE for six rats per group and are estimated using the ANOVA test. Asterisk indicates statistical significance (*p < 0.05) between each group.

Determination of the Relative Mitochondrial Copy Number

Genomic DNA was extracted and purified from approximately 30 mg of frozen muscle tissue using the G-spin Genomic DNA Extraction Kit (Intron). Mitochondrial DNA (mtDNA) content relative to the peroxisome proliferator-activated receptor-γ coactivator 1 R (PGC1-R) gene was measured using real-time PCR (Stratagene mx 3000p QPCR System).34 Primers for mtDNA were forward 50 ACACCAAAAGGACGAACCTG-30 and reverse 50 -ATGGGGAAGAAGCCCTAGAA-30 and for PGC1-R forward 50 -ATGAATGCAGCGGTCTTAGC-30 and reverse 50 -AACAATGGCAGGGTTTGTTC-30 . Statistical Analysis

All experimental results were compared by one-way analysis of variance (ANOVA) using the Statistical Package of Social Science (SPSS) program, and data were expressed as the mean ( SE (n = 6). Group means were considered significantly different at p < 0.05, as determined by the technique of protective least-significant difference (LSD) when ANOVA indicated an overall significant treatment effect.

’ RESULTS

Figure 2. Representative 2-DE gel image of silver-stained gastrocnemius muscle proteins of rats. Differentially regulated proteins are indicated by arrows together with identified major proteins in Table 2. Data are representative of at least four independent experiments.

HFD-Induced Phenotypes of OP and OR

Rats were randomly divided into two groups, with 8 rats fed an LFD and 37 rats fed an HFD, and the latter was subdivided into OP (n = 6) and OR rats (n = 6) according to their body weight gain. They represented two distinct distributions and the highest and lowest weight gainers existed at the tail ends of a normal distribution. However, some rats overlapped in body weight between groups; thus, we excluded them for reliable proteomic study when rats were divided into OP and OR groups. We also performed a power analysis for calculation of the minimum sample size for proteomic experiments beforehand and finally selected 6 rats in each group for this study. Changes in body

weight between individuals during the experimental period are shown in Figure 1. Body weights of rats were much the same at the beginning of this study but began to diverge after 4 weeks, such that OP rats were heavier (p < 0.01) than normal controls and OR rats at all subsequent time points. Accordingly, total body weight of the OP rats was higher by an average of approximately 25% than that of OR rats (Figure 1). No significant difference was observed in total food intake per total body weight of rats between rats of the OP and OR groups as well as normal rats (data not shown). These results prompted us to perform further proteomic studies. 1284



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Table 3. Expression Patterns of Proteins Determining Fastand Slow-Twitch Muscle Types of Rats before and after HFD Feeding fold changea protein

OP/Norb

OR/Norb

Cytoskeletal Structure desmin R B-crystallin

1.50

1.05

-1.28

-2.74

Metabolic Regulation glycogen phosphorylase

1.36

1.06

1.02 -1.15

1.00 1.00

GAPDH

1.09

1.09

aldolase

1.10

1.05

phosphofructikinase

1.04

1.04

ATP synthase subunit R

1.53

1.12

β-enolase pyruvate kinase

Contractile Apparatus myosin light chain 1

-1.09

-1.13

myosin light chain 2

-1.11

-1.16

1.80

1.16

tropomyosin R a

Proteins significantly up-regulated (g1.2-fold) in OP rats compared with normal and OR rats are highlighted in bold. b Notations for experimental group division: Nor, normal-diet rat group; OP, obesityprone rat group; OR, obesity-resistant rat group.

Separation and Identification of Skeletal Muscle Proteins

For identification of differentially expressed proteins, muscle protein samples were separated on 2-DE (Figure 2) and identified by MALDI-TOF-MS, as well as database searches with high confidence based on high score and sequence coverage. Using 2D Imagemaster software, more than 2000 spots were detected, ranging from 15 to 200 kDa masses between pH 3-10. Overall, the gastrocnemius protein map and identified proteins were almost matched with a protein database of rat skeletal muscle.19,35 Proteomic analysis demonstrated that 26 proteins among a total of 658 matched spots on the 2-DE map were differentially expressed, of which 23 proteins spots were identified as examined obesity associated proteins, and 3 spots were found to be unidentified by MS (Table 2). Alterations of Muscle Fiber Type Marker Proteins in Normal, OP, and OR Rats

