PPARγ agonist through the terminal differentiation phase is essential for adipogenic differentiation of fetal ovine preadipocytes
© The Author(s) 2017
Received: 12 January 2017
Accepted: 10 March 2017
Published: 23 March 2017
Although the 3T3-L1 preadipocyte cell line represents an informative model for in vitro adipogenesis research, primary cultured cells are often needed to understand particular human or animal metabolic phenotypes. As demonstrated by in vitro cultured preadipocytes from large mammalian species, primary cultured cells require specific adipogenic differentiation conditions different to that of the 3T3-L1 cell line. These conditions are also species-specific and require optimization steps. However, efficient protocols to differentiate primary preadipocytes using alternative species to rodents are scarce. Sheep represent an amenable animal model for fetal biology and developmental origins of health and disease studies. In this work, we present with the first detailed procedure to efficiently differentiate primary fetal and adult ovine preadipocytes.
Fetal and adult ovine adipose and skin tissue harvest, preadipocyte and fibroblast isolation, proliferation, and standardization and optimization of a new adipogenic differentiation protocol. Use of commercial cell lines (3T3-L1 and NIH-3T3) for validation purposes. Oil red O stain and gene expression were used to validate adipogenic differentiation. ANOVA and Fisher’s exact test were used to determine statistical significance.
Our optimized adipogenic differentiation method included a prolonged adipogenic cocktail exposure time from 2 to 8 days, higher insulin concentration, and supplementation with the peroxisome proliferator-activated receptor gamma (PPARγ) agonist, rosiglitazone. This protocol was optimized for both, fetal and adult preadipocytes.
Our protocol enables successful adipogenic differentiation of fetal and adult ovine preadipocytes. This work demonstrates that compared to the 3T3-L1 cell line, fetal ovine preadipocytes require a longer exposure to the differentiation cocktail, and the need for IMBX, dexamethasone, and/or the PPARγ agonist rosiglitazone through the terminal differentiation phase. They also require higher insulin concentration during differentiation to enhance lipid accumulation and similar to human primary preadipocytes, PPARγ agonist supplementation is also required for ovine adipogenic differentiation. This work highlights species-specific differences requirements for adipogenic differentiation and the need to develop standardized methods to investigate comparative adipocyte biology.
KeywordsSheep Fetal Preadipocyte Adipogenic differentiation
To study adipogenesis, adipogenic differentiation, and their regulation, investigators often resource to the commercially available preadipocyte cell line 3T3-L1 of murine origin . Results from these studies are not as useful for applications on human health as expected, given the physiological and metabolic differences among species . Human primary cultured cells are the best resource to understand human adipocyte biology in deranged metabolic states , but cannot inform about cell events that occur during prenatal life. The developmental origins of health and disease theory is pointing to early life events as one of the contributing factors towards the increased prevalence of metabolic diseases worldwide, including obesity [3–5]. To test hypotheses relative to fetal adipose tissue differentiation and fate and/or the effects of prenatal exposures (chemicals, stress, nutrition) on adult metabolic risk, alternative models are often needed . Other large mammalian species, such as feline and porcine primary cells, have recently been used as alternatives [7, 8]. The sheep is an outstanding animal model to understand how prenatal exposures can affect adult metabolic disease risk [9–12] because, as humans, sheep are a monotocous and precocial species, with majority of organs maturing before birth , including the adipose tissue . Identified barriers to the advance of our understanding on adipocyte biology include the great diversity of protocols available for some cell lines and the lack of standardized methods of adipogenic differentiation in other mammalian species . As we demonstrate in this study, standard murine 3T3-L1 differentiation protocols [15, 16] are not suitable for ovine preadipocyte adipogenic differentiation.
