Metabolic Differences between Subcutaneous and Visceral Adipocytes Differentiated with an Excess of Saturated and Monounsaturated Fatty Acids

Obesity is a major health problem in highly industrialized countries. High-fat diet (HFD) is one of the most common causes of obesity and obesity-related disorders. There are considerable differences between fat depots and the corresponding risks of metabolic disorders. We investigated the various effects of an excess of fatty acids (palmitic 16:0, stearic 18:0, and oleic acids 18:1n−9) on adipogenesis of subcutaneous- and visceral-derived mesenchymal stem cells (MSCs) and phenotypes of mature adipocytes. MSCs of white adipose tissue were acquired from adipose tissue biopsies obtained from subcutaneous and visceral fat depots from patients undergoing abdominal surgery. The MSCs were extracted and differentiated in vitro with the addition of fatty acids. Oleic acid stimulated adipogenesis, resulting in higher lipid content and larger adipocytes. Furthermore, oleic acid stimulated adipogenesis by increasing the expression of CCAAT enhancer binding protein β (CEBPB) and peroxisome proliferator activated receptor γ (PPARG). All of the examined fatty acids attenuated the insulin-signaling pathway and radically reduced glucose uptake following insulin stimulation. Visceral adipose tissue was shown to be more prone to generate inflammatory stages. The subcutaneous adipose tissue secreted a greater quantity of adipokines. To summarize, oleic acid showed the strongest effect on adipogenesis. Furthermore, all of the examined fatty acids attenuated insulin signaling and secretion of cytokines and adipokines.


Introduction
Obesity is characterized by a very rapid growth rate, mainly in highly industrialized countries [1]. It affects a wide array of health problems, such as metabolic disorders including insulin resistance and type 2 diabetes, cardiovascular diseases, or cancers [2]. Obesity is considered to be the result of the interaction between genetic and environmental factors; the latter mainly involve the result of increased energy intake combined with a low level of physical activity. Excessive food intake leads to adipose tissue expansion, either via enlargement of adipocytes (hypertrophy) or an increasing number of adipocytes (hyperplasia) [3].

Adipogenesis of Human MSC
A total of 24 h before adipogenesis induction, 0.5 mM fatty acids were added (palmitic 16:0, stearic 18:0 and oleic 18:1n−9 acids). For controls, an appropriate amount of BSA fatty acids-free was added.
The progress of adipogenesis was monitored at five time points: day 0 (D0)-the time of adipogenesis induction, day 0.5 (D0.5) 12 h after adipogenesis induction (the MCE stage), day 4 after adipogenesis induction (D4), day 7 (D7), and day 10 (D10)-the stage of fully mature adipocytes. At each selected time point, the cells were harvested and subjected to specific analysis.

Fatty Acids Preparation [25 mM]
The fatty acids were prepared as described previously [12]. Briefly, fatty acids were dissolved in 0.1 M NaOH (Sigma-Aldrich) and incubated for 30 min at 70 • C. Next, 10% of fatty acids-free bovine serum albumin (BSA, Sigma-Aldrich) was added and incubated further for 1 h at 55 • C with constant stirring. Finally, the fatty acids were filtered through a 0.2-µm syringe filter and stored at −20 • C.
Total cellular RNA was extracted using Tri-Reagent (Sigma-Aldrich, St. Louis, MO, USA). Following the cell lysis, chloroform was added and, after centrifugation at 4 • C, RNA was precipitated with isopropanol (Sigma-Aldrich, St. Louis, MO, USA). The RNA pellet was washed with 70% ethanol (StanLab, Lublin, Poland), dried in air, and finally dissolved in DEPC-treated water.

Gene Expression
The cDNA was synthesized with the use of a High-Capacity cDNA Reverse Transcription Kit (ThermoFisher Scientific, Waltham, MA, USA), using 1000 ng of total RNA. Gene expression was measured using the Fast SYBR Green Master Mix (ThermoFisher Scientific, Waltham, MA, USA). Primers were designed manually to flank two adjacent exons of mRNA. The primers sequences are located in Table 1. The primers specificity was investigated using Primer-BLAST (NCBI), further checked based on the denaturation curve. The possible secondary structures of designed primers were analyzed using OligoAnalyzer (IDT, Inc., Coralville, IA, USA). The primers efficiency was analyzed using the standard curve method. Gene expression was calculated using the delta-delta Ct (∆∆Ct) model, normalizing to the reference gene (β-actin gene).

