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Proteomic functional characterization of bovine stromal vascular cells from omental, subcutaneous and intramuscular adipose depots.


Adipose tissue is recognized not only as the main site of energy storage, but also as a complex, essential, and highly active metabolic and endocrine organ. For several decades, it has been appreciated that adipose tissue is regionally heterogeneous with respect to metabolic function (Arner, 1997; Kirkland et al., 1984, 1997). Adipocytes isolated from different depots differ in size, lipoprotein lipase release, lipid synthetic capacity, fatty acid incorporation and other characteristics (Hartman, 1985; Edens et al., 1993; Fried et al., 1993; Hube et al., 1996; Caserta et al., 2001). The interdepot variations observed solely a result of influences extrinsic to adipose cells (including their hormonal and paracrine micro environment, local nutrient availability, innervations, and anatomic constraints), or intrinsic differences in the innate characteristics of an adipocyte also contribute? Regional variations have been observed in replicative potential, fatty acid transfer and adipogenic differentiation of preadipocytes originating from various depots from the same individuals cultured under identical conditions (Hauner and Entenmann, 1991; Djian et al., 1993; Kirkland et al., 1996; Adams et al., 1997; Niesler et al., 1998). So, interdepot variations may not be solely a result of extrinsic influences, also inherent differences among the adipose cells likely to contribute.

The cellular development associated with adipose tissue growth involves both cellular hypertrophy (increase in size) and hyperplasia (increase in number). Hypertrophy is the result of excess triglyceride accumulation in existing adipocytes due to a positive energy balance (more intake and less energy expenditure). Hyperplasia (or "hypergenesis") is a general term referring to the proliferation of cells within an organ or tissue. Both these processes together are termed as adipogenesis that involves proliferation and differentiation of preadipocytes (adipose precursor cells). Preadipocytes are believed to be present throughout life (Prins and O'Rahilly, 1997) and are typically studied in vitro using both preadipocyte primary cultures (derived from the stromal vascular fraction of adipose tissue from various species including humans) (Loffler and Hauner, 1987; Novakofski and Hu, 1987; Gregoire et al., 1990; Litthauer and Serrero, 1992; Ramsay et al., 1993) and established cell lines, of murine origin, committed to the adipocyte lineage (Cornelius et al., 1994). Evidence from cell line studies suggests that the proliferation of preadipocytes occurs prior to differentiation (Smyth et al., 1993; Cornelius et al., 1994). Cell proliferation studies involving either by directly i.e., incorporation of 3H-thymidine or 5-bromo-2-dexoyuridine (BrdU) or indirectly (increase in the fat cell number) revealed the proliferation of adipose precursor cells rather than mature fat cells per se (Hausman et al., 2001). Also studies of de novo labeling of replicating preadipocytes in vivo showed that age and anatomical site or depot greatly influence the timing and rate of hyperplastic growth (Kirkland and Harris, 1980). There are many factors to consider in evaluating or defining preadipocyte proliferation, and they have been extensively studied (Cryer et al., 1992; Ramsay et al., 1993). In the present study, we have made an attempt to evaluate the different inherent factors of proliferating preadipocytes from different depots by the functional proteomic approach.



Five heads of Hanwoo (Korean cattle) steers were fed and managed in the feeding barn at Livestock Research Institute under the high quality beef production program (1997) and slaughtered at 24 months old. All experimental procedures and care of animals were conducted in accordance with the guidelines of the Animal Care and Use Committee (IACUC) of the National Institute of Animal Science in Korea.

Cell preparation

Immediately, after stunning and exsanguination, the muscle and fat portions between the 6th to 7th rib were removed, and the subcutaneous and intramuscular fat depots were sampled from this rib section aseptically. The omental adipose tissue was taken within the lesser curvature of the abomasum. All these tissue samples were kept in sterile saline (0.154 M NaCl, 37[degrees]C) for recovery of stromal vascular cells (Cianzio et al., 1982). The stromal vascular fraction of adipose tissue was prepared as described by Cryer et al. (1987). Tissue was sliced and cells were released by collagenase digestion in the Krebs Ringer Bicarbonate (KRB) buffer (1.22 mM CaCl2) for 1 h. The digested tissue was filtered through a nylon mesh to separate cells from undigested tissue fragments and debris. The filtrate was centrifuged at 2,500 rpm for 5 min at room temperature. The pellet was washed twice by centrifugation (2,500 rpm, 5 min) with Hank's Balanced Salt Solution (HBSS) and resuspended in a medium containing M199 supplemented with 10% Fetal Bovine Serum, penicillin (100 U/ml) and streptomycin (100 [micro]g/ml) and seeded in 10 mM petri-dish at a density of approximately 2,500 cells/[cm.sup.2]. The cells were incubated at 37[degrees]C in 5% C[O.sub.2] in an air, with a change in the culture medium on every second day.

Cell counting

Cell number was determined at day 2, 4, 6, 8 and 10 post plating. Cell cultures were washed three times with saline, then trypsinized with calcium and magnesium free Hank's solution containing 0.2% trypsin and finally counted in a "Countess Automated Cell Counter" from Invitrogen Ltd, USA. Cell viability was assessed by trypan blue exclusion. Regardless of anatomical origin, 95% of cells excluded trypan blue.

Protein preparation for 2-DE

Proteins from stromal vascular cells were extracted in 0.5 ml of lysis buffer (7 M Urea, 2M Thiourea, 4% CHAPS, 100 mM DTT, protease inhibitor cocktail (Roche Diagnostics GmbH, Mannheim, Germany). Following 15min centrifugation at 15,000 rpm, the supernatant was collected and precipitated with either one or three volumes of acetone at -20[degrees]C for 2 h. The pellets were collected by centrifugation and then completely dried using speed-vac. Dried samples were re-dissolved in the 2-DE sample buffer (7 M Urea, 2 M Thiourea, 2% CHAPS, 100 mM DTT, 0.5% pH3-10NL IPG buffer) for isoelectric focusing (IEF). The concentration of total protein in the sample was determined by Bradford's protein assay method.