Prior to comparison of differentially altered proteins between OP and OR rats, we first compared the protein levels of muscle fiber type markers in normal, OP, and OR rats. Table 3 summarizes identified spots associated with muscle type and their altered levels. Among these proteins, 4 proteins (desmin, glycogen phosphorylase, ATP synthase subunit R, and tropomyosin R) were up-regulated in the OP group, compared with the normal and OR group (Table 3, indicated in bold), whereas one protein (R B-crystallin) was down-regulated in both the OP and OR groups, compared with the normal group (Table 3). When extended to the results of immunoblot analysis, troponin I (a fast-twitch skeletal muscle isoform and a dominant protein in type II muscle fiber) was down-regulated, whereas myoglobin (a dominant protein in type I muscle fiber) was up-regulated in OR rats. This result suggests that these proteins played a pivotal role in determination of muscle phenotypes, thereby affecting HFD-induced obesity-resistance.

Alteration of Muscle Protein Levels between OP and OR Rats

Results from protein mapping by 2-DE showed that most of the identified proteins were up-regulated in OP rats upon HFD feeding, but not in the OR rat group. These included rCG43676-like (þ1.38, p = 0.0015), glycogen phosphorylase (þ1.28, not significant (NS)), ATP synthase subunit R (þ1.38, p = 0.0481), acyl-CoA thioesterase 2 (þ1.61, p = 0.0429), elongation factor 1-γ (þ1.54, p = 0.015), cytosolic aspartate aminotransferase (þ1.50, p = 0.0109), protein DJ-1 (þ1.59, p = 0.0278), electron transfer flavoprotein subunit R (þ1.54, p = 0.0443), R-enolase (þ1.47, p = 0.0273), rCG60221 isoform CRA_b (þ1.85, p = 0.0404), succinate dehydrogenase flavoprotein subunit (þ1.63, p = 0.0437), R B-Crystallin (þ1.69, p = 0.014), desmin (þ1.41, NS), pyruvate dehydorgenase E1 component subunit β (þ1.97, NS), tropomyosin R (þ1.55, NS), triose phosphateisomerase 1 (þ1.57, NS), NADH dehydogenase iron-sulfer protein 3 (þ1.79, p = 0.0401), vinculin (þ1.73, p = 0.0428), and 2-oxoglutarate dehydrogenase (þ1.41, NS) (Tables 2 and 3). In contrast, two proteins were down-regulated, including adenylosuccinate synthase like 1-like isoform 2 (-2.55, p = 0.043) and myoglobin (-1.67, p = 0.0445) in OP rats compared with OR rats (Tables 2 and 3). Our results provide the first evidence for differential expression of key muscle proteins (e.g., myoglobin, tropomyosin, and troponin I) in response to an HFD. These proteins showed significant differences in their expression levels between OP and OR rats. Myoglobin was significantly up-regulated, whereas tropomyosin R and troponin I were markedly down-regulated in OR rats, mimicking normal rats. These results provide the possibility that OR rats have higher locomotive activity than OP rats through enhanced regulation of muscle contraction, thereby leading to body weight reduction. Immunoblot Analysis for Some Muscular Proteins

Immunoblot analysis was performed for validation of proteomic results for three proteins of interest in muscle, including myoglobin, tropomyosin R, and glycogen phosphorylase. As shown in Figure 3, there was good agreement between the results of 2-DE and Western blot analysis. In addition, we also examined the differential patterns of some skeletal muscle proteins associated with mitochondrial uncoupling and antioxidative proteins. We obtained interesting results, which showed significantly higher expression levels of UCPs, Cyt C, Gpx, and SOD2 in HFD-induced OP rats, compared with rats fed a normal diet. However, expression patterns of these proteins in OR rats were quite different from those of OP rats, mimicking rats fed a normal diet. However, this was not the case with UCP3, which showed expression levels that were similar to those observed in OP rats (Figure 5A). These results drove us to hypothesize that mitochondrial contents or activity might be higher in OR rats; thus, we measured the mitochondrial contents of skeletal muscle. Figure 5B clearly shows that relative mitochondrial copy number was significantly increased after HFD feeding, particularly in OR rats. In addition, phosphorylation of AMPK was dramatically elevated in OR rats, although the total amount of AMPK protein did not change (Figure 6). Expression levels of GLUT4 and CPT1 were also increased in the OR rats group, while the level of phosphorylated ACC was decreased in OR rats upon HFD feeding (Figure 6). Collectively, these results suggest that OR rats have higher adaptive metabolic activity against HFD feeding and thus exhibited obesityresistance resulting from higher mitochondrial activity.

