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Differentiation of bovine intramuscular and subcutaneous stromal-vascular cells exposed to dexamethasone and troglitazone¹

A. C. Grant^*,2, G. Ortiz-Colón^*,3, M. E. Doumit^*,, R. J. Tempelman^* and D. D. Buskirk^*,4

^* Departments of Animal Science and Food Science and Human Nutrition, Michigan State University, East Lansing 48824-1225

	Abstract

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The objectives of these experiments were to compare differentiation of bovine stromal-vascular (S-V) cells isolated from i.m. and s.c. adipose tissues in response to a glucocorticoid and a peroxisome proliferator-activated receptor agonist. Stromal-vascular cells were isolated from i.m. and s.c. fat depots of 3 Angus steers and propagated in culture. Cells were exposed to differentiation media containing 0.25 µM dexamethasone (DEX), a glucocorticoid analog, and 40 µM troglitazone (TRO), a peroxisome proliferator-activated receptor agonist, or both. Cells treated with DEX and TRO had greater (P < 0.02) glycerol-3-phosphate dehydrogenase activity than control cells. No interactions between DEX, TRO, and depot (P > 0.59) or depot differences (P = 0.41) in glycerol-3-phosphate dehydrogenase activity were found. Morphological assessment of adipogenic colonies showed that DEX induced a 1.8-fold increase in the percentage of adipogenic colonies (P = 0.03), whereas TRO increased the proportion of adipogenic colonies by 1.9-fold (P = 0.02) compared with those not treated with DEX or TRO, respectively. Depots had a similar percentage of adipogenic colonies (P = 0.18); however, the percentage of differentiated cells within adipogenic colonies was found to be 6.4-fold greater in s.c. isolates compared with i.m. (P < 0.001). Addition of TRO increased the proportion of differentiated cells within colonies by 10-fold compared with those of nontreated colonies (P < 0.001), whereas the percentage of differentiated cells within adipogenic colonies only tended to be increased by DEX (P = 0.10). These data indicate that bovine i.m. and s.c. S-V cells are capable of enhanced differentiation in response to DEX and TRO, and these effects were additive. Most importantly, inherent differences in the capacity to differentiate exist between adipogenic bovine i.m. and s.c. S-V cells.

Key Words: bovine • differentiation • glycerol-3-phosphate dehydrogenase • glucocorticoid • peroxisome proliferator-activated receptor gamma • stromal-vascular

	INTRODUCTION

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Differentiation of preadipocytes into adipocytes is an important regulatory step in adipose development. Differentiation of preadipocytes is transcriptionally influenced by glucocorticoids (Wu et al., 1996) and peroxisome proliferator-activated receptor (PPAR) , which is expressed in bovine adipose tissue (Sundvold et al., 1997).

Glucocorticoids may function in differentiation via pathways that lead to the upregulation or activation of PPAR (Wu et al., 1996; Kliewer et al., 1997; Krey et al., 1997), which increases transcription of adipogenic genes (Tontonoz et al., 1994; Schoonjans et al., 1996). Glucocorticoids have been shown to increase differentiation of porcine (Ramsay et al., 1989; Tchoukalova et al., 2000), ovine (Soret et al., 1999), and bovine (Aso et al., 1995; Wu et al., 2000; Grant et al., 2008) stromal-vascular (S-V) cells. Greater differentiation in response to glucocorticoids was found in porcine s.c. compared with perirenal (Ramsay et al., 1989) and ovine s.c. compared with omental (Soret et al., 1999) S-V cells.

A PPAR ligand increased differentiation of perirenal and i.m. S-V cells isolated from Japanese Black cattle (Ohyama et al., 1998; Torii et al., 1998). Wu et al. (2000) found that a PPAR agonist induced a greater relative response in differentiation of bovine omental compared with s.c. S-V cells.

Comparisons of preadipocyte differentiation between cells from economically important adipose depots in the bovine are limited. The objective of these experiments was to determine the effects of dexamethasone (DEX), troglitazone (TRO), and their interaction on differentiation of bovine i.m. and s.c. S-V cells. We hypothesized that differentiation of bovine i.m. and s.c. S-V cells would be enhanced by a glucocorticoid and a PPAR agonist and that the relative response would be depot-dependent.

