Mouse 3T3-L1 cells were obtained from ATCC. Pre-adipocytes cells were cultured in DMEM containing 25 mmol/l glucose and supplemented with glutamax and without pyruvate, 10% (v/v) FBS, 1% penicillin/streptomycin (100 U/ml and 100 μg/ml), and 1% biotin/pantothenate. The cells were inoculated into either 6 multidishes at a density of 2 × 10⁴ cells/well for gene expression assays or 96 wells micro plate at a density of 5 × 10³ to evaluate lipid accumulation. Cells are maintained at 37°C under a humidified 5% CO₂ atmosphere. The numbers of cells were evaluated with a Neubauer hemocytometer, using 0.05% trypsin-EDTA dissociated cells, and cell viability was monitored by means of the trypan blue-dye exclusion test.

OP9 cells were cultured in MEM-α containing 20% FBS, 1% L-glutamine (2 mM), 1% penicillin and Streptomycin at 37°C in humid field containing 5% of CO₂. Cells are let to spontaneously differentiate during 2 weeks in DMEM + dexamethasone at 10⁻⁷ mol/L with or without AA at 100 μM.

Cells were cultured for 9 days in DMEM containing 25 mmol/l glucose, 10% (v/v) FBS, antibiotics and supplemented with dexamethasone (1 μmol/L), and insulin (1 μg/ml). IBMX was added or not to the medium. AA was added or not to the medium (dissolved in 1× PBS and then filtered). Medium was changed every 2 days.

Cells were rinsed by PBS and fixed with 10% (v/v) buffered formalin in Dulbecco's PBS for 10 min. Then formalin discarded and 2 ml of fresh formalin was added and incubate for 24 h. Following fixation, cells were washed by ddH₂O twice. The cells were let to dry at RT. Following drying, Oil Red-O solution (60% in distilled water) was added and cells incubated for 10 min. Cells were rinsed twice with ddH₂O. Pictures were acquired under the microscope.

Cells were seeded at a density of 5 × 10³ cells/well (96-wells plate), and then cultured in DMEM containing 10% FBS and 1% antibiotics and treated with dexamethasone (1 μmol/l), insulin (1 μg/ml), and various concentration of AA for 9 days. Thereafter, cells were rinsed with PBS. Then AdipoRed™ (Lonza Walkersville, USA) a dye that binds lipids, and, in different wells, SYBR^® Green I (Life Technology), that binds DNA (as an evaluation of cells number), were added. Plates were then read on a microplate reader (VICTOR™X4 from PerkinElmer^®) and the fluorescence of AdipoRed™ and SYBR^® Green I were measured with excitation 485 nm/emission 572 nm and excitation 494 nm/emission 521 nm respectively. Adipored fluorescence was normalized to DNA content.

Cell cultures were fixed in cold methanol for 15–20 min. After rinsing, the fixed cells were incubated with (5%) Silver Nitrate Solution (abcam product # ab150687) and crosslinked under UV light for 20–30 min, then rinsed twice with distilled water. The dishes were incubated with (5%) Sodium Thiosulfate Solution for 2–3 min, rinsed twice with distilled water; then finally incubated with Nuclear Fast Red Solution for 5 min, and rinsed several times with distilled water to remove excess stain.

Intracellular cAMP level was measured using cAMP-Glo™ Assay (Promega Corp.). Briefly, 100 μl of 3T3L1 cells solution (5 × 10⁴ cells/ml) was deposited in a sterile 96-wells microplate (culture treated) and incubated at 37°C in 5% CO₂/95% air. After cell adhesion (24 h), culture medium was replaced by 20 μl of our compounds solutions, prepared in “induction buffer.” Each solution dilution was tested in triplicate, and wells containing 3T3L1 cells in 20 μl of induction buffer alone were used as controls like basal level of intracellular cAMP. Then, assay was performed according to the manufacturer's instructions. At the end, plates were read using a microplate reader (Victor™ X4, PerkinElmer^®). According to kit instructions, “Induction Buffer” was composed with PBS containing 500 μM of 3-isobutyl-1-methylxanthine (IBMX) and 100 μM of 4-(3-butoxy-4-methoxybenzyl)imidazolidin-2-one (Ro20-1724). The IBMX and Ro20-1724 are inhibitors of phosphodiesterases and used to prevent cAMP hydrolysis during the assay. In summary, this assay evaluates cAMP production, as a consequence of adenylate cyclase activity.