’ DISCUSSION Since skeletal muscle is a major determinant of resting metabolic rate, it has been suggested that differences between 1285



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Figure 3. Comparison of expression patterns of various proteins determining fast- and slow-muscle types in normal, OP, and OR rats. (A) Zoom-in gel images for altered levels of muscle fiber marker proteins. (B) Western blot analysis of muscle fiber markers. Band density was digitized with software, and mean ( SE of three independent experiments are shown; statistical significance was determined by t-test where p-values are *p < 0.05 and **p < 0.01.

individuals in muscle oxidative capacity might play a role in the pathogenesis of obesity.17 Skeletal muscle composition is one of the important factors in regulation of body weight and is associated with DIO.6 An average daily respiratory quotient that was significantly lower than that of OR rats has been demonstrated in OP rats, suggesting greater daily fat oxidation in OR rats. A greater proportion of type I fibers has been known to be associated with a greater capacity for fat oxidation, which would favor resistance to body fat accumulation.6 In addition, diet-induced OP rats showed decreased content of type I muscle fibers and oxidative capacity.36-38 Other investigators have also proposed a negative relationship between adiposity and the relative percentage of type I muscle fibers and an increased percentage of type II muscle fibers in patients with type 2 diabetes.39,40 However, a contradictory result reported that genetically type II fiber dominant rats were more obesity-resistant, suggesting that muscle oxidative capacity rather than muscle fiber composition was a possible determinant of dietary obesity-resistance.13 In addition, no difference in muscle composition between OP and OR rats has been observed.41 Instead, the ratio of fat oxidation expressed as β-hydroxyacyl CoA dehydrogenase/citrate synthase

was significantly increased in OP rats, but not in OR rats. To clarify this issue, we compared expression patterns of several muscular marker proteins in normal, OP, and OR rats. Our proteomic data demonstrated that most glycolytic markers, including β-enolase, pyruvate kinase, GAPDH, aldolase, and phosphofructikinase did not show differential change between OP and OR rat muscle (Table 3). However, differential changes were observed in some contractile and structural proteins. Higher protein levels of desmin (þ1.50-fold, p < 0.05) and tropomyosin R (þ1.80-fold, p < 0.05) were detected in OP rats than in normal and OR rats (Table 2 and 3, Figure 3). This result is in line with the report by Okumura and co-workers, who found that the extensor digitorum longus (EDL) muscles (type II fiber) contained higher levels of structural (desmin) and contractile (tropomyosin R) proteins than the soleus muscles (type I fiber) in Wistar rats.42 We also found increased expression of myoglobin (dominant in type I muscle fiber) as well as decreased troponin I and glycogen phosphorylase (dominant in type II muscle fiber) in OR rats. This result indicates a shift in muscle fiber distribution from type II to type I due to resistance to developing obese states.36,43 Higher expression of PGC1R in OR 1286



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Figure 4. Differential expression levels of skeletal muscle proteins in normal, OP, and OR rats. Zoom-in gel images and their volume density for expression patterns of five muscle proteins. Spot density was digitized with software, and mean ( SE of four experiments are shown, *p < 0.05. Group normal versus Group OP and Group OP versus Group OR.