	MATERIALS AND METHODS

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Animal care was conducted according to procedures approved by the Michigan State University Committee on Animal Use and Care.

General Procedures

Subcutaneous and i.m. adipose tissues were collected from 3 Angus steers (age, 13.5 mo; HCW, 345 to 350 kg; s.c. fat thickness adjacent to the 12th rib, 12.7 to 21.6 mm). Stromal-vascular cells from s.c. and i.m. adipose tissues were isolated under sterile conditions according to procedures described previously (Grant et al., 2008). Briefly, adipose tissue samples were collected immediately after exsanguination from the left side of the carcass. Incisions were made dorsal to the 12th and 13th rib, and an approximately 10-cm³ sample containing a portion of both s.c. adipose tissue and LM was extracted. Upon collection, samples were immediately placed in sterile ice-cold PBS and transported to the laboratory. Subcutaneous and i.m. adipose tissues were separated from the LM, cut into sections, digested in collagenase, sequentially filtered, and centrifuged. Stromal-vascular cells were stored in liquid nitrogen before use. Stromal-vascular cells were thawed and propagated to confluence according to the procedures described by Grant et al. (2008). Experiments described here were performed on tertiary cultures. Unless otherwise stated, all reagents were of tissue culture grade and were purchased from Sigma (St. Louis, MO).

Differentiation media in these experiments consisted of base medium [Dulbecco’s modified Eagle’s medium, antibiotic-antimycotic (100 units of penicillin, 0.1 mg of streptomycin, and 0.25 µg of amphotericin B per milliliter), 50 µg/mL of gentamicin sulfate, 33 µM biotin, 17 µM pantothenate, and 100 µM ascorbate] plus 280 nM bovine insulin, 20 mM glucose, and 20 µL/mL of bovine serum lipids supplement (Ex-Cyte, Serologicals Corp., Norcross, GA) with treatment additions. Differentiation media treatment additions included 40 µM TRO, a thiazolidinedione PPAR agonist (Calbiochem, La Jolla, CA) and 0.25 µM DEX, a glucocorticoid analog, or both. The concentrations of media treatment additions were previously found to enhance differentiation of clonally derived bovine s.c. preadipocytes (Grant et al., 2008). Differentiation media were replaced with fresh media every 2 d. The TRO additions were present for the entire differentiation period, whereas DEX was supplemented only for the initial 48 h. Troglitazone was solubilized in dimethyl sulfoxide at a stock concentration of 22.7 mM before addition to media. Addition of dimethyl sulfoxide to differentiation media alone did not significantly affect differentiation (data not shown). All plates were washed twice with PBS before media replacement on d 2 of the differentiation period.

Experiment 1

Two independent trial replications were performed using i.m. and s.c. S-V cells from 3 steers. Subcutaneous and i.m. cells were seeded in 6-well tissue culture plates (35-mm diam.; Corning Inc., Corning, NY) at a density of 5,200 cells/cm² and incubated in growth medium at 37°C in a humidified atmosphere of 95% air and 5% CO₂. After reaching confluence, cells were washed twice with PBS, and differentiation media were added. Twelve days after differentiation, media treatments were applied, and glycerol-3-phosphate dehydrogenase (GPDH) enzyme activity, which is a biochemical marker of adipocyte differentiation, was quantified by the procedures described by Grant et al. (2008). All reactions measured were linear for at least 160 s, and GPDH activity was calculated from the linear range and expressed as nanomoles of NADH oxidized · min^–1·mg of protein^–1.