Pictures were acquired for stained cells in culture using a Zeiss microscope. Presence of mineralized nodules and deposited calcium were seen as dark brown to black spots using Von Kossa staining methods, while lipid accumulation droplets are colored in red by Oil Red O or appear as white droplet spots. The evaluation of lipid droplets and mineralization were quantified by means of ImageJ software version 1.47. Area presenting brown coloration, red coloration or white droplets is evaluated in fields of 7 mm². Ten fields have been evaluated.

Total RNA was extracted from cells using TRIZOL reagent (Invitrogen™) according to the manufacturer's protocol. Purified total RNA from adipocytes was reverse transcribed using Superscript II reverse transcriptase (Invitrogen™) to produce cDNA. Thereafter, quantitative real time-polymerase chain reaction (PCR) was performed using Light Cycler 480 Real-Time PCR System (Roche), using UPL probes. Specific primers were used and described in Table 1. Expression levels of target genes were normalized using β-actin (ACTB) as internal standard. The results were treated using the comparative C_T method, where the amount of the target, normalized to the endogenous reference and relative to a calibrator, is given by 2^−ΔΔCT.

We used Prism v5 software to perform statistical analysis of data. Data corresponding to an experiment were analyzed using ranking tests (Wilcoxon and Kruskal–Wallis). Results presenting a p-value < 0.05 were further analyzed using the Mann–Whitney two-tailed statistical significance test, with a confidence interval of 95%. We considered a p-value lower than 0.05 as significant. IC₅₀ has been evaluated using the dedicated section of Prism software.

In a first experiment, we cultured 3T3 L1 cells for 9 days in a medium that promotes adipocyte differentiation with IBMX, with and without AA. We observed that cells treated with 500 μmol/L AA present with less fully differentiated adipocytes, as depicted in Figures 1A,B. The experiment was repeated using increasing concentrations of AA to establish the dose-dependence of this effect. At low concentration of AA (50 μmol/L), we observed an increase in lipid accumulation (Figure 1C). However, when concentration of AA increased, we observed inhibition of lipid accumulation. This result could be explained by the competition between the effects of IBMX and AA. Since IBMX increases cAMP levels (by inhibiting phosphodiesterases), while AA inhibits cAMP production (Kaya et al., 2007, 2008a), it becomes obvious that an increase in AA concentrations counteracts the effect of IBMX. In order to determine if there is a critical period for AA action, cells were treated for 3 days with IBMX and AA at a range of concentrations (Figure 1D) and left to differentiate until day 9. We observed that 3 days of treatment were sufficient to inhibit lipids accumulation. In a separate experiment, cells were treated for 3 days with IBMX without any AA, followed by incubation without IBMX, but with increasing doses of AA. We observed (Figure 1E) that lipid accumulation in cells that underwent this treatment was not inhibited by AA, except at a concentration of 500 μmol/L. A schematic representation of these experiments is presented in Supplementary Figure 1. Our findings suggest the existence of a critical period for AA action, extending from day 0–3.

In order to avoid competition between the IBMX-induced increase in cAMP levels, and AA-mediated decrease, we cultured 3T3 L1 pre-adipocytes in a medium without IBMX for 9 days with or without AA. In these conditions, the percentage of cells that differentiated into mature adipocytes was found to be lower, but we did observe that cells could differentiate into adipocytes in cultures without AA (Figure 2A). The next question was to determine whether inhibition of lipids accumulation by AA is dose-dependent. As presented in Figures 2A–F, we observed that lipid accumulation is inhibited in a dose-dependent manner when cells are incubated with increasing doses of AA. In a further experiment, cells were cultured in 96-well microplates in a medium without IBMX and with increasing doses of AA. After 9 days, lipid accumulation was evaluated using an assay developed in our laboratory (see Materials and Methods), with the results presented in Figure 2G. These data demonstrated that inhibition of 3T3 L1 cells differentiation by AA is dose-dependent, with IC₅₀ about 150 μmol/L.

3T3-L1 cells were cultured for 9 days in a medium without IBMX with a range of concentrations of AA. RNA was extracted and expression of specific markers of adipocyte differentiation evaluated by qPCR. Results are presented in Figure 3. We observed that expression of genes associated with adipocyte differentiation (CEBP-α and -β, PPAR-γ, and SREB) was lower in the cells treated with AA compared to that in the cells incubated without AA, and this inhibition was dose-dependent. Expression of adiponectin, a marker of late stages of differentiation, was also inhibited by AA. Additionally, expression of TIMP3, which is normally reduced during adipocyte differentiation (Bernot et al., 2010), was elevated in cells treated with AA (Figure 3F). Expression of these differentiation markers is in agreement with the proposed inhibitory effect of AA on adipocyte differentiation.