rats supports this result, because PGC1R is an essential factor for both mitochondrial biogenesis and muscle fiber type transformation.44 Differentiation into type I muscle fiber accompanied by a decrease in the amount of tropomyosin R has been demonstrated.45 These results are consistent with the notion that type I muscle fiber has higher oxidative activity while type II fiber has higher glycolytic activity.42 Taken together, our results provide conclusive proof that HFD-induced obesity-resistance resulted, at least in part, from shifting of muscle fiber distribution from type II to type I muscle fibers. Of particular interest in this proteomics study was the upregulation of vinculin in skeletal muscle of OP rats and not in normal and OR rats (Figure 4). Vinculin is localized at the cytoplasmic surface and closely associated with cell adhesion.46 This protein is ubiquitously expressed in various tissues and plays a role in biological processes, including cell motility, migration, development, and wound healing.47 Several studies have indicated that vinculin had a tumor suppressor function by inhibition of tumorigenic and metastatic potential.48 In addition, Barcelo-Batllori et al.49 reported that protein expression of vinculin was increased in adipose tissue of diet-induced obese rats. However, we failed to interpret the result of overexpression of vinculin in muscle of OP rats; thus, further study will be needed for clarification of this result. A very exciting finding in the present study was the identification of protein DJ-1, which was significantly down-regulated in OR rat muscle in response to an HFD. Earlier studies showed that the DJ-1 protein, which is ubiquitously expressed in various tissues, has a role in protection against oxidative stress.50-53 Indeed, DJ-1 protein has been shown to stabilize Nrf2, which is a master regulator of antioxidant gene responses.54 To date, no evidence has linked protein DJ-1 to obesity. However, one possible explanation for the reduced levels of this protein in OR rat muscle may be that OR rats do not require increased

levels of antioxidant proteins. This result was supported by reduced levels of other antioxidant proteins, such as cytochrome c (Cyt C), glutathione peroxidase (Gpx), and superoxide dismutase 2 (SOD2), as will be discussed later. Of particular interest, we found reduced levels of acyl-CoA thioesterases (ACOT) in OR rat muscle. ACOT play important roles in β-oxidation and fatty acid overload.55 This enzyme hydrolyzes acyl-CoA esters to the free fatty acid and coenzyme A (CoASH) for β-oxidation and is ubiquitously expressed in cytosol, mitochondria, and peroxisomes.56 Highly expressed mitochondrial ACOT are regulated by peroxisome proliferator-activated receptor R (PPARR), resulting in increased activity of β-oxidation.56,57 In addition, moderate overexpression of uncoupling protein 3 (UCP3) in skeletal muscle led to increased expression levels of mitochondrial ACOT-1 mRNA.58 The role of increased mitochondrial ACOT-1 expression has been considered in abnormal conditions, such as fasting and excess of intramyocellular lipid facilitate β-oxidation with UCP3 by recycling of CoASH and fatty acid at the expense of ATP.56,58 In the present study, mitochondrial ACOT-2 showed marked up-regulation in OP rat muscle, compared with normal and OR rats (Figure 4). This result is in line with upregulation of UCP3 in HFD-induced obese rats (Figure 5). Similarly, muscular electron transfer flavoprotein (ETF) was down-regulated in OR rats. β-Oxidation of fatty acid in mitochondria requires several dehydrogenases and ETF.59 ETF serves as an obligatory electron acceptor for fatty acid oxidation and is then transferred to the respiratory chain.60 It has been recognized that expression of ETF-R and several dehydogenases involved in β-oxidation are regulated by various factors, including development, hormones, and nutrition.61 Therefore, expression level of ETF is an important indicator for capacity of β-oxidation. A previous study revealed that the ratio of β-oxidation was increased in HFDinduced OP rats, but not in OR rats.41 Taken together, reduced 1287



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Figure 5. Effects of HFD on expression of UCPs and some antioxidant proteins (A) as well as mitochondrial contents (B) in normal, OP, and OR rats. (A) Band density was digitized with software, and mean ( SE of three independent experiments are shown for each group, where values not sharing a common letter are statistically different by least-significance difference post-hoc comparison (p < 0.05). (B) Relative mitochondrial copy number in skeletal muscle as determined by quantitative PCR (n = 4, for each group). Statistical significance was determined by t-test, where p-values are *p < 0.05 and **p < 0.01.

levels of ACOT and ETF in OR rat muscle is supposed to reflect lower levels of accumulated lipid in OR rat muscle, thereby resulting in no requirement for an increase in the levels of these proteins. Otherwise, reduced expressions of ACOT and ETF were presumably a consequent result of a higher capacity for lipid oxidation in OR rats. Another interesting finding in this study is up-regulation of elongation factor-1 (EF-1). EF-1 is an important factor involved in protein synthesis by the transfer of amnoacyl-tRNA to ribosomes and is composed of four subunits: R, β, γ, and δ.62 In a previous study, it has been demonstrated that levels of EF-1R mRNA were increased in diabetic skeletal muscle; however, EF1β and γ subunits were not changed. Unbalanced expression of different subunits was proven to contribute to altered protein synthesis in a diabetic state.63 Of particular interest, the mRNA level of EF-1γ was up-regulated in an obese state induced by long-term HFD feeding.64 We observed for the first time that EF1γ was significantly elevated in HFD-induced OP rat muscle, but not in normal and OR rats (Figure 4). It is well recognized that UCP1 can mediate adaptive thermogensis by proton leaks, thereby reducing the efficiency of ATP synthesis, particularly in brown adipose tissue.65,66 In contrast, UCP1

