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Journal of Animal Science - Animal Growth, Physiology, and Reproduction

Factors influencing the differentiation of bovine preadipocytes in vitro1

 

This article in JAS

  1. Vol. 88 No. 6, p. 1999-2008
     
    Received: Aug 27, 2009
    Accepted: Feb 09, 2010
    Published: December 4, 2014


    2 Corresponding author(s): bcorl@vt.edu
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doi:10.2527/jas.2009-2439
  1. A. J. Lengi and
  2. B. A. Corl 2
  1. Department of Dairy Science, Virginia Polytechnic Institute and State University, Blacksburg 24061-0315

ABSTRACT

Our objectives were to isolate bovine stromal-vascular cells using explants and to determine media components that promote differentiation into mature adipocytes for studies of lipogenic enzyme regulation. Stromal-vascular cells were grown from explants and treated with differentiation media for 8 d after reaching confluence. Differentiation was assessed by measuring radiolabeled acetate incorporation into lipids, glycerol-3-phosphate dehydrogenase activity, and the mRNA expression of fatty acid binding protein-4, PPAR-γ, and acetyl-CoA carboxylase-α (ACCα). After 8 d of differentiation, medium containing 10 μg/mL of insulin, 0.25 μM dexamethasone, 0.5 mM isobutylmethylxanthine, 1 mM octanoate, and 2% Intralipid (Fisher Scientific, Suwanee, GA) produced greater acetate incorporation (P < 0.001) and glycerol-3-phosphate dehydrogenase activity (P < 0.001) compared with other media tested. This differentiation medium also increased mRNA expression of fatty acid binding protein-4, PPARγ, and ACCα by 180-, 7-, and 3-fold, respectively, compared with undifferentiated control cells (P < 0.05). To further improve the differentiation protocol, the effects of Intralipid, rosiglitazone, and troglitazone were examined. Removal of 2% Intralipid did not improve any differentiation measures. Addition of rosiglitazone (1 μM), a PPAR-γ agonist, increased acetate incorporation and ACCα mRNA (P < 0.01). Addition of troglitazone (5 μM), another PPAR-γ agonist, increased acetate incorporation to a similar extent as rosiglitazone and produced the greatest expression of ACCα mRNA (P < 0.01), but was not superior to medium that included rosiglitazone for any other differentiation measures. Cell-seeding density influences the cell divisions required to reach confluence, and increased plating density (2 × 104 cells/cm2 vs. 6.7 × 103 cells/cm2) increased acetate incorporation by 100% (P < 0.001). Differentiating stromal-vascular cells in the presence of trans-10, cis-12 CLA inhibited differentiation of stromal-vascular cells into mature adipocytes, reducing radiolabeled acetate incorporation into lipids (P < 0.001), stearoyl-CoA desaturase-1 mRNA (P < 0.05) and protein abundance (P < 0.05), and ACCα protein abundance (P < 0.05). We have developed a method to differentiate primary bovine adipocytes, which will allow us to study the regulation of lipogenic enzymes by nutrient and endocrine factors.



INTRODUCTION

Intramuscular adipose tissue (marbling) improves beef quality and is the basis for quality grading (Platter et al., 2005). In contrast, excess subcutaneous adipose tissue is discarded during carcass processing and wastes feed resources. Data from the USDA indicate a trend for production of fatter carcasses with less marbling (Drouillard and Reinhardt, 2008), contrary to the objectives of the beef industry. Developing a greater understanding of bovine adipocyte biology may lead to strategies to shift fat deposition to depots that add value and away from depots that reduce returns.

In vitro cell culture methods are used to study adipocyte biology under controlled conditions. Procedures to isolate preadipocytes, or stromal-vascular cells, from adipose tissue can require significant amounts of tissue and collagenase. After isolation, stromal-vascular cells are cultured with specialized media containing a cocktail of chemicals to stimulate differentiation into mature adipocytes. However, the specific media components required to stimulate differentiation differ by species. Factors affecting the differentiation of porcine and ovine stromal-vascular cells in vitro have been described (Suryawan et al., 1997; Soret et al., 2006). Methods for stromal-vascular cell isolation and adipocyte differentiation have facilitated detailed studies of the effects of hormonal and nutritional factors on adipocytes, including bovine adipocytes (Aso et al., 1995; Suryawan et al., 1997; Wu et al., 2000; Soret et al., 2006; Hirai et al., 2007).