Experiment 2

Two independent trial replications using i.m. and s.c. S-V cells, derived from 3 steers, were exposed to differentiation media treatments with or without DEX and TRO in a 2 x 2 factorial arrangement of treatments. Cells were seeded in forty-eight 10-cm-diam. plates at clonal densities (400 cells/plate) and incubated in growth medium (base medium containing 10% fetal bovine serum) undisturbed for 8 d to allow distinct colonies to arise. Plates were washed twice with PBS, and differentiation treatments were applied. After a 10-d differentiation period, cell differentiation was morphologically assessed by observation of cells containing lipid droplets stained by oil red O with cell nuclei counterstained using Giemsa stain as described by Grant et al. (2008). Cells were visualized within 24 h of staining. The percentage of colonies with differentiated cells was determined by microscopic analysis, and the proportion of differentiated cells within colonies was determined by analysis of digital photomicrographs, which were obtained using a Nikon CoolPix 5000 digital camera (Nikon Inc., Melville, NY) fitted to a Zeiss inverted microscope (Carl Zeiss Inc., Thornwood, NY). A differentiated cell was defined as a cell having 1 or more lipid droplets 10 µm in diameter. A colony with at least 1 differentiated cell was defined as adipogenic. Any colony presumed to be derived from more than 1 cell was omitted from analysis. All distinct colonies observed were scored as adipogenic or nonadipogenic, and the percentage of adipogenic colonies on each plate was determined. Photomicrographs of 10 randomly selected adipogenic colonies on each of 24 plates were taken to determine the proportion of differentiated cells within adipogenic colonies. Three to 7 field-of-view photomicrographs of each colony were taken across the diameter of the colony to capture a representative cross-section of cells at 200x magnification. All enumerations were conducted by an evaluator blinded to depot and media treatment.

Statistical Analysis

Data were analyzed using the mixed model analysis procedure (PROC MIXED; SAS Inst. Inc., Cary, NC). For experiment 1, two independent trial replications were performed using i.m. and s.c. S-V cells from each of 3 steers. Steer was considered the experimental unit. The GPDH data were analyzed using 2 wells of a 6-well plate as the observational unit. To satisfy the conditions of normality and homogeneity of variance, GPDH data were log_e-transformed. Least squares means were calculated for the fixed effects of depot, DEX, TRO, and their 2- and 3-way interactions, with steer, steer x depot, steer x DEX, and steer x TRO included as random effects. For experiment 2, two independent trial replications were performed using i.m. and s.c. S-V cells from each of 3 steers. Plate served as the observational unit in the morphological determination of the percentage of adipogenic colonies, as well as for data collected on differentiated cells within colony. The logit transformation, as described by Ramsey and Schafer (2002), of each percentage response value was used to satisfy conditions of normality and homogeneity of variance. Least squares means for the percentage of differentiated colonies, and differentiated cells within colony, were calculated for the fixed effects of depot, DEX, TRO, and their 2- and 3-way interactions. When analyzing colony data, replication, steer, steer x depot, steer x DEX, and steer x TRO were included as random effects to appropriately define steers as the experimental units. Plates within steer were additionally included as random effects when differentiated cells within a colony were analyzed. No interactions were determined to be significant in the experiments described herein (P > 0.27). Main effects were considered significant at P < 0.05. When main effects were determined to be significant, differences between means were investigated using Tukey’s multiple comparison test.

	RESULTS

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Dexamethasone and TRO initiated increases in GPDH activity over cells not treated with DEX (P = 0.01) or TRO (P = 0.02), respectively (Figure 1). There was no interaction between DEX and TRO (P = 0.53), and the combination of compounds increased GPDH activity above that of either compound alone (P < 0.05). No significant depot differences in differentiation of i.m. and s.c. cells were observed (P = 0.40; Figure 1).

View larger version (12K): [in this window] [in a new window]   Figure 1. Effect of differentiation media with no additions (control), addition of 0.25 µM dexamethasone (DEX) for the first 48 h, 40 µM troglitazone (TRO) for 12 d, or their combination on loge glycerol-3-phosphate dehydrogenase (GPDH) activity of bovine intramuscular (IM) and subcutaneous (SC) stromal-vascular cells from steers (n = 3). Values are least squares means and SEM for 2 replicate trials. DEX: P = 0.01; TRO: P < 0.02; depot: P = 0.40; DEX x TRO: P = 0.53. *P < 0.05; **P < 0.01; ***P < 0.001.