Glucose Uptake
Measurement of glucose uptake was carried out on the mature adipocytes (on day 10 of differentiation) using a Glucose Uptake-Glo Assay (Promega, Madison, WI, USA) according to protocol. Before the experiment, the cells were starved in a serum-free medium overnight. On the day of the experiment, the medium containing glucose was removed, and cells were washed twice with sterile PBS to wash out any remaining glucose. Next, cells were stimulated with 1 µM insulin for Genes 2020, 11, 1092 6 of 16 20 min, followed by incubation with 1 mM of 2-deoxyglucose (2DG) for an additional 20 min. Finally, the reaction mixture was added, and the signal was detected by luminescence readers.

Assessment of the Accumulated Lipids
The amount of accumulated lipids was measured at the end of adipogenesis (D10 of adipogenesis induction) using Oil Red-O (Sigma-Aldrich, St. Louis, MO, USA). The cells were fixed with 4% paraformaldehyde (PFA, Sigma-Aldrich, St. Louis, MO, USA) for 10 min at RT. The PFA was removed, cells were washed with water, and further incubated with 60% isopropanol (Sigma-Aldrich, St. Louis, MO, USA) for an additional 5 min. The staining solution (Oil Red-O Stain, Sigma-Aldrich, St. Louis, MO, USA) was added and incubated at RT for 30 min. Next, the staining solution was discarded, cells were washed several times with water. The accumulated Oil Red-O was extracted with 100% isopropanol. The absorbance was measured at 492 nm.

Assessment of the Size of Adipocytes
The size of adipocytes was determined using Olympus IX81 using CellSens Dimension software.

Statistical Analysis
Statistical analysis was done with the use of Statistica 13.1 (StatSoft, Tulsa, OK, USA). The numerical values between studied groups were compared using one-way and/or multifactor ANOVA/MANOVA and the post-hoc test (the Fisher's Least Significant Difference-LSD test). The differences between SAT and VAT were estimated using a t-test. Statistical significance was set at p < 0.05

Morphological Changes and Lipid Accumulation
We observed that the MSCs differentiated with an excess of oleic acid (18:1n−9) faster than either control cells or two other experimental cells (differentiated with palmitic (16:0) and stearic (18:0) acids). On day 0.5 (D0.5) following the addition of differentiation medium, cells cultured with oleic acid began to change their structure into round cells with lipid droplets located around the cytoplasm (Figure 1). In control cells and cells cultured with saturated fat, the described changes began to appear around D4.
At the end of adipogenesis, total lipid accumulation and the size of adipocytes were analyzed. We observed that oleic acid stimulated total lipid accumulation both in SAT and VAT (p = 0.0000); however, total lipid content was two times higher in cells obtained from SAT than from VAT (p = 0.0000). We also observed statistically increased lipid content in adipocytes collected from SAT treated with stearic acid (p = 0.0002), but not in VAT (p = 0.0610, Figure 2A). Total lipid accumulation corresponded to the size of adipocytes. Adipocytes differentiated with an excess of oleic acid were significantly larger than control cells (p = 0.0000, both SAT and VAT: Figure 2B). Similarly, adipocytes differentiated with an excess of stearic acids were larger than controls (p = 0.0031 for SAT and p = 0.0003 for VAT). The palmitic acid influenced the size of adipocytes collected from VAT (p = 0.0063, Figure 2B). At the end of adipogenesis, total lipid accumulation and the size of adipocytes were analyzed. We observed that oleic acid stimulated total lipid accumulation both in SAT and VAT (p = 0.0000); however, total lipid content was two times higher in cells obtained from SAT than from VAT (p = 0.0000). We also observed statistically increased lipid content in adipocytes collected from SAT treated with stearic acid (p = 0.0002), but not in VAT (p = 0.0610, Figure 2A). Total lipid accumulation corresponded to the size of adipocytes. Adipocytes differentiated with an excess of oleic acid were significantly larger than control cells (p = 0.0000, both SAT and VAT: Figure 2B). Similarly, adipocytes differentiated with an excess of stearic acids were larger than controls (p = 0.0031 for SAT and p = 0.0003 for VAT). The palmitic acid influenced the size of adipocytes collected from VAT (p = 0.0063, Figure 2B).