We demonstrated above that AA inhibits differentiation of mesenchymal cells into mature adipocytes. To gain information regarding the action of AA on mature adipocytes, we cultured 3T3-L1 cells in a medium allowing their differentiation into mature adipocytes, in the presence of IBMX. When maturation was observed following 7 days of incubation, the cells were incubated in a medium without IBMX and the culture continued for 7 days with or without AA (0.1 of 0.5 mmol/L). At the end of the final incubation, lipid accumulation was recorded. As presented in Figures 4A–C, we observed that lipid accumulation continued in the absence of AA, but was decreased when AA is added to the medium.

These results suggest that AA is able to act on mature adipocytes, reversing lipid accumulation. However, it will be interesting to confirm these results using stromal vascular fraction (SVF) of adipose tissue and testing lipid accumulation and gene expression.

AA has been described primarily as an antioxidant, but our recent work (Kaya et al., 2007, 2008a,b) identified a new function: a global regulator of cAMP levels through competitive inhibition of adenylate cyclase. To investigate whether the observed effect of AA on cell differentiation was due to its direct action on the cAMP pool or an outcome of its antioxidant properties, we used K873, an analog of AA recently synthesized in our laboratory. This molecule did not present any antioxidant properties but, like AA, it was shown to reduce the cAMP levels, although at lower concentrations (Bordignon et al., 2013). Treatment of 3T3-L1 with K873 during adipocyte differentiation inhibited the expression of adipocyte markers (C/EBPβ, PPAR-γ, and adiponectin), as observed in Figure 5. This result indicates that inhibition of adipocyte differentiation by treatment of pre-adipocytes with K873 is likely to result from its effect on cAMP levels.

While we have previously described a reduction of cAMP levels following treatment with AA in different cell types, this effect has never been evaluated in mesenchymal pre-adipocytes. We tested 3T3L1 incubated without AA or with increasing concentrations of AA, and evaluated the intracellular cAMP concentration. Results are presented in Figure 6A. We observed that AA decreased intracellular cAMP concentration in a dose-dependent manner. Moreover, we incubated 3T3L1 with dibutyril-cAMP (db-cAMP) alone or in the presence of IBMX without adding AA. We observed that addition of these compounds increased lipid accumulation, as was previously shown. Additionally, we treated 3T3L1 cells with db-cAMP and added AA to the medium. Adding AA inhibited lipid accumulation stimulated by db-cAMP (Figure 6B).

Taken together, our data confirm that increasing intracellular cAMP concentration promotes adipogenesis, while lowering intracellular cAMP levels through treatment with AA inhibits adipogenesis.

In order to rule out the possibility that the observed properties of AA are specifically linked to the 3T3 L1 cell line, we analyzed the effect of AA on differentiation of OP9 cells. This cell line has adipogenic capacity, but could also differentiate into osteogenic cells, depending on the culture conditions (Gao et al., 2010). In addition to evaluating the impact of AA on adipogenic differentiation, this analysis provided us with data regarding the impact of AA on osteogenic differentiation, another mesodermic lineage. We treated OP9 cells with a medium promoting adipocyte differentiation, with or without AA. After 21 days of culture, cells were stained either with Oil Red O to label lipids or with Von Kossa staining to highlight osteogenic differentiation. As shown in Figure 7, we observed that OP9 cells without AA treatment differentiate into adipocytes. Conversely, cells treated with AA poorly differentiated into adipocytes but exhibited clear signs of osteogenic differentiation. These data suggests that AA drives adult mesodermic mesenchymal cells to differentiate following the osteogenic pathway and inhibits adipogenic differentiation.

Little is known about a putative receptor of AA. However, the presence of the transport/receptor of AA, SVCT2, was shown to be required for AA signaling in various conditions (Sotiriou et al., 2002). SVCT2 is a transmembrane protein present in a number of cell types that exhibits high specificity for AA and could act as its receptor. Therefore, we evaluated the expression of the gene coding for this protein during adipocyte differentiation. We observed that the expression of this gene decreased during differentiation (Figure 8A), with almost no expression of this gene detected in mature adipocytes. Additionally, we observed that treatment with AA increased SVCT2 expression in a dose-dependent manner (Figure 8B). Several articles (Reidling et al., 2008; Wu et al., 2008; Bordignon et al., 2013; Hong et al., 2013) report that a decrease in gene expression is correlated with a decrease in concentration of the corresponding protein SVCT2. In addition, these articles report that variations in expression of the gene coding for SVCT2 have an impact on various biological processes.

In conclusion, mature adipocytes, under normal conditions, could probably not receive signals from AA present outside cells. Pre-adipocytes treated with AA, however, continue to receive the AA signal, as evidenced by the removal of repression of SVCT2 expression.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.