View larger version (89K): [in this window] [in a new window]   Figure 2. Representative photomicrographs of intramuscular and subcutaneous adipogenic stromal-vascular cell colonies exposed for 10 d to differentiation media. Treatments included no additions (control), addition of 0.25 µM dexamethasone (DEX) for the first 48 h, 40 µM troglitazone (TRO) for 10-d, or their combination. Cells are stained with oil red O and lightly counterstained with Giemsa stain. Bar = 100 µm.

	DISCUSSION

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Peroxisome proliferator-activated receptor has been described as a critical transcriptional regulator in preadipocyte differentiation (Spiegelman, 1998) and is primarily expressed in adipose tissues of most species (Chawla et al., 1994; Braissant et al., 1996) including cattle (Sundvold et al., 1997). Peroxisome proliferator-activated receptor is activated by endogenous (Kliewer et al., 1997; Krey et al., 1997) and synthetic (Lehmann et al., 1995; Spiegelman, 1998) ligands. Once ligand-bound, PPAR dimerizes with a retinoid X receptor. This dimer is then capable of binding to PPAR response elements in the promoters of adipogenic genes, which stimulate differentiation (Kliewer et al., 1992). Undifferentiated bovine (Torii et al., 1998) and porcine (Kim et al., 2000) S-V cells express PPAR protein. Therefore, differentiation in S-V cells may be stimulated through agonist binding to PPAR (Ding et al., 2003). Thiazolidinediones, such as TRO, are known to be high-affinity PPAR ligands (Lehmann et al., 1995) and have been used to enhance differentiation of bovine (Grant et al., 2008), ovine (Soret et al., 1999), and porcine (Tchoukalova et al., 2000) S-V cells.

Glucocorticoids can influence adipocyte differentiation through the upregulation of CCAAT-enhancer binding protein β expression in 3T3-L1 cells (Yeh et al., 1995; Wu et al., 1996; Tomlinson et al., 2006). In addition, glucocorticoids have been found to increase arachidonic acid metabolism, with subsequent PG production, in Ob1771 preadipocytes (Gaillard et al., 1991). These pathways may lead to the upregulation of PPAR protein or increased ligand binding to PPAR respectively (Wu et al., 1996; Kliewer et al., 1997; Krey et al., 1997), resulting in the transcription of adipogenic genes (Tontonoz et al., 1994; Schoonjans et al., 1996). Glucocorticoids have been shown to increase adipose conversion in ovine (Soret et al., 1999), porcine (Ramsay et al., 1989; Tchoukalova et al., 2000), as well as in bovine i.m. (Aso et al., 1995; Sato et al., 1996; Grant et al., 2008), s.c. (Wu et al., 2000; Grant et al., 2008), and omental (Wu et al., 2000) S-V cells.

Both PPAR ligands and glucocorticoids may have adipose depot-specific effects on differentiation of S-V cells, but comparisons of these compounds on S-V cells derived from different bovine adipose tissue depots are lacking. Thiazolidinediones, which are potent PPAR ligands, stimulate differentiation of human S-V cells isolated from s.c. fat to a greater extent than those isolated from omental fat (Adams et al., 1997; Sewter et al., 2002; Tchkonia et al., 2002). In contrast, omental S-V cells isolated from wether lambs (Soret et al., 1999) and Holstein cows (Wu et al., 2000) were stimulated to differentiate to a greater extent by PPAR ligands than s.c. S-V cells. These authors suggested that omental cells may have a lower capacity to produce endogenous PPAR ligands than s.c. cells. Contrary to our hypothesis, TRO increased differentiation similarly in both i.m. and s.c. S-V cells. Troglitazone increased both the number of colonies that contained differentiated adipocytes and greatly increased the relative percentage of differentiated adipocytes within adipogenic colonies from both i.m. and s.c. depots. Collectively, these data indicate that TRO triggered preadipocyte differentiation through PPAR activation. These effects were paralleled by increased GPDH activity for preadipocytes treated with TRO in mass culture.