Transcription Factors Regulating Adipogenesis
To assess the progress of adipogenesis, we investigated the expression of the main adipogenic transcription factors: two early (CEBPB and CEBPD) and two late (CEBPA and PPARG) transcription factors. The expression of CEBPB was higher in cells from SAT differentiated with an abundant

Transcription Factors Regulating Adipogenesis
To assess the progress of adipogenesis, we investigated the expression of the main adipogenic transcription factors: two early (CEBPB and CEBPD) and two late (CEBPA and PPARG) transcription factors. The expression of CEBPB was higher in cells from SAT differentiated with an abundant concentration of oleic acid at D0 and D0.5 (p = 0.0016 and p = 0.0013, respectively) compared to control cells. Similarly, oleic acid stimulated the expression of CEBPB in VAT-derived adipocytes (D4 p = 0.0135: Figure 3).

The Phenotype of Mature Adipocytes
The phenotype of adipocytes differentiated with the excess of fatty acids was investigated at the end of adipogenesis (on mature adipocytes). The metabolic markers of insulin signaling, the secretion rate of cytokines and adipokines, and the lipid metabolism were measured. Finally, the metabolic differences between cells from SAT and VAT were assessed. In the last-named case, comparisons were made between control cells only, differentiated at standard conditions without the excess of fatty acids.

Insulin Signaling Pathway
To assess insulin sensitivity, the expression of main genes belonging to the insulin-signaling pathway was measured.
In adipocytes collected from SAT, no significant changes were noted in the expression of insulin receptor (INSR) and phosphoinositide-3-kinase regulatory subunit 1 (PIK3R1) genes within the investigated cells; however, the expression of solute carrier family 2 member 4 (SLC2A4) was downregulated in adipocytes treated with oleic acid (p = 0.0492) ( Figure 4A). PPARG was overexpressed in VAT-derived MSCs differentiated with the addition of oleic acid through the entire adipogenesis process; however, a significant increase was noted at D0 (p = 0.0281), D7 (p = 0.0194), and D10 (p = 0.0335) ( Figure 3B). Similarly, in SAT-derived cells, the expression of PPARG was considerably greater throughout the adipogenesis process, with significant differences at D0 (p = 0.0432), D4 (p = 0.0325), and D7 (p = 0.0435) of differentiation ( Figure 3A). We also observed a considerable increase in the expression of PPARG in preadipocytes treated with stearic acid; however, the increase was significant only at D0 of differentiation (p = 0.0354).
We also investigated potential differences in the expression rate of transcription factors during the adipogenesis of the MSCs collected from visceral and subcutaneous fat. The early transcription factors (CEBPB and CEBPD) were expressed at a higher rate in cells collected from SAT. Among the late transcription factors, PPARG was expressed at a higher level in VAT-derived adipocytes throughout the Genes 2020, 11, 1092 9 of 16 process of adipogenesis (D0, p = 0.0013; D0.5, p = 0.0571; D4, p = 0.0437; D7, p = 0.0001; D10, p = 0.0154, Figure 3C).

The Phenotype of Mature Adipocytes
The phenotype of adipocytes differentiated with the excess of fatty acids was investigated at the end of adipogenesis (on mature adipocytes). The metabolic markers of insulin signaling, the secretion rate of cytokines and adipokines, and the lipid metabolism were measured. Finally, the metabolic differences between cells from SAT and VAT were assessed. In the last-named case, comparisons were made between control cells only, differentiated at standard conditions without the excess of fatty acids.