Our overall goal is to define mechanisms controlling bovine adipocyte differentiation and filling. In this work, our first objective was to develop a method for the harvest of bovine stromal-vascular cells from small amounts of adipose tissue and to stimulate differentiation into mature adipocytes. Our second objective was to examine the influence of trans-10, cis-12 CLA on bovine adipocyte differentiation.


MATERIALS AND METHODS

Tissues were collected postmortem from animals slaughtered for food and not for research; therefore, Institutional Animal Care and Use Committee approval was not required.

Cell Culture Supplies and Media Additives

Sodium acetate, dexamethasone, 3-isobutyl-1-methylxanthine, insulin, octanoate, Dulbecco’s modified Eagle’s medium (DMEM), DMEM/Ham’s nutrient mixture F12, Hanks’ balanced salt solution, and 100× antibiotic-antimycotic solution were all purchased from Sigma-Aldrich (St. Louis, MO). Fetal bovine serum was purchased from Atlanta Biologicals (Lawrenceville, GA). Trypsin-EDTA was purchased from Mediatech Inc. (Herndon, VA). Rosiglitazone and troglitazone were purchased from Cayman Chemical (Ann Arbor, MI). Linoleic acid was purchased from Nu-Chek Prep (Elysian, MN). Trans-10, cis-12 CLA was purchased from Matreya LLC (Pleasant Gap, PA). Intralipid (20%) was purchased from Fisher Scientific (Suwanee, GA).

Animals

Adipose tissue for cell isolation was collected from beef cattle slaughtered at a local abattoir or by the Department of Food Science and Technology at Virginia Tech. Most of the cattle were Angus or Angus-cross steers, but the background of some animals was unknown. Subcutaneous adipose tissue was collected from the flank just above the lymph node immediately after exposure. Adipose tissue was transported to the laboratory in prewarmed growth medium (DMEM/Ham’s nutrient mixture F12 supplemented with 10% fetal bovine serum and antibiotics). Transport time was less than 15 min for tissue collected at the Food Science Department and 45 min for tissue collected at the abattoir.

Stromal-Vascular Cell Isolation Procedure

On return to the laboratory, tissue was transferred into a sterile cell culture hood and briefly, but vigorously, rinsed in a solution of 20% betadine in growth medium, after which all procedures were performed under aseptic conditions. Tissue was rinsed again in fresh sterile growth medium, and minced into pieces of approximately 1 mm3. Five drops of high-vacuum grease were placed in a 10-cm cell culture Petri dish. Four pieces of tissue were arranged around each drop of sterile grease. With forceps, a coverslip was carefully pressed on top of the tissue pieces and was held in place by vacuum grease, as shown in Figure 1A. Coverslips and high-vacuum grease were sterilized by autoclaving. After all 5 coverslips were added to the Petri dish, 15 mL of growth medium was added, with care taken not to dislodge the coverslips. Explants were cultured at 37°C and 5% CO2, changing medium every 3 to 4 d. Within a few days, adherent stromal-vascular cells were observed growing out from the explants on both the culture dish and the coverslips (Figure 1B). After 10 to 14 d, coverslips were removed and transferred to separate culture dishes, and cell monolayers and coverslips were washed with Hanks’ balanced salt solution. Cells were then harvested from the culture dishes and coverslips by trypsinization, collected by centrifugation at 250 × g for 10 min at room temperature, counted, and reseeded into fresh 6-well plates. New sets of explants, isolated from different animals, were used for each experiment.

Figure 1.
Figure 1.

Bovine stromal-vascular cells were grown from adipose tissue explants. Adipose tissue explants were compressed under coverslips held in place by a central drop of vacuum grease (A). Stromal-vascular cells were apparent at d 4 (B). Stromal-vascular cells continued to grow on the surface of the Petri dish and on the coverslip as shown on d 7 (C) and d 10 (D).

 

Experiments

Differentiation Experiments.