2-DE and image analysis

500 [micro]g of protein was loaded onto the immobiline dry strips pH 3-10 NL (GE Health care). The rehydrated strips were focused on IEF system (AP Biotech, Sweden) for ~80 kVh at a maximum of 8,000 V in a rapid ramping mode with maximum current per strip of 50 | A. Equilibration of the immobilized pH gradient strips was performed in two steps: reduction followed by alkylation (Ahmed and Bergsten, 2005). The second dimension was run on 12.5% polyacrylamide sodium dodecyl sulphate gels (26x20 cm) with a constant voltage of 100 V for 30 min, 250 V for 6 h using the EttanDALT II system (Amersham Bioscience, Piscataway, USA). The proteins were visualized using a Coomassie Brilliant Blue (CBB) G-250 staining method. The CBB-stained gels were scanned using a GS-800 scanner (Bio-Rad) at an optical resolution of 300 dpi. Spot detection, quantification and matching were performed using Image Master Version 7.0 (GE healthcare). A match set consisting of three images, each from one depot was created. To correct for variability due to CBB staining and reflect the quantitative variations of protein spot, the individual spot volumes were normalized by dividing their optical density (OD) values by the total OD values of all the spots present in the gel. The significance of the expression difference of protein between three depots was estimated by Student's t-test, p<0.05 and was done using Image Master (ver 7.0) software.

Protein identification

The CBB-stained protein spots were excised from gels using a punch and placed in 500 Lil Eppendorf tubes. The proteins were digested in-gel with trypsin as described by Hellmann et al. (1995). Briefly, each spot was destained with 50 [micro]l 50% acetonitrile (ACN) in 50 mM N[H.sub.4] HC[O.sub.3], incubated at 37[degrees]C for 30 min and repeated once. Then the gels were reduced and alkylated. The gel pieces were digested overnight with trypsin (20 [micro]g/[micro]l) in 50 mM N[H.sub.4] HC[O.sub.3] containing 10% ACN. The digest was then vortexed for 30 min and dried using speed vac. The dried extracted peptides were resuspended in a 1 [micro]l solution containing pure water:ACN:trifluoracetic acid (TFA) (93:5:2).

Solution-phase nitrocellulose target preparation was used according to the method reported by Landry et al. (2000), [alpha]-cyano-4-hydroxycinnanic acid (CHCA) (40 mg/ml) and nitrocellulose (20 mg/ml) were prepared separately in acetone and mixed with 2-propanol at a ratio of 2:1:1. The matrix solution was mixed with the sample at a ratio 1:1, 0.5-0.3 [micro]l was spotted onto the target and dried. The immobilized samples were washed with 1% formic acid twice and samples were then dried for a second time prior to the MALDI-TOF-MS/MS analysis.

Sample peptide masses were obtained using the Applied Biosystems 4700 Proteomics analyzer MALDI-TOF/TOF mass spectrometer (Applied Biosystems) in the positive ion reflector mode. MS/MS analysis was performed on the 5 most abundant ions and the proteins identified by searching the SWISS-PROT and National Center for Biotechnology Information databases using the Mascot programs (Matrix Science, London, UK). Mass accuracy was considered to be within 50 ppm for peptide mass analysis and within 100 ppm for MS/MS analysis. For protein identification, known contamination peaks such as those of keratin and auto proteolytic were removed, and molecular weight, pI and protein scores were considered.

Western blot analysis

Western blot analysis was carried out for a subset of four differentially expressed proteins. For western blotting, 40 [micro]g of sample proteins were separated on SDS-PAGE and gels were transferred to PVDF membranes (Millipore) in the ice-cold transfer buffer (25 mM Tris-Cl, pH 8.3, 1.4% glycine, 20% methanol) at 250 mA for 90 min. Membranes were 1 treated with blocking buffer containing 5% non-fat milk (Becton, Dickinson and Company, MD, USA) in TBS/T (10 mM Tris-HCl, pH 8.0, 150 mM NaCl, and 0.1% Tween 20) and incubated overnight at 4[degrees]C. Primary goat mouse-HSPB1 (sc-1049), goat-anti-CFL (sc-32158) and goat-anti-ACTB (sc-47778) antibodies, all from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA), at 1:400 dilutions and goat-anti-PRDX 1 (NBPI-006064; Novus Biologicals), at 1:5,000 were used in TBS/T with 5% non-fat milk. Following 2 h of incubation with primary antibodies, membranes were washed three times for 10 min each with 10 ml of TBS/T. Horseradish peroxidase-labeled (HRP) anti-goat and anti-mouse secondary antibody (sc-2020; sc-2005) was diluted 1:5,000 in TBS/T with 5% nonfat milk and incubated with the membranes for 1 h. After three 10-min washes, membranes were visualized using a chemiluminescent HRP substrate (Millipore) and a VersaDoc imaging system (Bio-Rad). All experiments were repeated in triplicate.


Depot origin affects the proliferation rate

The stromal vascular cells of adipose tissue were prepared from three depots, omental, subcutaneous and intramuscular (Figure 1A, B and C). Cells were proliferated in vitro in M199 supplemented 10% FBS with antibiotics (Figure 1D, E and F). During the initial days of cultivation, proliferation rate and pattern of preadipocytes from all the three depots were similar. As the cells reached confluence (day 10), preadipocytes from the intramuscular depot were showing higher proliferative rates in comparison to other two depots (Figure 1G). Although preadipocytes from all the three depots were showing different proliferation rate, the pattern of proliferation was found to be similar.