Insulin Signaling Pathway
To assess insulin sensitivity, the expression of main genes belonging to the insulin-signaling pathway was measured.
In adipocytes collected from SAT, no significant changes were noted in the expression of insulin receptor (INSR) and phosphoinositide-3-kinase regulatory subunit 1 (PIK3R1) genes within the investigated cells; however, the expression of solute carrier family 2 member 4 (SLC2A4) was downregulated in adipocytes treated with oleic acid (p = 0.0492) ( Figure 4A).
Genes 2020, 11, x FOR PEER REVIEW 10 of 16 The expression of insulin signaling genes was similar between SAT and VAT adipocytes. However, we observed different effects of the increased concentration of fatty acids on the expression of SLC2A4 in VAT-derived cells, which was increased by investigated fatty acids (p = 0.0061, p = 0.0020, and p < 0.0000 for palmitic (16:0), stearic (18:0), and oleic acids (18:1n−9), respectively: Figure  4B).
Next, we measured insulin-stimulated glucose uptake in both types of mature adipocytes. In both types of control cells, we showed an increase in glucose uptake rate following insulin stimulation (p < 0.0000 for both SAT and VAT); on the other hand, we detected no increase in glucose uptake following insulin stimulation in cells treated with fatty acids throughout the adipogenesis ( Figure 4C). The rate of glucose uptake in the basal stage was the same as that following insulin stimulation. A similar effect was observed regardless of the type of fatty acids.

Cytokine and Adipokine Expression and Secretion
Adipose tissue, apart from its basic role of energy storage, plays an enormous role as an endocrine organ. Accordingly, we were interested in how the excess of fatty acids influence adipokines secretion and inflammatory-stage induction.
We observed no effect of free fatty acids on inflammatory induction. Rates of IL-6 and IL-10 expression and IL-6 secretion were unchanged following the addition of fatty acids, which may suggest that free fatty acids do not induce inflammation during adipogenesis, although they may do so later in the life of adipocytes. In VAT-derived adipocytes, no significant changes were noted in the expression of INSR and PIK3R1 genes within the investigated cells. On the other hand, SLC2A4 was considerably overexpressed in all cells treated with fatty acids (p = 0.0340, p = 0.0352, and p = 0.0360 for palmitic (16:0), stearic (18:0), and oleic acids (18:1n−9), respectively: Figure 4A).
The expression of insulin signaling genes was similar between SAT and VAT adipocytes. However, we observed different effects of the increased concentration of fatty acids on the expression of SLC2A4 in VAT-derived cells, which was increased by investigated fatty acids (p = 0.0061, p = 0.0020, and p < 0.0000 for palmitic (16:0), stearic (18:0), and oleic acids (18:1n−9), respectively: Figure 4B).
Next, we measured insulin-stimulated glucose uptake in both types of mature adipocytes. In both types of control cells, we showed an increase in glucose uptake rate following insulin stimulation (p < 0.0000 for both SAT and VAT); on the other hand, we detected no increase in glucose uptake following insulin stimulation in cells treated with fatty acids throughout the adipogenesis ( Figure 4C). The rate of glucose uptake in the basal stage was the same as that following insulin stimulation. A similar effect was observed regardless of the type of fatty acids.