Adipocyte differentiation is a complex process by which preadipocytes transition into lipid-filled, insulin-responsive adipocytes. Adipocyte fate is controlled by transcription factors, including peroxisome proliferator-activated receptor gamma (PPARγ), CCAAT/enhancer-binding proteins (C/EBPs), and sterol regulatory element binding protein (SREBP). Preadipocyte differentiation is routinely initiated by a 48 h exposure to a basic 3T3-L1 differentiation induction cocktail containing 3-isobutyl-1-methyxantine (IBMX), dexamethasone, and insulin . IBMX regulates C/EBPβ , enhances 3T3-L1 differentiation upon longer exposures , and alone  or in combination with dexamethasone , regulates PPARγ activity. PPARγ, the master regulator of adipogenesis, cooperates with C/EBPα to initiate adipogenic differentiation, is required in human preadipocyte differentiation [21, 22], and can increase glycerol-3-phosphate dehydrogenase (GPDH) activity in sheep preadipocytes , an enzyme involved in lipid biosynthesis. PPARγ agonists, such as thiazolidinediones, have been used to enhance adipogenic differentiation in 3T3-L1 and primary preadipocytes of large mammals [7, 14, 17]. In addition, supplementation with insulin promotes preadipocyte differentiation , but the insulin dose is among the most variable media components of 3T3-L1 differentiation protocols .
In this work, we aimed to standardize a protocol for effective ovine adipogenic differentiation using fetal and adult primary cultured preadipocyte that will help understand basic adipocyte biology knowledge in a relevant meat-producing species, as well as events occurring during fetal adipose tissue remodeling. To enable ovine adipogenic differentiation, we applied several media modifications. Here, we have only presented the key modifications that resulted in significant improvements in ovine preadipocyte adipogenic differentiation.
Materials and methods
Fetal and adult tissue harvest
All procedures used in this study were approved by the Institutional Animal Care and Use Committee of Michigan State University (MSU) and are consistent with the National Research Council’s Guide for the Care and Use of Laboratory Animals and the Animal Welfare Act. The study was conducted at the MSU Research Facility (East Lansing, MI; 42.7° N, 84.4° W) using an in-house flock of multiparous Polypay x Dorsett breed of sheep. Female sheep were bred using a time mated pregnancy strategy. At gestational day 120 and after humane sacrifice, fetal perirenal adipose tissue and skin from the abdominal midline were harvested from female fetuses to isolate fetal ovine preadipocytes (oPADs) and skin fibroblasts. Immediately after, tissues were placed in transport medium (Omental Preadipocyte Medium, Zen Bio, USA) and processed within two hours. Adipose tissue was also collected from the subcutaneous adipose depot of three adult females after humane sacrifice.
Generation of fetal and adult ovine primary preadipocytes
Preadipocyte isolation method was conducted following a common standard protocol used in isolation of human adipogenic precursor cells . This isolation protocol followed by adipogenic differentiation allows an average cell differentiation rate of 50-60% . In brief, fetal and adult oPADs were isolated by rinsing a 1–2 g of adipose tissue with pre-warmed Dulbecco’s phosphate-buffered saline (DPBS) with antibiotic-antimycotic (Invitrogen), removing any visible blood clots and connective tissue using sterile forceps and then minced into small pieces. Collagenase-I (C0130, Sigma, 1 mg/ml) was used to digest tissue for 40 min in a 37 °C water bath. The mixture was filtered through a mesh filter (250 μm, U-CMYBK-250, Component Supply Company, Fort Meade, USA) and centrifuged at 1,200 rpm for 5 min. The cell pellet was washed with fresh omental preadipocyte medium and seeded into 6-well plates. After six days of culture, cells were frozen (Preadipocyte Cryopreservation Medium, FM-1-100, Zen-Bio) and stored in liquid nitrogen until further use. All experiments were performed using fetal oPAD primary cells (passage 3) from one female and validated with fetal and adult oPADs from 2 and 3 additional females, respectively. The 3T3-L1 (preadipocyte) and NIH-3T3 (fibroblastic) cell lines were used as positive and negative control of adipogenic cells, respectively. Ovine fetal primary fibroblasts were also used as a negative control of adipogenesis.