Two experiments were conducted to examine media components required for stimulation of stromal-vascular cell differentiation into mature adipocytes. After stromal-vascular cell isolation, cells were seeded at approximately 6.7 × 103 cells/cm2. On reaching confluence, growth medium was removed and replaced with 3 mL of one of the differentiation media described in Tables 1 and 2. One treatment group included cells that remained in growth medium (undifferentiated controls). Media were changed every other day. Eight days after the initiation of differentiation, radiolabeled acetate incorporation into lipids and glycerol-3-phosphate dehydrogenase (G3PDH) activity were measured, and RNA was isolated.

Table 1.

Please see the pdf to view this table.

 
Table 2.

Please see the pdf to view this table.

 

Seeding Density Experiment.

Stromal-vascular cells were seeded at either approximately 6.7 × 103 cells/cm2 (low density) or approximately 2 × 104 cells/cm2 (high density). After reaching confluence, growth medium was removed and replaced with 3 mL of differentiation medium 2.2 (Table 2). Stromal-vascular cells seeded at high density typically reached confluence 2 to 3 d before stromal-vascular cells seeded at low density. After 8 d in differentiation medium, incorporation of radiolabeled acetate into lipids was determined.

CLA Experiment.

After stromal-vascular cell isolation, cells were seeded at approximately 2 × 104 cells/cm2 and allowed to reach confluence. Growth medium was then removed and replaced with 3 mL of differentiation medium 2.2 (Table 2). One of 3 treatment supplements was added. Linoleic acid (cis-9, cis-12 18:2; 50 μM) and trans-10, cis-12 CLA (50 μM) were added as fatty acid complexes with BSA (2.5:1 molar ratio). For complexes, sodium salts of fatty acids were prepared by combining fatty acids and 50 mM sodium hydroxide solution warmed to 70°C followed by continued warming and vigorous mixing until solutions were clear. Fatty acid-free BSA was dissolved in culture medium followed by addition of sodium salts of fatty acids and mixing to produce complexes. Control treated cells received BSA (20 μM). After 8 d in supplemented differentiation media, radiolabeled acetate incorporation into lipids was measured. Cells were also harvested for RNA isolation and immunoblotting.

Laboratory Analyses

Radiolabeled Acetate Incorporation Assay.

Fatty acid synthesis was measured as radiolabeled acetate incorporation into lipids. A 500-μL quantity of medium was removed from a well of a 6-well plate containing 3 mL of medium and replaced with 500 μL of DMEM containing 1 μCi of [1,2-14C]acetate (ARC Inc., St. Louis, MO). Dishes were returned to the incubator for 4 h. Cells were then washed twice with 1 mL of PBS and lysed in 1 mL of 0.1% SDS. Lysates were extracted with 3 mL of 3:2 hexane:isopropanol (Hara and Radin, 1978). The organic phase containing labeled fatty acids was transferred to a scintillation vial, combined with scintillation cocktail (ScintiSafe 30%, Fisher Scientific), and subjected to liquid scintillation counting (LS 6000LL, Beckman Coulter Inc., Brea, CA).

G3PDH Activity Assay.

Glycerol-3-phosphate dehydrogenase activity was assayed (Wise and Green, 1979). Briefly, cells from 1 well of a 6-well plate were harvested in 200 µL of buffer (50 mM Tris-HCl, 1 mM EDTA, 1 mM β-mercaptoethanol, pH 7.5) and sonicated (2 × 5 s, power setting 3; Sonicator Cell Disruptor, Model W 185 F, Plainview, NY). Duplicate wells were assayed for each treatment. The suspension was then cleared by centrifugation (10,000 × g, 30 min, 4°C), and the supernatant was used immediately for assays and determination of protein concentration by Bradford assay (Bio-Rad, Hercules, CA). Assays were conducted using a microplate reader (Bio-Tek Instruments, Winooski, VT). Total reaction volume was 200 μL and contained 100 mM triethanolamine-HCl (pH 7.5), 2.5 mM EDTA, 0.12 mM NADH, 0.2 mM dihydroxy acetone phosphate, 0.1 mM β-mercaptoethanol, and between 7 and 35 μg of protein. Reactions were conducted at 25°C and initiated by the addition of protein. Absorbance at 340 nm was measured.

Real-Time PCR.