Mapping proteins expressed in preadipocytes from different depots

Proteome of the proliferating preadipocytes was analyzed by 2D gel coupled with MALDI TOF/TOF. Gels with separated proteins from omental (Figure 2A), subcutaneous (Figure 2B) and intramuscular (Figure 2C) depots are displayed in Figure 2. Further, to identify the differentially expressed proteins, a set of 5 gels were produced from each experiment and all the gels associated with the same collection were completely super imposable. Each spot detected by the Image Master (ver 7.0) was assigned a unique number to identify spots in a gel matching process. Methodical replicates of silver stained gels of the same sample showed high reproducibility (95%) by comparison using Image Master ver 7.0 software. Spots were isolated and identified from CBB-stained gels in order to generate a 2-DE-proteome map. Using the 2-DE technology, 252 spots were mapped and 138 spots out of them were identified (Table 1). To classify identified proteins, 16 functional categories were established based on information from Gene Ontology database and additional information from ExPasy ( About 41% of the proteins were related to cell structure, 9% to carbohydrate metabolism, 6% stress response, 5% of proteins in energy generation, followed by the groups of proteins involved in translation, lipid metabolism, cell signaling and others comprise of 10% of total identified proteins (Figure 3).



Differential protein expression of preadipocytes from three depots

To evaluate the differential protein expression in proliferating preadipocytes from omental, subcutaneous and intramuscular depots, we used a 2 fold cutoff (p<0.05) from our image analysis studies to designate up and down regulated proteins. Figure 4A, B and C represent the differential protein expression of omental versus subcutaneous, subcutaneous versus intramuscular and omental versus intramuscular depots respectively. This comparative analysis revealed the presence of 16 different proteins, and they were categorized into 6 functional groups based on the gene ontology function: heat shock proteins/chaperones (HSPB 1, HSPA 9 and HSPA 5); redox (PRDX 6, 4 and 1); cytoskeleton (ACTB, CFL1, TAGLN and GSN); cell processes (ANXA 6, 5 and 4); metabolism (LDHB and ALDH1A1) (Table 2).

Our image analysis along with in-depth spot analysis (lower panel of Figure 4A, B and C) revealed omental depot with higher expressions of ANXA 6, HSPB1, TAGLN, ALDH1A1 and CALD 1, whereas in the subcutaneous depot, expressions of PRDX 6 and GSN were found to be high. Differential protein expression analysis between omental and intramuscular depots showed higher expressions of HSPA 5, HSPA 9, CALD 1, PRDX 4, ALDH1A1 and TAGLN in omental, while ANAX 4 and ACTB in intramuscular depots. Results from subcutaneous versus intramuscular depots revealed higher expression of ANAX 5, PRDX 1 and LDHB in intramuscular, whereas CFL 1 was found to be high in the subcutaneous depot.

Western blot analyses

For western blot analyses, immunoblots prepared with the protein extracts from proliferating preadipocytes from all the three depots used in the proteomic analysis. These immunoblots were further probed with antibodies for detection of a subset of four proteins (CFL 1, ACTB, HSPB 1 and PRDX 1) that are differentially expressed in our proteomic analyses (Figure 5). Our immunoblot results were found be corroborating with our proteomic analyses, further confirming the differential expression of proteins during preadipocyte proliferation.


The basis for depot specific differences in proliferating preadipocytes has not been studied. Most of the studies until now are concentrated on the regulation of adipocyte differentiation, as it is considered to be the most important event during adipogenesis. In the present study, proliferation rate of preadipocytes although was same in the initial stages of growth, as cells reached confluence, the preadipocytes from the intramuscular depot were found to have higher proliferation rates. It has been demonstrated that preadipocytes from different depots in rats (Kirkland et al., 1992; Lacasa et al., 1997) porcine (Samulin et al., 2008) and humans (Tchkonia et al., 2002; Van Harmelen et al., 2004) vary in proliferation rate. The depot-specific differences in proliferation rate observed in our study can be attributed to the inherent changes in the protein expression that control the proliferation. This can be considered as a valid reason as the preadipocytes were grown in similar in vitro conditions. Also, the reason for us to select preadipocytes for this study is, despite exposure to hormonal manipulations in vivo, such as estrogen treatment, hypophysectomy, or castration, preadipocytes cultured from various depots retain distinct cell-dynamic and biochemical responses relative to other depots (Kirkland et al., 1992; Lacasa et al., 1997).


Until now, proteomic comparative analyses of proliferating preadipocytes from different adipose depots of bovine are lacking. But, proteomic analysis of the adipocyte was performed using cell lines such as 3T3-L1 preadipocytes. Many studies involving the effect of hormones (Fasshauera et al., 2002), apopototic factors (Renes et al., 2005) and oligosaccharides (Rahman et al., 2008) on differentiating 3T3-L1 preadipocytes are in literature. Our proteomic analyses displayed about 16 differently expressed proteins, and they belong to similar categories observed in proteomic analysis of primary cultures of human adipose-derived stem cells (James et al., 2005) . Further, our immunoblot results for a sub set of four different proteins were found to be corroborating with our proteomic analyses, further confirming the differential expression of proteins during proliferation. In vivo studies demonstrated that preadipocyte proliferation, differentiation and their interrelations are very complex and reported to be defined by a number of factors including age, hormones, species and depot (Hartman, 1985). Most of the proteins that were differentially expressed during proliferation in this study were also reported during differentiation of 3T3-L1 cells (Adachi et al., 2007; Barcelo-Batllori and Gomis, 2009) . Although studies from Petrak et al. (2008) and Wang et al. (2009) have shown that, many of our differentially expressed proteins as common cellular stress proteins, but most of them were already reported to have a role in cell proliferation and lipid metabolism. Hence, we intend to discuss the role of differently expressed proteins in our subsequent sections.