Generation of fetal ovine primary skin fibroblasts
Ovine fetal primary fibroblasts were isolated as follows. After hair and connective tissue removal using sterile forceps, fetal skin tissue was rinsed twice with pre-warmed DPBS supplemented with antibiotic and antimycotic. Tissue (1 g) was minced into small pieces and distributed into 6-well plates with 0.2 ml of serum per well. After 6 h incubation, 2 ml of basal medium consisting of DMEM/F12 medium (Invitrogen) supplemented with 1% penicillin-streptomycin, 10 mM HEPES, and 10% fetal bovine serum (Corning, Manassas, VA, USA) was added into the plate and primary cells cultured for 4 to 6 days until confluency. Cells were then trypsinized and passaged as primary fibroblasts for further study.
Ovine primary preadipocytes proliferation
Primary cultured oPADs were trypsin digested (Invitrogen, 0.05%) for subculture upon 90% confluency. Proliferation ability for each cultured cell (passage 3) was assessed by growth curve analysis. Cells were seeded at a density of 10,000 cells per well in 24-well plates and the counts were performed in haemocytometer chamber. Cell counts were performed every 24 h for 8 days. Triplicate wells for each time point per cultured cell were used.
Cell culture and adipocyte differentiation
Oil Red O stain
Preadipocyte differentiation into mature adipocytes was determined by Oil Red O (ORO) stain, as a marker of lipid accumulation. In brief, ORO (0.3%) was dissolved in isopropanol diluted in water (0.18%). Adipocytes at differentiation day 8, were washed with DPBS twice then fixed in 10% formalin for 30 min at room temperature. Cells were washed with DPBS and incubated 5 min in 60% isopropanol followed by incubation in ORO working solution. After multiple DPBS washes, bright field images were captured on an inverted microscope. Five images of each treatment were used to calculate ORO positive area using the Image J software .
Quantitative real time PCR (qRT-PCR)
Primers for quantitative real time PCR
oADIPOQ - Forward
oADIPOQ - Reverse
oβ-ACTIN - Forward
oβ-ACTIN - Reverse
oC/EBPα - Forward
oC/EBPα - Reverse
oDLK1 - Forward
oDLK1 - Reverse
oFABP4 - Forward
oFABP4 - Reverse
oGAPDH - Forward
oGAPDH - Reverse
mGAPDH - Forward
mGAPDH - Reverse
oPPARγ - Forward
oPPARγ - Reverse
oRPL27 - Forward
oRPL27 - Reversr
oZFP423 - Forward
oZFP423 - Reverse
mZFP423 - Forward
mZFP423 - Reverse
All data are presented as mean ± SEM. All experiments (adipogenic differentiation in fetal and adult oPADs and oSFs, and qRT-PCR) were conducted in triplicate. Appropriate transformations were applied, as needed, to account for normality of data. ANOVA was used to compare gene expression data among differentiation time points followed by Dunnett’s posthoc test. For comparing percent differences Fisher’s exact test was used. Statistical software used was PASW Statistics for Windows release 18.0.1. Differences were considered significant at P < 0.05.
Results and discussion
We first tested the ability of the 3T3-L1 differentiation protocol (Fig. 1c) to induce differentiation in fetal oPADs and compared it to the differentiation ability in the 3T3-L1 cell line (Fig. 1). After 8 days of differentiation, over 90% of 3T3-L1 cells differentiated in fully mature adipocytes as shown by ORO stain (Fig. 1a and b1). However, the 3T3-L1 protocol was not able to induce terminal adipogenic differentiation in oPADs (Fig. 1a and b2). The ≥ 16-fold difference between 3T3-L1 and fetal oPADs differentiation was similar to reports standardizing adipogenic differentiation with human primary preadipocytes [22, 30].