Cytokine and Adipokine Expression and Secretion
Adipose tissue, apart from its basic role of energy storage, plays an enormous role as an endocrine organ. Accordingly, we were interested in how the excess of fatty acids influence adipokines secretion and inflammatory-stage induction.
We observed no effect of free fatty acids on inflammatory induction. Rates of IL-6 and IL-10 expression and IL-6 secretion were unchanged following the addition of fatty acids, which may suggest that free fatty acids do not induce inflammation during adipogenesis, although they may do so later in the life of adipocytes.
However, adipokines showed divergences in both expression and secretion rates under the influence of fatty acids. Both the expression and secretion of leptin were stimulated by fatty acids in adipocytes collected from SAT. The expression of the Leptin (LEP) gene was three times higher in adipocytes differentiated with palmitic acid, 16:0 (p = 0.0202), and oleic acid, 18:1n−9 (p = 0.0247) and twice as high in adipocytes cultured with an excess of stearic acid, 18:0 (p = 0.0505) compared to controls. Similarly, leptin secretion was induced, particularly by palmitic (p = 0.0271) and stearic (p = 0.0298) acids ( Figure 5A). Similarly, fatty acids increased the expression of the LEP gene in adipocytes collected from VAT. In particular, palmitic and stearic acids increased the expression of the LEP gene approximately six-fold compared to controls (p = 0.0147 and p = 0.0202, respectively). Oleate increased the expression of the LEP gene approximately three-fold compared to controls and was near-significant (p = 0.0506). The secretion of leptin was increased in adipocytes differentiated with the addition of palmitate (p = 0.0298) and stearate (p = 0.0371).
The adiponectin level decreased in cells differentiated with an excess of fatty acids collected from the SAT, in terms of both expression and secretion levels. The drop in expression was similar for all investigated fatty acids; however, due to a very high standard deviation, it did not reach the level of significance (p = 0.1012, p = 0.1287, and p = 0.1641 for palmitic (16:0), stearic (18:0), and oleic acids (18:1n−9), respectively). The secretion of adiponectin was considerably reduced in cells differentiated with an excess of all of the investigated fatty acids (p = 0.0291, p = 0.0188, and p = 0.0189 for palmitic (16:0), stearic (18:0), and oleic acids (18:1n−9), respectively). VAT-derived cells showed no changes in either the expression of the ADIPOQ gene or the amount of secreted adiponectin in terms of fatty acids ( Figure 5A).
Comparing two fat depots, we observed a significant increase in IL-6 gene expression (p < 0.0000) and IL-6 secretion (p < 0.0000) in VAT-derived adipocytes ( Figure 5B) compared to cells obtained from SAT. On the other hand, LEP gene expression and Leptin secretion were considerably increased in SAT-derived adipocytes (p = 0.0394 and 0.0152, respectively: Figure 5B). either the expression of the ADIPOQ gene or the amount of secreted adiponectin in terms of fatty acids ( Figure 5A).
Comparing two fat depots, we observed a significant increase in IL-6 gene expression (p < 0.0000) and IL-6 secretion (p < 0.0000) in VAT-derived adipocytes ( Figure 5B) compared to cells obtained from SAT. On the other hand, LEP gene expression and Leptin secretion were considerably increased in SAT-derived adipocytes (p = 0.0394 and 0.0152, respectively: Figure 5B).

Lipid Metabolism
The influence of fatty acids on lipid metabolism was measured by the expression of lipid metabolism genes, that is one lipolysis (LPL-lipoprotein lipase) and three lipogenesis (ACACA-acetyl-CoA carboxylase α, FASN-fatty acid synthase, and SCD1-stearoyl-CoA desaturase 1). In cells collected from SAT, no changes in expression in experimental cells were detected compared to controls, except SCD1 gene overexpression in cells treated with stearic acid (p = 0.0247, Figure 6A). However, in cells collected from VAT, numerous genes were overexpressed, including LPL, FASN, and SCD1 in experimental cells compared to controls ( Figure 6B). The LPL gene was overexpressed about 2.5 times more in cells cultured with palmitic (p = 0.0341) and stearic (p = 0.0314) acids compared with controls. Oleic acid stimulated the expression of LPL approximately two-fold more compared to controls (p = 0.0545). The expression of FASN was significantly overexpressed in cells cultured with stearic (p = 0.0086) and oleic (p = 0.0026) acids, whereas the SCD1 gene was overexpressed in cells differentiated with the addition of palmitic (p = 0.0124) and stearic (p = 0.0008) acids.
Furthermore, we analyzed potential differences in lipid metabolism between cells collected from the two different fat depots. In particular, VAT adipocytes were characterized by significantly higher expression of the lipolytic gene (LPL, p = 0.0119), whereas lipogenesis genes were highly expressed in cells obtained from SAT; however, the difference was only nearly statistically significant (p = 0.0965, p = 0.0734, p = 0.0858 for ACACA, FASN, and SCD-1, respectively, Figure 6C). Furthermore, an additive effect of the examined fatty acids in FASN expression in cells collected from VAT compared to SAT was shown (16:0, p = 0.0459; 18:0, p = 0.0004; 18:1n−9, p < 0.0000, Figure 6D). the two different fat depots. In particular, VAT adipocytes were characterized by significantly higher expression of the lipolytic gene (LPL, p = 0.0119), whereas lipogenesis genes were highly expressed in cells obtained from SAT; however, the difference was only nearly statistically significant (p = 0.0965, p = 0.0734, p = 0.0858 for ACACA, FASN, and SCD-1, respectively, Figure 6C). Furthermore, an additive effect of the examined fatty acids in FASN expression in cells collected from VAT compared to SAT was shown (16:0, p = 0.0459; 18:0, p = 0.0004; 18:1n−9, p < 0.0000, Figure 6D).