homologues UCP2 and UCP3 do not have a primary role in adaptive thermogenesis.66 Instead, earlier studies indicated that UCP2 and UCP3 may act as protection against cellular damage caused by production of excess ROS in many tissues.67-69 In addition, UCP3 export the fatty acid anion to the cytosol, resulting in increments of β-oxidation efficiency in the face of any oversupply of fatty acids.69,70 Several lines of evidence suggest that an HFD can induce thermogenesis in oxidative muscle.16,71 Kus and colleagues16 reported that muscle nonshivering thermogenesis could be stimulated by HFD in OR mice (A/J) through UCP1 activation; however, this is still a matter of long-lasting controversy. In addition, UCP3 expression also increased with HFD feeding and plays a role in mitochondrial uncoupling, thereby resulting in resistance to DIO.71,72 Li et al.73 demonstrated that expression of UCP in skeletal muscle of transgenic mice enhanced respiratory uncoupling and glucose transport, thereby preventing HFD-induced obesity. On the basis of these observations, we compared expression patterns of UCPs in normal, OP, and OR rats. Our immunoblot analysis revealed that all UCPs were significantly increased in HFD-induced OP rats, indicating that both ROS production and adaptive thermogenesis were obviously induced by an HFD (Figure 5A). This is supported by data demonstrating that alterations in ROS production are also 1288



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Figure 6. Changes in expression of proteins associated with energy expenditure and lipid metabolism in rat skeletal muscle. Band density was digitized with software, and mean ( SE of three independent experiments are shown. Statistical significance was determined by t-test, where p-values are *p < 0.05 and **p < 0.01.

clearly reflected by up-regulation of notable antioxidant enzymes, such as cyt C, Gpx, and SOD2 (Figure 5A). However, expression levels of UCP1 and UCP2 decreased in OR rats compared with OP rats. However, of particular interest, UCP3 was highly regulated in both OP and OR rat muscle, suggesting that UCP3 is likely to play a different role in OR rat muscle, compared with UCP1 and UCP2 (Figure 5A). Taken together with reduced expression of antioxidant proteins (e.g., Cyt C, Gpx, and SOD2), UCP2 and UCP3 in OR rats were unlikely to have involvement in antioxidative roles in HFD-fed rat muscle. Instead, reduced expression of UCP2 is likely involved in enhanced oxidation of lipids in OR rats, but UCP3 is not. A similar finding has been reported, showing that the higher skeletal muscle UCP2 content in obesity was positively correlated with percent of fat mass.74 Mitochondrial dysfunction during feeding of high caloric diets is regarded as an important factor causing DIO. van den Broek and coworkers demonstrated that skeletal muscle in HFD-induced obese rats required a progressively larger mitochondrial pool size for maintenance of normal oxidative capacity and increased mitochondrial content rescued muscle oxidative capacity in long-term HFD-fed rats.75 Similarly, our finding indicated that OP rats had higher mitochondrial contents than rats in the normal group. More important, OR rats had significantly increased mitochondrial contents, compared with OP rats, suggesting higher oxidative capacity (Figure 5B).