Differential expression of proteins during preadipocyte proliferation from different depots

Heat shock proteins/chaperones-Preadipocyte proliferation has resulted in the induction of three different heat shock proteins/chaperones (HSPB1, HSPA9 and HSPA5). Apart from proteomic analyses, higher expression of HSPB1 was confirmed in our immunoblot analyses (Figure 5). HSPB1 was already reported to be high in omental preadipocytes (Perez-Perez et al., 2009), and it has been shown to be involved in the modulation of adipocyte differentiation and metabolism. It was reported that HSPB1 interacts with insulin-like growth factor receptor 1 and its signal transducer, the serine/theronine kinase Akt, which together modulate adipocyte metabolism (Rane et al., 2003; Shan et al., 2003).

HSPA9 protein is highly expressed in proliferating omental preadipocytes and this protein is very well shown to be involved in adipogenesis. In fact, omental fat was already reported to have over expression of heat shock proteins (Perez-Perez et al., 2009). Moreover, there is a growing body of literature linking chaperone-like molecules to adipogenesis, obesity, and diabetes (Cherian and Abraham, 1995; Kurucz et al., 2002; Kumar et al., 2004; Ozcan et al., 2004), For example, adipogenesis in 3T3-L1 cells is accompanied by increased expression of the chaperone-related immunophilin, FK-binding protein 51 (Yeh et al., 1995). Moreover, the nuclear hormone receptors that control adipogenic transcription, the glucocorticoid receptor, and the peroxisome proliferation-activated receptor, are sequestered in the cytosol as a complex with HSPA 9 prior to ligand activation (Hache et al., 1995; Young et al., 2005). It is interesting to note that clinical studies have linked polymorphisms in HSPA 9 to an increased risk for obesity and type 2 diabetes (Chouchane et al., 2001; Zouari Bouassida et al., 2004). It has been also reported that preadipocytes from the omental depot are more susceptible to apoptosis (Niesler et al., 1998). Presence of HSPA 9 in omental preadipocytes may protect them from apoptosis, because HSPA9 also called as mortalin and is known to have antiapoptotic role. Mortalin was shown to inhibit apoptosis by inactivating p53 (Wadhwa et al., 1998; Kaul et al., 2001).


HSPA 5 also known as glucose-regulated protein 78 kDa (GRP 78) was found to be absent in intramuscular depot. GRP 78, is well known to positively modulate the adipogenesis. This protein is critical to the regulation of a transcription factor, X-box binding protein 1 (XBP1) (Shi et al., 2009) and this XBP 1 was shown to play a key role in adipocyte differentiation by acting as a critical regulator of the morphological and functional transformations during adipogenesis (Sha et al., 2009). Hence, the absence of this protein expression in the intramuscular depot may be one of the factors that contribute to the low level of fat seen in this depot from adult animal.

Redox : Three anti-oxidative proteins peroxiredoxin1, 4 and 6 (PRDX 1, 4, 6) are found to be differently expressed in our proteomic analysis. PRDX 1, 4 and, 6 proteins were highly expressed in proliferating preadipocytes from intramuscular, omental and, subcutaneous depots respectively. It has been reported that proliferating cells are always at the risk of the oxidative damage (Villani et al., 2000). So, all the proliferating preadipocytes from three depots are been guarded by one or the other anti-oxidant protein. More specifically, PRDX 1 was reported to be over expressed to the high proliferative signals (Prosperi et al., 1998) and the presence of this protein in high levels from the intramuscular depot also supports our observation of high proliferative rate among the preadipocytes from this depot. High levels of PRDX 4 in omental preadipocytes are related to the anti-apoptotic function and as already mentioned that preadipocytes from the omental depot are more sensitive to apoptotic stimuli. Anti-apoptotic nature of PRDX 4 is by activating NF-kB (Jin et al., 1997). PRDX 6 plays a role in protecting cells from cell death by its antioxidant property (Hoehn et al., 2003).

Cytoskeleton : Actin (ACTB), cofilin 1 (CFL 1) and transgelin (TAGLN) are the three proteins that have a role in cytoskeleton development are found to be differently expressed across the depot specific proliferating preadipocytes. ACTB was shown to play a critical role in cell architecture/mobility and is essential for various life cyclic processes (Weil et al., 2009). In fact, mechanical tension, acting through the actin filament complex, can control the differentiation status of adult stromal stem cells (Takenouchi et al., 2004). Furthermore, ACTB was gradually shown to increase during adipose conversion of bovine intramuscular preadipocyte cells (McBeath et al., 2004), suggesting that a rearrangement of cytoskeletal proteins has a role in the intracellular accumulation of lipid droplets (Luegmayr et al., 1996). Cofilins are a family of actin-binding proteins and are expressed in all eukaryotic cells. Presence of CFL 1 in subcutaneous adipose tissue is already reported (Choi et al., 2003). TAGLN, also called as SM22[alpha] although is a structural protein, we here implicate this protein to involve in adipogenic differentiation. Transcriptional up regulation of SM22[alpha] down regulates protein kinase C (PKC) (Mayr et al., 2004). This down regulation of PCK has a direct effect on 3T3-F442A cell adipogenic differentiation (Fleming et al., 1999). In the case of gelsolin (GSN), which is absent in the omental depot may have a role as an anti-apoptotic factor. GSN inhibits apoptosis by inhibiting the TNF-[alpha], which is one of the proteins that actively participate in apoptosis (Sezen et al., 2009).