Discussion
In the present paper, we displayed the effect of fatty acids excess on adipocytes differentiation and the phenotype of mature adipocytes. All of the investigated fatty acids were shown to influence the phenotype of mature adipocytes via various effects, mainly through impairment of insulin signaling and dysregulation of adipokine secretion and expression of lipid metabolic genes.
We showed that all of the examined fatty acids, if supplied in excess amounts, may lead to obesity and may increase the risk of obesity-related disorders such as insulin resistance. First of all, we observed that an excess of oleic acid enhanced adipogenesis, as estimated based on the expression of transcription factors, total lipid accumulation, and changes in morphological structures of the cell during adipogenesis. We particularly observed changes regarding total lipid accumulation in mature adipocytes that was five times greater than in controls. Our results confirmed the adipogenic effect of oleic acid described previously by us and by others using the 3T3-L1 cell line and hen or bovine adipocytes [10,12,21,22]. Previously, it had been suggested that oleic acid aggravates adipogenesis via stimulation of PPARG [10,12], which is consistent with our results. We showed that oleic acid stimulated expression of almost all transcription factors. Indeed, oleic acid and other polyunsaturated fatty acids: Docosahexaenoic acid (DHA) and Eicosapentaenoic acid (EPA) were shown to act as natural ligands for PPARG, stimulating the expression of PPARG, CEBPA, and adiponectin [15,23]. On the other hand, inhibition of PPARG attenuates lipid formation and adipogenesis [24]. These results may explain the beneficial effect of oleic acid on overall glucose and lipid metabolism through increasing the plasticity of adipose tissues and promoting lipid accumulation and storage, thus reducing lipid accumulation in other tissue or organs, as well as overall lipotoxicity [25]. In addition to oleic acid, stearic acid was also shown to increase the expression of transcription factors as well as total lipid accumulation and the size of mature adipocytes. The effect may be indirect, as we showed increased expression of the SCD-1 gene. Eventually, this could lead to the increased content of endogenous oleic acid; however, no evaluation of the lipid fraction was performed. The above finding can be supported by previous results, where an increased content of oleic acid was observed in 3T3-L1 cells with stable overexpression of the SCD-1 gene [26]. On the other hand, it needs to be mentioned that both oleic and stearic acids impaired insulin-stimulated glucose uptake; thus, the beneficial effect of oleic acid should be more fully evaluated in terms of the insulin-signaling pathway in adipose tissue.
Considerable differences were observed in the process of adipogenesis between preadipocytes collected from the two main fat depots. The SAT-derived mature adipocytes accumulated a greater amount of total lipids than those collected from VAT. Similarly, the expression of early transcription factors was greater in adipocytes from SAT than from VAT, which is consistent with other reports [27]. Furthermore, as has been shown previously, adipocytes from subcutaneous fat are larger and accumulate more lipids than those from visceral fat in obese or overweight subjects [28], which is also consistent with our results. PPARG expression was much greater in adipocytes collected from VAT throughout the process of differentiation, especially in cells treated with oleate (18:1n−9) during adipogenesis.