Ramsay et al. (1989) found that glucocorticoid administration stimulated GPDH activity in porcine s.c. S-V cells, whereas perirenal cells did not respond. Similarly, Soret et al. (1999) found greater GPDH activity in DEX-stimulated ovine s.c. S-V cells than in omental cells. Contrary to our hypothesis, our results demonstrate that the addition of 0.25 µM DEX to differentiation media increased GPDH activity in bovine i.m. and s.c. preadipocytes to an equal extent. Likewise, DEX affected differentiation of the proportion of colonies and cells within colonies similarly between depots, although the effects were less pronounced than with TRO.

Given that DEX may increase the expression of PPAR, and that TRO appears capable of activating PPAR, these compounds may interact to stimulate bovine preadipocyte differentiation. However, studies investigating possible interactions between the 2 compounds, and their potential depot-specific effects on bovine S-V cell differentiation, have not been reported. Tchoukalova et al. (2000) found that DEX and a thiazolidinedione enhanced GPDH activity in porcine S-V cells when added individually to differentiation media. However, the combination of compounds did not result in an additive effect. Using S-V cells isolated from wether lambs, Soret et al. (1999) found that addition of DEX in combination with a PPAR agonist had additive effects on GPDH activity of both omental and s.c. cells, with no depot differences reported. Although no depot x treatment interactions in GPDH activity were found in our present experiment, we showed that TRO and DEX individually stimulated GPDH activity in bovine S-V cells, and the combination of compounds was additive. This is similar to results in primary human preadipocytes where DEX and TRO have been shown to sequentially stimulate differentiation (Tomlinson et al., 2006).

Approximately 50% of the colonies evaluated by clonal analysis were adipogenic under the various treatment conditions. The fact that a similar percentage of adipogenic colonies was observed in i.m. and s.c. S-V cultures indicates that our isolation procedures yield a similar proportion of preadipocytes from both adipose tissue depots. Importantly, we found that s.c. cells exhibited a greater inherent ability to differentiate than those from i.m. when exposed to these culture conditions. We recognize that by using clonal analysis we may have underestimated the number of i.m. colonies that would ultimately be adipogenic, because they differentiate to a lesser degree compared with colonies from s.c. However, this potential bias would make the observed differences in proportion of differentiated cells between i.m. and s.c. cells even more conservative. We detected depot differences in differentiation using clonal analysis but did not detect differences in enzyme activity using mass culture. The contrasting results were likely due to the difference in sensitivity between the 2 techniques. The mass culture technique may have been constrained by variability in GPDH activity among cells from different animals and the relatively low percentage of i.m. preadipocytes that differentiated. Activity of GPDH in the control treatments frequently proved to be below the sensitivity of the GPDH assay. Clonal analysis appears to be a more sensitive method of detecting differences in differentiation between preadipocytes from these depots than measurement of GPDH activity.

We conclude that bovine S-V cells isolated from i.m. and s.c. depots are capable of enhanced differentiation in response to DEX and TRO. Although no depot-specific responses due to treatment were found, we are the first to report that bovine s.c. preadipocytes differentiate more extensively than i.m. preadipocytes when cultured in similar environments. This suggests that inherent differences in the capacity for adipose differentiation between bovine i.m. and s.c. preadipocytes exist. A better understanding of factors that differentially influence differentiation of preadipocytes derived from i.m. and s.c. adipose tissues will aid in development of strategies to improve i.m. fat deposition and decrease excess s.c. fat accumulation or both in beef carcasses.

	Footnotes

 
¹ This research was supported by the Michigan Agricultural Experiment Station and was partially funded through a grant from the Michigan Animal Agricultural Initiative. We thank the staff of the Michigan State University Beef Cattle Teaching and Research Center for care of the animals, the staff of the Michigan State University Meats Laboratory and E. Helman and C. Allison in the Department of Animal Science at Michigan State University for assistance with sample collection and laboratory assistance..

² Current address: Nutrition Service Associates, PO Box 839, Okotoks, Alberta, T1S1A9, Canada.

³ Current address: University of Puerto Rico, Mayagüez, Department of Animal Sciences, PO Box 9030, Mayagüez, Puerto Rico 00681-9030.

⁴ Corresponding author: buskirk{at}msu.edu

Received for publication January 10, 2008. Accepted for publication May 22, 2008.

	LITERATURE CITED

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