Cellular processes : Annexins are the family of calcium-dependent phospholipid binding proteins. In our study, we found three annexins (VI, V and IV) differently expressed among the depots. The preadipocytes from the intramuscular depot were found to express more ANXA 5, and adipocytes from this depot was shown to have less amount of fat than omental. Studies have shown that ANXA 5 inhibits two important proteins that have the direct regulation on fat synthesis. Protein kinase C (PKC) is inhibited by ANXA 5 via a mechanism of phospholipids (PS) sequestration, as annexin proteins are known to have domains that are involved in phospholipid binding. This sequestration of phospholipids by ANXA 5 may lead to a reduction in the amounts of PS that is required for PKC activation (Dubios et al., 1998). In fact, PKC promotes adipogenic commitment and is essential for the terminal differentiation of 3T3-F442A preadipocytes (Webb et al., 2003). PKC is also involved in the phosphorylation of CCAAT-enhancer-binding protein (C/EBP) family and these proteins are transcription factors known to have direct interaction with adipocyte differentiation genes (Darlington et al., 1998). ANXA 5 also inhibits the A2 (PLA2) activity by inhibiting PKC, which is required for activating PLA2 through phosphorylation (Mira et al., 1997). A study from Gao and coworkers has shown that PLA2 is a differentiation specific enzyme activity in adipogenic cell line and adipocyte precursors in primary culture (Gao and Serrero, 1990). Hence, inhibition of both proteins by ANXA 5 in the intramuscular depot will definitely play a major role in adipogenic differentiation and lipogenic pathways.

Metabolism : Adipogenesis in preadipocytes is accompanied by the induction of proteins associated with glycolysis and other enzymes that participate in the metabolism of the cell. In this category, we found two metabolically active enzymes that are differentially expressed in the preadipocytes. Aldehyde dehydrogenase 1 (ALDH1A1) was found to be in its higher expression in omental preadipocytes in comparison to the other two depots. There is also a study reporting the increased levels of ALDH1A1 in omental fat (Perez-Perez et al., 2009). Moreover, mice lacking ALDH1A1were preserved from developing diet-induced obesity and insulin resistance (Ziouzenkova et al., 2007), and it was also shown that increased levels of ALDH1A1 in the obese omental fat might involve in fat accumulation (Perez-Perez et al., 2009).

Lactate dehydrogenase B (LDHB) is another important enzyme highly expressed in the preadipocytes of intramuscular depot. LDHB is an important enzyme in the glycolytic pathway as it converts pyruvate, the final product of glycolysis to lactic acid. It also performs the reverse reaction during the cori cycle in the liver generating ATP. LDHB is seen to be more expressed in cells that are involved in fast proliferation (Pan et al., 1991). This justifies our observation of fast proliferation of intramuscular preadipocytes and high expression of LDHB.

Most of the proteins that are differentially expressed in proliferating preadipocytes like, a number of annexins, cytoskeletal and heat shock proteins have already been reported in the proteome analysis of differentiating preadipocytes (Aboulaich et al., 2004; James et al., 2005). Hence, the proteins discussed in the current study will have their profound effect either on differentiation or metabolism of adult adipocytes. In addition, further studies like protein specific gene regulation for these proteins will throw light on their hidden pathways in adipocyte development.


This study was supported by the National Institute of Animal Science research project, "Proteomic characterization of animal adipose and muscle tissue in relation to growth" (Project No. 200806I01011066).


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Ramanna Valmiki Rajesh, Seong-Kon Kim, Mi-Rim Park, Jin-Seon Nam, Nam-Kuk Kim, Seulemina Kwon, Duhak Yoon, Tae-Hun Kim and Hyun-Jeong Lee *

Division of Animal Genomics and Bioinformatics, National Institute of Animal Science, Rural Development Administration, #564 Omockchun-dong, Suwon, Korea

* Corresponding Author: Hyun-Jeong Lee. Tel: +82-31-290-1594, Fax: +82-31-290-1594, E-mail:

Received March 22, 2010; Accepted May 18, 2010
Table 1. i) Common proteins identified from the proliferating
preadipocytes from omental, subcutaneous and intramuscular depots *