Cells were plated in 6-well plates with 3 mL of medium, with duplicate wells for each treatment. Cells were scraped in 1 mL of TRI Reagent (Molecular Research Center, Cincinnati, OH) per well, and total RNA was isolated according to the instructions of the manufacturer. The RNA was reverse transcribed (500 ng/reaction) into cDNA using an Omniscript Reverse Transcription kit (Qiagen, Valencia, CA) according to the instructions of the manufacturer. Real-time PCR reactions were performed using a Quantitect SYBR Green PCR kit (Qiagen) and an Applied Biosystems 7300 Real-Time PCR instrument (Applied Biosystems, Foster City, CA). Each reaction was performed in duplicate wells. Beta-actin was used as an endogenous control gene, and undifferentiated (DMEM) cells were used as the calibrator for making relative comparisons between treatments. Primer pairs are shown in Supplemental Table 1. Reaction conditions used were 40 cycles of 94°C for 30 s, 58°C for 30 s, and 72°C for 1 min. For statistical analysis, the difference between the target transcript cycle threshold (Ct) and housekeeper transcript Ct value (ΔCt) was computed, and used for normality and homogeneity of variance testing and to determine the presence of significant treatment effects. Fold change is presented in figures and was calculated using the 2−ΔΔCt method, as described previously (Livak and Schmittgen, 2001).

Immunoblot.

Whole cell lysates were mixed with an equal volume of 2× Laemmli sample buffer and boiled for 5 to 10 min. Samples were electrophoresed on a 12 or 7.5% SDS-PAGE gel and transferred to a polyvinylidene difluoride membrane. Membranes were blocked with Tris buffered saline (TBS) with 0.1% Tween-20 and 5% nonfat dried milk for 1 h at room temperature on a shaking platform. Membranes were then incubated with antibodies reactive against acetyl-CoA carboxylase-α (ACCα; Cell Signaling Technology, Danvers, MA; 1:1,000), stearoyl-CoA desaturase-1 (SCD1; custom rabbit anti-bovine SCD1, Pacific Immunology, Ramona, CA; 1:1000), or β-actin (Sigma; 1:4000) diluted in TBS with 0.1% Tween-20 and 5% nonfat dried milk overnight at 4°C on a shaking platform. Membranes were washed and then incubated with the appropriate horseradish peroxidase-conjugated secondary antibody (Santa Cruz Biotechnology, Santa Cruz, CA) diluted in TBS with 0.1% Tween-20 and 5% nonfat dried milk for 1 h at room temperature on a shaking platform. Membranes were washed again and then developed using ECL-Plus Reagents (Amersham, Piscataway, NJ). Bands were visualized and quantified using a ChemiDoc XRS Digital Imaging System (Bio-Rad).

Statistical Analyses

Data were statistically analyzed using the mixed model procedure (SAS Inst. Inc., Cary, NC). Individual treatments were applied to cell culture dish wells, and this was the experimental unit. The model included set and treatment. When the effect of treatment was significant, treatment means were separated using Tukey’s multiple comparison adjustment. Differences in treatment means were considered different when P < 0.05.


RESULTS

Explant Method for Growing Preadipocytes

We developed an explant-based method for growing bovine stromal-vascular cells from adipose tissue based on procedures from Singer et al. (1985). Figure 1A shows small adipose explants compressed by sterile coverslips held by sterile vacuum grease in a Petri dish. Figure 1B, 1C, and 1D show adherent preadipocyte cells growing out from an explant onto the Petri dish surface after 4, 7, and 10 d, respectively. The proportion of explants that produced cells and the number of cells growing from each explant varied by animal.

Optimizing Preadipocyte Differentiation

We initially tested several previously published differentiation media preparations for differentiating bovine preadipocytes, as shown in Table 1. Stromal-vascular cells were grown to confluence in 6-well tissue culture dishes after seeding at a density of approximately 6.7 × 103 cells/cm2. After 8 d in differentiation media, the degree of differentiation induced by each medium was measured by several assays: acetate incorporation, G3PDH activity, and mRNA expression of fatty acid binding protein-4 (aP2), PPARγ, and ACCα, measured by quantitative real-time PCR (Figure 2). Differentiating cells with differentiation medium 2 resulted in greater radiolabeled acetate incorporation and G3PDH activity than in any other medium tested (P < 0.001). This medium did not result in different aP2, PPARγ, or ACCα mRNA expression compared with differentiation medium 1 or 1.1, but did result in greater aP2 and PPARγ mRNA expression compared with differentiation medium 3 (P < 0.001). We chose to use differentiation medium 2 for further experiments.