In addition to their effect on the adipogenesis process, fatty acids impacted the metabolism of mature adipocytes. A constant excess of fatty acids during adipogenesis disrupted correct carbohydrate metabolism. Based on a gene expression study, insulin-signaling genes were not aborted; however, utilization of insulin-stimulated glucose by adipocytes differentiated, with an excess of all of the examined fatty acids discontinued. A similar effect was seen for all of the examined fatty acids, irrespective of the origin of the adipocytes. Treatment of cells with oleic acids reduced the rate of SLC2A4 expression in SAT-derived adipocytes. On the other hand, we observed an increase in SLC2A4 expression in VAT-derived adipocytes, which may be a compensatory effect of newly developed insulin resistance. We, and other researchers, have recently shown considerable reduction in the expression of SLC2A4 in 3T3-L1 adipocytes [12] and bovine adipocytes [22] treated with oleic acid. However, further research needs to be conducted to evaluate the reason for the differential profile of SLC2A4 expression between two fat depots influenced by oleic acid.
In SAT-derived adipocytes, the examined fatty acids displayed no divergences in the expression of enzymes regulating lipid metabolism, apart from overexpression of SCD-1 in cells cultured with the addition of stearic acid, which may be due to increased access to the substrate for SCD-1. We observed a similar effect regarding 3T3-L1 adipocytes [12]. On the other hand, we showed dysregulation in the expression of lipid metabolism genes in VAT-derived adipocytes, including LPL, FASN, and SCD1, proving that VAT is metabolically more active than SAT. It had been concluded previously, based on results obtained in vivo and in vitro, that visceral fat was characterized by a higher level of lipolytic activity compared to subcutaneous fat [4]. This also explains the increased degree of lipid accumulation in SAT-derived adipocytes during adipogenesis compared to VAT-derived adipocytes, which could be explained in turn by the greater lipogenesis activity of SAT adipocytes and much lower levels of lipolysis activities in those cells. Contrary to SAT, VAT-derived adipocytes were characterized by a significantly higher level of lipolytic activity, which is consistent with other reports [4,29].
It is obvious that obesity is related to low-grade inflammation; accordingly, we were interested as to whether fatty acids were capable of influencing cytokine secretion. Generally, VAT is related to the development of inflammatory states mainly through the production of pro-inflammatory cytokines such as IL-6 [26]. Indeed, we showed that VAT-derived adipocytes expressed and secreted IL-6 at a higher rate than SAT-derived adipocytes; however, based on our study, fatty acids did not influence cytokine secretion. Our results contradict others that showed cytokine overexpression (IL-6, TNFα (Tumor Necrisis Factor α)) following palmitic acid treatment [30]. The increase in cytokine expression and secretion is likely visible at a later stage in the life of adipocytes. SAT and VAT also differ in terms of profiles of adipokine secretion. It had been previously reported that adipokines are expressed and secreted at a high level by SAT [31,32]. We, like others, showed significantly greater expression and secretion of leptin and adiponectin in SAT-derived adipocytes [33,34]. Furthermore, the influence of fatty acids was considerable, mainly on SAT. Generally, we reported that, in SAT-derived adipocytes, secretion of leptin was stimulated, and adiponectin secretion was inhibited by fatty acids. These results confirmed that insulin resistance developed in experimental cells, as adiponectin, irrespective of its other roles, was shown to increase insulin-stimulated glucose uptake [35]. We detected no changes in the expression or secretion rate of adiponectin in VAT-derived adipocytes, suggesting a minor role for VAT depots in the secretion of adiponectin. Indeed, the VAT-derived cells expressed and secreted less adiponectin than the adipocytes obtained from SAT.
In all cases, the above differences prove that SAT and VAT are metabolically different tissues; furthermore, dietary factors such as fatty acids have a different effect on both adipogenesis and the phenotype of mature adipocytes. Oleic acid was shown to stimulate adipogenesis of both SAT and VAT-derived MSCs and to increase total lipid accumulation. All of the examined fatty acids affected the phenotype and metabolism of mature adipocytes, albeit with a different effect on the origin of adipocytes.