No.      Protein name

  6    78 kDa glucose-regulated protein precursor (GRP 78)
  7    Protein disulfide-isomerase precursor (EC (PDI)
  8    Vimentin
 10    Tubulin beta-2 chain (Beta-tubulin class-II)
 14    ATP synthase subunit beta, mitochondrial precursor (EC
 15    Protein disulfide-isomerase A6 precursor (EC
 22    Cis-2,3-dihydrobiphenyl-2,3-diol dehydrogenase (EC
 26    4-hydroxy-3-methylbut-2-en-1-yl diphosphate synthase (EC
 30    HERV-K_1p13.3 provirus ancestral Env polyprotein
 32    Hippocalcin-like protein 1 (Visinin-like protein 3) (VILIP-3)
 33    Non-muscle caldesmon
 43    Annexin A5 (Annexin V) (Lipocortin V) (Endonexin II)
 44    Calpain small subunit 1 (CSS1)
 48    Heat shock cognate 71 kDa protein (Heat shock 70 kDa
       protein 8)
 49    60 kDa heat shock protein, mitochondrial precursor (Hsp60)
 57    Prolyl 4-hydroxylase alpha-2 subunit precursor (EC
 58    Stress-70 protein, mitochondrial precursor
 60    Endoplasmin precursor (Heat shock protein 90 kDa beta
       member 1)
 64    Actin, alpha sarcomeric/skeletal (Actin alpha 3)
 70    Vacuolar ATP synthase catalytic subunit A, ubiquitous isoform
 73    Annexin A6 (Annexin VI) (Lipocortin VI) (P68) (P70)
 79    Tryptophanyl-tRNA synthetase (Tryptophan--tRNA ligase)
 80    Gelsolin
 81    Vinculin (Metavinculin)
 82    T-complex protein 1 subunit alpha (TCP-1-alpha) (CCT-alpha)
 86    Vacuolar ATP synthase subunit B, brain isoform (EC
 89    Protein disulfide-isomerase A3 precursor (EC
100    WD repeat protein 1 (Actin-interacting protein 1) (AIP1)
101    WD repeat protein 1 (Actin-interacting protein 1) (AIP1)
102    Phosphoenolpyruvate carboxykinase [GTP], mitochondrial
103    Transketolase (EC (TK)
104    4-aminobutyrate aminotransferase (EC
105    Acetyl-coenzyme A carboxylase carboxyl transferase subunit
106    Stress-induced-phosphoprotein 1 (STI1)
107    Glutaminase kidney isoform, mitochondrial precursor (GLS)
108    Retinal dehydrogenase 1 (EC (RalDH1) (RALDH 1)
109    Lamin-A/C (70 kDa lamin) (NY-REN-32 antigen)
112    T-complex protein 1 subunit zeta (TCP-1-zeta) (CCT-zeta)
113    Putative HTH-type transcriptional regulator protein MJ0300
114    Lamin-A/C
118    T-complex protein 1 subunit beta (TCP-1-beta) (CCT-beta)
121    T-complex protein 1 subunit eta (TCP-1-eta) (CCT-eta)
122    Pyruvate kinase isozyme M2 (EC
126    ATP synthase subunit alpha heart isoform, mitochondrial
127    Glutamate dehydrogenase 1, mitochondrial precursor (EC
128    Serine hydroxymethyltransferase, mitochondrial precursor
129    Septin-11
131    Transcriptional regulator IE63 homolog
132    Alpha-enolase (EC (NNE)
134    Alpha-centractin (Centractin) (Actin-RPV) (ARP1)
135    Elongation factor Tu, mitochondrial precursor (EF-Tu)
136    Isocitrate dehydrogenase [NADP] cytoplasmic (EC
137    Septin-2
139    Citrate synthase, mitochondrial precursor (EC
140    Synaptotagmin-15 (Synaptotagmin XV) (SytXV)
141    Phosphoglycerate kinase 1 (EC
143    Fructose-bisphosphate aldolase A (EC (Muscle-type
145    Elongation factor 1-alpha 1 (EF-1-alpha-1) (Elongation factor
       1 A-1)
146    Annexin A2 (Annexin II) (Lipocortin II) (Calpactin I heavy
147    Aspartate aminotransferase, mitochondrial precursor (EC
148    Glyceraldehyde-3-phosphate dehydrogenase (EC
149    Heterogeneous nuclear ribonucleoproteins A2/B1
152    L-lactate dehydrogenase A chain (EC (LDH-A)
153    Malate dehydrogenase, mitochondrial precursor (EC
154    Heterogeneous nuclear ribonucleoproteins A2/B1
155    Calponin-1 (Calponin H1, smooth muscle) (Basic calponin)
158    Heterogeneous nuclear ribonucleoprotein A1 (Helix-
       destabilizing protein)
160    Voltage-dependent anion-selective channel protein 1 (VDAC-1)
161    Non-structural RNA-binding protein 53
162    Iron sulfur cluster assembly protein 1, mitochondrial precursor
163    Potassium voltage-agted channel subfamily D member 1
165    Calponin (Calponin H2, smooth muscle)
168    Proteasome subunit alpha type 7
169    Transgelin (smooth muscle protein 22-alpha)
170    Peroxiredoxin 1
171    Transgelin 2
172    Peptidyl-propyl cis-trans isomerase B precursor (EC
174    Peptidyl-propyl cis-trans isomerase A precursor (EC
175    UPF0343 protein MK0485
179    GLuthathione S-transferase P (EC
180    Triosephosphate isomerase (EC
181    Phosphoglycerate mutase 1 (EC
183    Peroxiredoxin 6
184    Heat shock protein beta-1 (HSP 27)
185    DNA-directed RNA polymerase beta chain
187    Purine nucleoside phosphorylase
186    Cofilin (non-muscle isoform)
188    Proteasome subunit alpha type 1 (EC
189    Heterogeneous nuclear ribonucleoprotein M
191    Annexin A1 (Lipocortin I)
192    Glutamyl-tRNA (GLn) amidotransferase subunit A
193    26S proteasome non-ATPase regulatory subunit 14
194    Isocitrate dehydrogenase (NAD) subunit, mitochondrial precursor
195    Cell division control protein 2 homolog (p34 protein kinase)
197    60S acidic ribosomal protein P0
198    26S Proteasome non-ATPase regulatory subunit 13
199    Actin alpha sarcomeric/cardiac
200    Actin, cytoplasmic 2
201    Tubulin beta 2 chain
202    Peroxiredoxin 4
205    Annexin A4
206    F-actin capping protein subunit beta isoforms 1 and 2
207    Chloride intracellular channel protein 4
208    Prohibitin
211    Proteasome subunit beta type 4 (EC
212    Actin, cytoplasmic 1
213    Proteasome subunit beta type 3 (EC
228    Collagen alpha-2 (I) chain precursor
236    Actin, cytoplasmic I
237    Ena/VASP-like protein
238    Rho GDP-disassociation inhibitor 1 (Rho-GD1 alpha)
242    Phosphoglycerate mutase I (EC
243    Annexin A2 (Lipocortin II)
250    26S protease regulatory subunit 7 (protein MSSI)
251    Prolyl4-hydoxylyase alpha-1 subunit precursor
252    Elongation factor 2 (EF-2)

Spot   Protein   Protein score   Swiss Prot       MW         pI
No.     score       (C.I %)        Number