Figure 2.
Figure 2.

Differentiation of stromal-vascular cells into adipocytes. Stromal-vascular cells were induced to differentiate using differentiation media (Diff 1, 1.1, 2, 3) or grown in medium without differentiation components [Dulbecco’s modified Eagle’s medium (DMEM), Sigma-Aldrich, St. Louis, MO]. Differentiation measurements were executed on d 8 after initiation of differentiation and included incorporation of radiolabeled acetate into lipids (A) and activity of glycerol-3-phosphate dehydrogenase (G3PDH; B). The mRNA abundance of fatty acid-binding protein 4 (aP2; C), PPARγ (D), and acetyl-CoA carboxylase-α (ACCα; E) was measured by real-time PCR, with DMEM serving as the comparator. Error bars are SEM. Means lacking a common letter (a–c) are different (P < 0.05). This experiment was repeated 3 times with cells isolated from different animals, and duplicate wells were used for each measurement (n = 6).

 

We next tested the effect of several factors on bovine preadipocyte differentiation, including the lipid supplement Intralipid and the PPARγ agonists rosiglitazone and troglitazone. Medium was removed from confluent stromal-vascular cells and replaced with differentiation medium (Table 2). After culturing for 8 d, the degree of differentiation induced by each medium was assessed by acetate incorporation, G3PDH activity, and mRNA expression of aP2, PPARγ, and ACCα, measured by quantitative real-time PCR (Figure 3). Inclusion of Intralipid in differentiation medium 2 had no effect on acetate incorporation or G3PDH activity compared with medium with no Intralipid (differentiation medium 2.1). Expression of aP2, PPARγ, and ACCα mRNA was not different in media with or without Intralipid. The addition of 1 μM rosiglitazone to the differentiation medium (differentiation medium 2.2) resulted in increases in radiolabeled acetate incorporation and ACCα mRNA expression compared with media without rosiglitazone (P < 0.01), whereas G3PDH activity, aP2, and PPARγ expression mRNA were not different. The medium containing rosiglitazone resulted in the greatest induction of aP2 and PPARγ mRNA expression, resulting in greater than 200-fold and greater than 8-fold increases in the expression of aP2 and PPARγ, respectively, compared with undifferentiated cells (P < 0.001). The addition of 5 μM troglitazone to the differentiation medium (differentiation medium 2.3) did not result in increases in radiolabeled acetate incorporation, G3PDH activity, aP2 mRNA expression, or PPARγ expression compared with medium without troglitazone (differentiation medium 2). The addition of troglitazone did result in greater ACCα mRNA expression than any other differentiation medium tested (P < 0.001); however, G3PDH activity (P < 0.01), aP2 mRNA expression (P < 0.01), and PPARγ mRNA expression (P < 0.05) were less compared with the medium containing rosiglitazone. Therefore, we chose to use differentiation medium 2.2, containing 1 μM rosiglitazone and 2% Intralipid, for further experiments.

Figure 3.
Figure 3.

Differentiation of stromal-vascular cells into adipocytes. Stromal-vascular cells were induced to differentiate using differentiation media (Diff 2, 2.1, 2.2, 2.3) or grown in medium without differentiation components [Dulbecco’s modified Eagle’s medium (DMEM), Sigma-Aldrich, St. Louis, MO]. Differentiation measurements were executed on d 8 after initiation and included incorporation of radiolabeled acetate into lipids (A) and activity of glycerol-3-phosphate dehydrogenase (G3PDH; B). The mRNA abundance of fatty acid-binding protein 4 (aP2; C), PPARγ (D), and acetyl-CoA carboxylase-α (ACCα; E) was measured by real-time PCR, with DMEM serving as the comparator. Error bars are SEM. Means lacking a common letter (a–d) are different (P < 0.05). This experiment was repeated 3 times with cells isolated from different animals, and duplicate wells were used for each measurement (n = 6).