Because insulin concentration is among the adipogenic media supplements with the largest variation , we tested if increasing the insulin concentration would improve accumulation of lipid droplets during adipogenic differentiation. A 10-fold increase in insulin concentration (10 μg/ml) induced the formation of small and middle-sized lipid droplets in fetal oPADs (data not shown). 3T3-L1 cells do not require adipogenic induction with a PPARγ agonist, however differentiation protocols for large mammalian species [7, 8, 14] preadipocyte and human mesenchymal stem cells into adipocytes often include this modification [31, 32]. To test if supplementation with a PPARγ agonist results in enhanced fetal ovine preadipocyte differentiation, rosiglitazone was selected over other PPARγ agonists, such as troglitazone, because troglitazone is only a partial PPARγ agonist . Rosiglitazone (20 μM) was included into the differentiation medium (DM2a; Fig. 2c) and cells exposed for 2 days. Thereafter, differentiation medium was replaced with DM2b (Fig. 2c) and fetal oPADs cultured for 6 additional days. Addition of a PPARγ agonist for 48 h improved differentiation, increasing medium-sized lipid droplet formation (Fig. 2b1) and suggesting that similar to feline , porcine , and human  preadipocytes, ovine preadipocytes have lower sensitivity to insulin than murine cell lines and PPARγ agonist may be a more stringent requirement for adipogenic differentiation in these species.
Since previous studies have shown that longer exposure (>2 days) to the differentiation cocktail can enhance adipogenesis [35, 36], we tested if a longer exposure to the DM2a increased ovine adipogenic differentiation. Exposure to the DM2a for 8 days resulted in an enhanced adipogenic differentiation of oPADs and formation of medium to large sized lipid droplets (Fig. 2b2). With this differentiation protocol, lipid accumulation is readily visible under light microscope starting at day 3 of culture. The final protocol (8 days of DM2a exposure) was tested in two additional fetal oPADs confirming differentiation results observed in the first fetal oPAD cultured cells. We also tested our optimized adipogenic differentiation system in three different ovine adult primary cultured preadipocytes. Adult subcutaneous preadipocytes were used and a similar differentiation rate to that seen in fetal oPADs was observed (Fig. 2a and b3). Because PPARγ agonists can induce lipid droplet accumulation in non-adipogenic cells, we used ovine fetal skin fibroblasts to test if rosiglitazone could induce lipid droplet accumulation. Fetal fibroblasts originated from the same animals as the oPADs. When subjected to the final protocol (DM2a for 8 days), fetal fibroblasts were not able to accumulate lipid droplets (Fig. 3c).
In conclusion, our protocol enables successful adipogenic differentiation of fetal and adult ovine preadipocytes. It also demonstrates that compared to the 3T3-L1 cell line, fetal ovine preadipocytes require a longer exposure to the differentiation cocktail, and suggests the need for IMBX, dexamethasone, and/or the PPARγ agonist rosiglitazone through the terminal differentiation phase. They also require higher insulin concentration during differentiation to enhance lipid accumulation and similar to human primary preadipocytes [21, 22], PPARγ agonist supplementation is also required for ovine adipogenic differentiation. This work highlights species-specific differences requirements for adipogenic differentiation [7, 14, 31, 34] and the need to develop standardized methods to investigate comparative adipocyte biology.
- ADIPOQ :
American Type Culture Collection
- C/EBP :
- DLK1 :
Delta like non-canonical Notch ligand 1
Dulbecco’s modified Eagle’s medium
Dulbecco’s phosphate-buffered saline
- FABP4 :
Fatty acid binding protein 4
- GAPDH :
Glyceraldehyde 3-phosphate dehydrogenase
- GPDH :
Ovine fetal preadipocytes
Oil Red O
- PPARγ :
Peroxisome proliferator-activated receptor γ
Quantitative real time PCR
- ZFP423 :
Zinc finger protein 423
We thank the support of the Michigan State University Sheep Teaching and Research Farm and the technical help of Mr. Jeremy Gingrich and Dr. Steve Suhr.
This research was supported by Michigan State University (MSU) General Funds, AgBio Research and the United States Department of Agriculture (USDA) National Institute of Food and Agriculture, Hatch project MICL02383, and 1K22ES026208 (NIEHS/NIH).
Availability of data and material
AV-L and YP conceived the study, carried out the experiments, and analyzed the data. AV-L wrote the manuscript draft and AV-L and YP revised and had final approval of the submitted and published versions.
The authors declare that they have no competing interests.
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