To address molecular mechanisms for phenotypic differences between muscles of OP and OR rats in terms of lipid oxidation, immunoblot analysis was extended to several relevant proteins that have not been detected in 2-DE analysis. AMP-activated protein kinase (AMPK) has emerged as a key player in regulation of energy homeostasis, influencing glucose and lipid metabolism in skeletal muscle tissue.76,77 Activated AMPK inhibits acetylCoA carboxylse (ACC) by phosphorylation, hence reducing concentration of malonyl-CoA and fatty acid synthase (FAS).77 Reduction of malonyl-CoA led to import of fatty acid into the mitochondria for β-oxidation by activation of carnitine palmitoyltransferase 1 (CPT1).77,78 In addition, activated AMPK stimulates glucose uptake into the cell by increase of glucose transporter 4 (GLUT4).78,79 The present data confirmed differential protein levels of AMPK, ACC, FAS, CPT-1, and GLUT4 among each group (Figure 6). In OP rats, excessive energy fuel substrates accumulated in skeletal muscle during HFD feeding, resulting in reduction of GLUT4 and FAS levels (Figure 6). It is well established that defects in skeletal muscle glucose uptake and disposal in rats result in the greatest susceptibility to HFD-induced obesity due to decreased insulin action. Both glucose oxidation and glycogen synthesis were significantly decreased in OP rats.80 Of particular interest, in contrast, levels of GLUT4 and FAS were markedly increased in the OR rat group (Figure 6). Several earlier studies showed that glucose transport and levels of GLUT4 were increased in skeletal muscle when ATP production was impaired by mitochondrial uncoupling 1289


Journal of Proteome Research or was elevated by expenditure of ATP by muscle contraction.81-83 In addition, activation of AMPK and phosphatidylinositol 3-kinase (PI3K) signaling by leptin-stimulated theromogenesis in skeletal muscle associated with substrate cycling between de novo fatty acid synthesis and oxidation has been demonstrated.84 Collectively, increased levels of AMPK, CPT1, FAS, and GLUT4, as well as decreased levels of ACC, suggest enhanced metabolic activity in OR rats. Finally, special attention was paid to key proteins associated with muscle contraction through proteomic and immunoblot analyses. Our results provide the first evidence for differential expression of proteins involved in muscle contraction (e.g., myoglobin, tropomyosin, and troponin I) in response to a HFD. These proteins showed significant differences in their expression levels between OP and OR rats. Myoglobin was significantly upregulated, whereas tropomyosin R and troponin I were markedly down-regulated in OR rats, mimicking normal rats. Troponin I is a part of the troponin complex, which binds to actin in thin myofilaments to hold the actin-tropomyosin complex in place. Because of its inhibitory character in muscle contraction, we hypothesized that reduced protein levels of troponin I in OR rats might affect enhancement of muscle contractile activity. In addition, higher levels of myoglobin are likely involved in promotion of muscle contractile activity in OR rats because myoglobin facilitates oxygen transport from red blood cells to mitochondria during periods of increased metabolic activity. These results imply that OR rats might have locomotive activity higher than that of OP rats through enhanced regulation of muscle contraction, thereby leading to body weight reduction. This postulation is plausible when compared with the results of Jensen et al.,85 who found that glycogen phosphorylase activation is decreased in muscles with low glycogen levels during exercise. To conclude, most of the candidate proteins identified herein by differential proteomics were previously unrecognized in skeletal muscle in response to HFD, especially in obesity-susceptibility. Our muscle proteomics study conclusively proved that enhanced regulation of proteins involved in lipid metabolism and muscle contraction, as well as increased expression of determining proteins for oxidative muscle type (type I), contributed to obesity-resistance; however, antioxidative proteins did not.

’ AUTHOR INFORMATION Corresponding Author

*Fax: þ82-53-850-6559. E-mail: [email protected].

’ ACKNOWLEDGMENT This research was supported by the Midcareer Researcher Program (grant number R01-2008-000-10277-0) and SRC program (Center for Food & Nutritional Genomics: grant number 2010-0001888) of the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology. ’ ABBREVIATIONS 2-DE, 2-dimensional electrophoresis; ACC, acetyl-CoA carboxylase; ACOT, acyl-CoA thioesterase; AMPK, AMP-activated protein kinase; CPT, carnitine palmitoyltransferase; Cyt C, cytochrome c; DIO, diet-induced obesity; EF, elongation factor; ETF, electron transfer flavoprotein; FAS, fatty acid synthase; GLUT, glucose transporter; Gpx, glutathione peroxidase; HFD,

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high-fat diet; MALDI-TOF, matrix-assisted laser desorption/ ionization time-of-flight; OP, obesity-prone; OR, obesityresistant; PGC1-R, proliferator-activated receptor-γ coactivator 1 R; PMF, peptide mass fingerprinting; ROS, reactive oxygen species; SOD, superoxide dismutase; UCP, uncoupling protein

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Changes in Expression of Skeletal Muscle Proteins between Obesity

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