 

The Effect of Seeding Density on Bovine Preadipocyte Differentiation

Next, we examined the effect of seeding density on the ability of bovine preadipocytes to differentiate because cells seeded at a greater density require fewer rounds of cell division to reach confluence. Stromal-vascular cells were grown from explants, harvested, and seeded into 6-well plates at a low (6.7 × 103 cells/cm2) or high density (2 × 104 cells/cm2). After reaching confluence, cells were differentiated for 8 d in differentiation medium 2.2, after which the extent of differentiation was assayed by radiolabeled acetate incorporation (Figure 4). Cultures seeded at the greater density incorporated 100% more acetate than those seeded at a smaller cell density (P < 0.001); therefore, we chose the greater seeding density for subsequent experiments.

Figure 4.
Figure 4.

Increasing seeding density increases adipocyte differentiation potential of stromal-vascular cells. Stromal-vascular cells were seeded at a low (6.7 × 103 cells/cm2) or high (2 × 104 cells/cm2) seeding density and cultured in the differentiation medium (Diff 2.2) or medium without differentiation components [Dulbecco’s modified Eagle’s medium (DMEM), Sigma-Aldrich, St. Louis, MO] for 8 d. Differentiation was assessed by incorporation of radiolabeled acetate into lipids. Error bars are SEM. Means lacking a common letter (a–c) are different (P < 0.05). This experiment was repeated 4 times with cells isolated from different animals, and duplicate wells were used for each measurement (n = 8).

 

Effect of CLA on Bovine Preadipocyte Differentiation

Finally, we examined the effect of CLA on the ability of bovine preadipocytes to differentiate. After cells grew to confluence, medium was removed and replaced with differentiation medium 2.2 containing either 50 μM linoleic acid, 50 μM CLA, or 20 μM BSA, as the control. After culturing for 8 d, the effects of treatment differentiation media were assessed by acetate incorporation; mRNA expression of aP2, PPARγ, ACCα, and SCD1, measured by quantitative real-time PCR; and SCD1 and ACCα protein expression, measured by immunoblot. Differentiating bovine preadipocytes in the presence of CLA resulted in a reduction in radiolabeled acetate incorporation (P < 0.001) compared with both the linoleic acid-treated cells and the no fatty acid (BSA) control (Figure 5). There was no effect of CLA on the mRNA expression of aP2, PPARγ, or ACCα, but there was a reduction in the mRNA expression of SCD1 (P < 0.05) compared with cells cultured with linoleic acid and the no fatty acid (BSA) control. Both ACCα and SCD1 protein expression were reduced (P < 0.05) by differentiating preadipocytes in the presence of CLA compared with linoleic acid and a BSA control (Figure 6).

Figure 5.
Figure 5.

Conjugated linoleic acid reduces adipocyte differentiation of bovine stromal-vascular cells. Stromal-vascular cells were induced to differentiate in the presence of BSA [no fatty acid (No FA)], 50 μM linoleic acid (18:2), or 50 μM trans-10, cis-12 CLA. Differentiation measurements were executed on d 8 after initiation and included incorporation of radiolabeled acetate into lipids (A). The mRNA abundance of fatty acid-binding protein 4 (aP2; B), PPARγ (C), acetyl-CoA carboxylase-α (ACCα; D), and stearoyl-CoA desaturase-1 (SCD1; E) was measured by real-time PCR, with No FA serving as the comparator. Error bars are SEM. Means lacking a common letter (a–c) are different (P < 0.05). This experiment was repeated 3 times with cells isolated from different animals (n = 3).

 
Figure 6.
Figure 6.

Conjugated linoleic acid reduces stearoyl-CoA desaturase-1 (SCD1) and acetyl-CoA carboxylase-α (ACCα) in bovine adipocytes differentiated from stromal-vascular cells. Stromal-vascular cells were induced to differentiate in the presence of BSA [no fatty acid (No FA)], 50 μM linoleic acid (18:2), or 50 μM trans-10, cis-12 CLA. The protein abundance of SCD1 and ACCα was determined by immunoblotting, with actin indicating equal loading. Error bars are SEM. Means lacking a common letter (a, b) are different (P < 0.05). This experiment was repeated 3 times with cells isolated from different animals, and pooled duplicate wells were used for each measurement (n = 3).