  6      228          100          P11021      72,332.96    5.07
  7      111          100          P09103       57,143.6    4.19
  8       93          100          P48616       53,727.8    5.06
 10      165          100          P32882      49,953.06    4.78
 14      192          100          P00829      56,283.53    5.15
 15       87          100          Q922R8      48,100.39    5
 22       58           64          P72220      28,906.96    5.44
 26       62           85          Q886Z0      39,763.85    6.36
 30       62           84          P61568      21,461.53    6.29
 32       59           67          P62749      22,338.24    5.32
 33       62           91          Q27976      60,547.75    6.34
 43      139          100          P81287      36,088.86    4.86
 44      103          100          P13135      27,931.39    5.06
 48       81          100          P19378      70,804.92    5.24
 49      141          100          P18687      60,988.56    5.84
 57       67           95          Q60716      61,002.13    5.55
 58      128          100          P38646      73,680.50    5.81
 60       72           98          Q95M18      92,429.70    4.76
 64       89          100          P04752      41,983.85    5.22
 70       89          100          P31404      68,344.11    5.41
 73       72           98          P08133      75,873.27    5.42
 79      106          100          P17248      53,812.05    5.49
 80       89           99          Q3SX14      80,645.69    5.58
 81      190          100          Q64727     116,714.36    5.77
 82       80          100          P28480      60,359.65    5.86
 86       84          100          P21281      56,500.73    5.57
 89       84          100          P38657      56,929.56    5.86
100      120          100          O75083      66,193.52    6.17
101      123          100          O88342      66,406.70    6.11
102       76           99          Q8BH04      70,527.90    6.92
103      101          100          Q6B855      67,905.81    7.56
104       63           88          P14010      55,489.33    8.92
105       67           95          Q73Q55      33,417.44    8.66
106      103          100          Q60864      62,582.11    6.4
107       65           92          O94925      73,461.10    7.85
108      115          100          P48644      54,805.76    6.24
109      114          100          P02545      74,139.49    6.59
112       66           95          Q3MHL7      57,956.17    6.32
113       61           80          Q57748      33,111.20    6.98
114      130          100          P48678      30,776.86    8.98
118      162          100          P80314      57,477.24    5.94
121      122          100          Q2NKZ1      59,442.79    6.78
122       88          100          P52480      57,844.89    7.17
126      192          100          P19483      59,719.64    9.21
127      129          100          P00366      61,511.97    7.25
128      135          100          Q3SZ20      55,605.50    7.67
129       68           96          Q5R8U3      49,033.00    6.89
131       60           75          P28939      51,320.88    9.65
132       97          100          Q9XSJ4      47,326.13    6.37
134      114          100          P61163      42,613.74    6.19
135      171          100          P49410      49,398.26    6.72
136      151          100          Q9XSG3      46,785.42    6.13
137       83          100          Q5RA66      41,503.47    6.15
139       97          100          Q29RK1      51,772.54    8.16
140       67           95          Q8C6N3      47,270.32    8.18
141      129          100          P00559      44,602.68    8.68
143      101          100          P00883      39,342.89    8.31
145       87          100          P62629      50,113.84    9.1
146       91          100          P07355      38,604.04    7.57
147       79          100          P12344      47,513.53    9.19
148       90          100          P00355      35,836.05    8.51
149      152          100          P22626      37,429.70    8.97
152      124          100          P19858      36,597.64    8.12
153       87          100          P00346      35,595.63    8.93
154      179          100          O88569      37,402.67    8.97
155       87          100          Q08091      33,355.59    9.05
158      197          100          P04256      34,212.26    9.2
160      139          100          P21796      30,772.60    8.62
161       65           93          P30212      57,645.33    8.10
162       63           89          Q6FJY3      22,900.85    9.51
163       61           82          Q03719      71,697.79    8.56
165       75           99          Q5RFN6      33,697.05    6.94
168       75           99          O14818      27,886.85    8.60
169      166          100          Q9TS87      22,598.85    8.87
170      114           95          Q5E947      22,195.39    8.59
171       69           97          Q5E9F5      22,426.49    8.40
172      104          100          P80311      23,746.52    9.33
174       64           90          P62935      17,869.35    8.34
175       63           90          Q8TY21      37,111.78    6.00
179       79           99          P28801      23,613.19    6.89
180       93           99          Q5E956      26,689.51    6.45
181       69           97          Q3SZ62      28,851.99    6.67
183      125          100          O77834      24,920.01    6.02
184      105          100          Q3T149      22,379.33    5.98
185       62           86          Q4L3K4     135,006.84    6.14
187      105          100          P55859      32,036.54    5.92
186       87           99          Q5E9F7      18,587.70    8.26
188       77           99          P25786      29,555.59    6.15
189       67           96          P52272      77,515.53    8.84
191      139          100          P46193      38,951.68    6.38
192       65           93          O06491      52,663.74    5.39
193       91           99          O35593      34,577.04    6.06
194      110          100          P41563      39,667.84    6.76
195       64           92          Q9DGA5      34,605.13    8.60
197       92           99          Q95140      34,370.62    5.72
198       92           99          Q5E964      42,866.34    5.44
199       62           85          P20399      42,033.00    5.23
200       79           99          P63256      41,877.95    5.39
201      123          100          P83130      49,955.05    4.77
202      112          100          Q9BG12      30,721.98    6.01
205      182          100          P13214      35,735.06    5.55
206       64           90          P14315      30,610.64    5.69
207       75           99          Q9XSA7      28,727.07    5.60
208      207           99          P35232      29,804.10    5.57
211       60           95          Q3T108      29,031.08    5.52
212       58           94          O18840      41,736.73    5.29
213       92           99          Q58DU5      28,405.17    5.19
228       79           99          P02465     129,063.58    9.23
236      122          100          Q71FK5      41,736.73    5.29
237       55           87          O08719      42,094.88    8.74
238       97          100          P19803      23,421.40    5.12
242       58           93          Q9DBJ1      28,831.99    6.67
243       61           97          P04272      38,612.07    6.92
250      124          100          P46471      48,647.88    5.72
251      102          100          Q5RAG8      61,049.30    5.70
252       81           99          P13639      95,338.14    6.41

* Proteins (isoforms) that appeared in three or more spots are
Septin-11 and Retinal dehydrogenase (3 spots); Lamin A/C (6
spots); Vimentin (10 spots).