 


DISCUSSION

With this report, we describe a novel method for growing bovine stromal-vascular cells using small adipose explants, based on procedures developed for growing thymic epithelial cells (Singer et al., 1985). This procedure offers some advantages over other stromal-vascular cell isolation methods. Compared with commonly used collagenase-based procedures, this explant procedure requires less time and expense to perform. It also requires less tissue, offering the possibility of using small amounts of tissue, such as intramuscular fat or an adipose biopsy, for the isolation of stromal-vascular cells. Additionally, this method enables the use of primary cells for experiments, rather than immortalized cell lines or clonally derived cells, which may have unexpected or unknown alterations in the signaling pathways of interest. Isolating cells from different animals for each experiment may better represent natural variation in broader populations, and this procedure facilitates isolation of cells from larger numbers of individual animals.

We observed a great deal of animal-to-animal variability regarding how well stromal-vascular cells grew from explants, as well as in some measures of adipocyte differentiation. It is likely that many factors, including breed, sex, age, reproductive status, and body condition of individual animals, can influence the growth and differentiation potential of stromal-vascular cells. We also observed that different production lots of fetal bovine serum used in the growth medium varied widely in their ability to support stromal-vascular cell growth from explants.

Differentiation of adipocytes is often assessed by their lipogenic capacity or the mRNA abundance of enzymes, proteins, or both that serve as markers of mature adipocytes. Indeed, we use the same types of differentiation markers in this report. Radiolabeled acetate incorporation into lipid and G3PDH activity are both measures of lipogenic capacity. We measured the mRNA abundance of PPAR-γ, ACCα, and aP2 as markers of mature adipocytes. The aP2 transcript has been used extensively as a marker of adipocyte differentiation (MacDougald and Lane, 1995; Gomez et al., 2003; McNeel and Mersmann, 2003; Darimont et al., 2006), as has G3PDH activity (Wu et al., 2000, 2001; Hirai et al., 2007; Grant et al., 2008). Most of these markers are used on a relative basis, however, and there is no threshold value for fatty acid synthesis or enzyme transcripts that define mature adipocytes.

In the present study, stromal-vascular cells were isolated exclusively from subcutaneous adipose tissue. It is probable that stromal-vascular cells isolated from different adipose depots will vary in their ability to differentiate in vitro. For example, a previous study using canine adipose tissue showed marked differences in the ability of cells isolated from different adipose depots to differentiate in culture (Wu et al., 2001). Similarly, in the bovine it has recently been shown that the extent of stromal-vascular cell differentiation in culture differs according to the originating adipose depot (Ortiz-Colón et al., 2009).

In this report, we found that including the PPARγ agonist rosiglitazone in the differentiation medium resulted in greater induction of acetate incorporation and greater ACCα mRNA expression compared with medium without rosiglitazone. Furthermore, rosiglitazone resulted in greater G3PDH activity and greater aP2 and PPARγ expression than medium containing troglitazone, another PPARγ agonist. Others have shown that troglitazone stimulated differentiation of bovine stromal-vascular cells compared with cells cultured without troglitazone, if cultured at a greater concentration (Grant et al., 2008). In addition, cells isolated from perirenal adipose tissue were shown to differentiate in medium containing troglitazone (Hirai et al., 2007), but neither of these studies included a direct comparison with rosiglitazone. These data also suggest that there may be adipose depot differences in the responsiveness of PPARγ to synthetic ligands. Differences in the transactivation potency of various synthetic PPARγ ligands have been observed among different species (Willson et al., 2000), but the activity of different PPARγ agonists in the bovine has not been evaluated. Despite the apparent superiority of rosiglitazone over troglitazone in most differentiation measures used in these experiments, troglitazone yielded similar or superior responses for ACCα mRNA expression. These results may reflect differences in the ability of PPARγ ligands to induce interaction with distinct transactivating cofactors, leading to ligand-specific PPARγ-mediated gene expression (Kodera et al., 2000).

Intralipid is a lipid emulsion that is stable in aqueous media. Consisting of phospholipids and triglycerides, it may supply exogenous fatty acids to developing adipocytes when included in cell culture media. No differences between cells cultured with or without Intralipid were observed for any differentiation measures (differentiation medium 2 vs. 2.1); however, differentiation medium containing Intralipid did increase G3PDH activity of differentiated omental adipose tissue-derived stromal-vascular cells compared with control cells in another report (Wu et al., 2000). In the present study, cells differentiated in the presence of Intralipid appeared to have larger lipid droplets (data not shown) compared with cells cultured in the absence of Intralipid, suggesting that lipid uptake from the medium may be important for lipid droplet expansion. In support of this, Wu et al. (2000) observed increases in G3PDH activity and the proportion of differentiated cells when stromal-vascular cells were cultured with exogenous very low density lipoprotein, but not with low-density or high-density lipoprotein.