Table 2. Comparison of differentially expressed proteins from
depots proliferating preadipocytes of omental, subcutaneous and

Function       Name protein                           OM vs. SC

Cell           Annexin A6 (ANXA 6)              Low in SC/High in OM
  processes    Annexin A5 (ANXA 5)                       --
               Annexin A4 (ANXA 4)                       --
Heat shock     Heat shock 27 kDa protein 1      Low in SC /High in OM
  /chaperone     (HSPB 1)
               Heat shock 70 kDa protein 9               --
                 (HSPA 9)
               Heat shock 70 kDa protein 5               --
                 (HSPA 5)
Redox          Peroxiredoxin 6 (PRDX 6)         Low in OM/High in SC
               Peroxiredoxin 4 (PRDX 4)                  --
               Peroxiredoxin 1 (PRDX 1)                  --
Cytoskeleton   Actin, beta (ACTB)                        --
               Cofilin 1 [non-muscle] (CFL 1)            --
               Transgelin (TAGLN)               Low in SC/High in OM
               Caldesmon 1 (CALD 1)                   ND in SC
               Gelsolin (GSN)                   Low in OM/High in SC
Metabolism     Lactate dehydrogenase B (LDHB)            --
               Aldehyde dehydrogenase 1         Low in SC/High in OM
                 (ALDH1A 1)

Function       Name protein                          OM vs. IM

Cell           Annexin A6 (ANXA 6)                       --
  processes    Annexin A5 (ANXA 5)                       --
               Annexin A4 (ANXA 4)              Low in OM/High in IM
Heat shock     Heat shock 27 kDa protein 1               --
  /chaperone     (HSPB 1)
               Heat shock 70 kDa protein 9      Low in IM/High in OM
                 (HSPA 9)
               Heat shock 70 kDa protein 5            ND in IM
                 (HSPA 5)
Redox          Peroxiredoxin 6 (PRDX 6)                  --
               Peroxiredoxin 4 (PRDX 4)         Low in IM/High in OM
               Peroxiredoxin 1 (PRDX 1)                  --
Cytoskeleton   Actin, beta (ACTB)               Low in OM/High in IM
               Cofilin 1 [non-muscle] (CFL 1)            --
               Transgelin (TAGLN)               Low in IM/High in OM
               Caldesmon 1 (CALD 1)                   ND in IM
               Gelsolin (GSN)                            --
Metabolism     Lactate dehydrogenase B (LDHB)            --
               Aldehyde dehydrogenase 1         Low in IM/High in OM
                 (ALDH1A 1)

Function       Name protein                          SC vs. IM

Cell           Annexin A6 (ANXA 6)                       --
  processes    Annexin A5 (ANXA 5)              Low in SC/High in IM
               Annexin A4 (ANXA 4)                       --
Heat shock     Heat shock 27 kDa protein 1               --
  /chaperone     (HSPB 1)
               Heat shock 70 kDa protein 9               --
                 (HSPA 9)
               Heat shock 70 kDa protein 5               --
                 (HSPA 5)
Redox          Peroxiredoxin 6 (PRDX 6)                  --
               Peroxiredoxin 4 (PRDX 4)                  --
               Peroxiredoxin 1 (PRDX 1)         Low in SC/High in IM
Cytoskeleton   Actin, beta (ACTB)                        --
               Cofilin 1 [non-muscle] (CFL 1)   High in SC/Low in IM
               Transgelin (TAGLN)                        --
               Caldesmon 1 (CALD 1)                      --
               Gelsolin (GSN)                            --
Metabolism     Lactate dehydrogenase B (LDHB)   Low in SC/High in IM
               Aldehyde dehydrogenase 1                  --
                 (ALDH1A 1)

OM = Omental; SC = Subcutaneous; IM = Intramuscular; ND = Not

The differential expression was determined from the in-depth
protein spot analysis along with 3D analysis of the spots (please
refer bottom panel of Figure 4A, 4B, 4C for OM vs. SC, SC vs. IM
and OM vs. IM respectively).

Figure 3. Functionality of common identified proteins in
proliferating preadipocytes from three depots. The 16 functional
protein categories found were based on the n = 138 individual
proteins and are largely assigned according to the gene ontology
database with additional help from Swissprot. The largest group
comprising 41% was related to cell structure and next being
proteins of carbohydrate metabolism (9%). The proteome of the
proliferating preadipocytes was categorized based on the
information contained in Swiss-Prot. The "Other" function category
(10%) includes cytokine, ion channel, iron-binding protein, signal
transduction, and transcription.

Cell strcture                 41%
Cell growth                    1%
Proteins of TCA
Carbohydrate metabolism        9%
Energy generation              5%
Translation                    4%
Stress response                6%
Cell signaling                 3%
Small molecule transport       2%
Lipid metabolism               2%
Oxidation/Reduction            2%
Amincacid metabolism           2%
Others                        10%
Subcellular organells          3%
Nucleus/Ribosomal protein      6%

Note: Table made from pie graph.
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Article Details
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Author:Rajesh, Ramanna Valmiki; Kim, Seong-Kon; Park, Mi-Rim; Nam, Jin-Seon; Kim, Nam-Kuk; Kwon, Seulemina;
Publication:Asian - Australasian Journal of Animal Sciences
Article Type:Report
Geographic Code:9SOUT
Date:Jan 1, 2011
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