Conjugated linoleic acid has been shown to have a suppressive effect on adiposity in many animal models (House et al., 2005), as well as a suppressive effect on milk fat synthesis in lactating dairy cows (Bauman et al., 2008). In this study, culturing differentiating bovine preadipocytes in the presence of CLA reduced acetate incorporation into lipids, indicating reductions in lipogenesis and inhibition of differentiation. Culturing bovine preadipocytes with CLA reduced SCD1 mRNA expression and protein abundance during differentiation in this experiment, in agreement with a previous report (reviewed by Smith et al., 2006). This response has also been repeatedly observed in vivo in many species. Mice fed trans-10, cis-12 CLA had reduced SCD activity (Park et al., 2000) and mRNA expression (Lee et al., 1998). Feeding CLA has also been shown to inhibit SCD activity and mRNA expression in chickens (Shang et al., 2005) and SCD1 activity in pigs (Gatlin et al., 2002; Smith et al., 2002). In addition, the bovine SCD1 gene promoter has been shown to be downregulated by CLA in the bovine mammary epithelial MAC-T cell line (Keating et al., 2006).

Interestingly, whereas CLA resulted in a reduction in SCD1 mRNA, it had no impact on other mRNA expression markers of differentiation, including aP2. Differentiating bovine preadipocytes in the presence of CLA did reduce protein concentrations of both SCD1 and ACCα. Changes in mRNA abundance often indicate changes in the protein abundance of a gene product, but the relationship of transcript to protein is not always 1:1, as was observed with ACCα mRNA and protein abundance in this experiment. An explanation for the disagreement between ACCα mRNA and protein abundance is unclear. The data would tend to indicate reduced translation or increased degradation of the protein, but there is no evidence in the literature to support this mechanism. Inhibition of differentiating preadipocytes by CLA has been repeatedly observed with 3T3 cells, human preadipocytes, and porcine stromal-vascular cells (Brandebourg and Hu, 2005; House et al., 2005 Hausman et al., 2009) as well as bovine stromal-vascular cells (reviewed by Smith et al., 2006). Many mechanisms have been proposed to explain the observed effects of CLA, including changes in lipolysis, β-oxidation, and energy expenditure; inhibition of fatty acid synthesis and uptake; inhibition of differentiation; and induction of dedifferentiation and apoptosis of adipocytes, but the exact mechanism remains unresolved (House et al., 2005; Bauman et al., 2008; Hausman et al., 2009).

The in vivo response of bovine adipose tissue to CLA has not been thoroughly examined. The dose of CLA required to reduce milk fat synthesis in dairy cows is much less than the dose required to reduce body fat accretion in other species (Bauman et al., 2008). The in vivo response of finishing beef cattle to CLA feeding has been examined in only 1 study. In that study, feeding a rumen-protected CLA supplement did not obviously influence any measures of fatness (Gillis et al., 2004). This may indicate that adiposity in cattle is unresponsive to CLA supplementation in general, that cattle are unresponsive at that age, or that an ineffective amount of CLA was absorbed because others have shown that the PUFA in a calcium-salt product are not fully inert in the rumen (Castañeda-Gutiérrez et al., 2007). The response of bovine preadipocytes in vitro in the current study is similar to the response of differentiating preadipocytes from other species known to respond to CLA supplementation in vivo, including rodents and pigs (Brandebourg and Hu, 2005; House et al., 2005; Bauman et al., 2008; Hausman et al., 2009).

Conclusions

With this report, we demonstrate a new approach for the isolation of bovine stromal-vascular cells, reducing the time and labor spent isolating these cells. In evaluating differentiating media, several factors promoting differentiation of subcutaneous preadipocytes were identified, including rosiglitazone and Intralipid. Finally, trans-10, cis-12 CLA reduced differentiation of preadipocytes when included with the differentiation medium. This resulted from inhibition of lipogenesis and may be related to inhibition of SCD1 expression.

 

References

Footnotes


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