Molecular and Functional Characterization of Human SW 872 Adipocytes as a Model System for Testing Nutraceutical Products ^†

Abstract

Background: The availability of characterized human adipocyte cell models for in vitro studies is currently limited. They may be useful to better understand the role of dysfunctional adipocytes in the pathophysiology of cardio-metabolic diseases and to evaluate the metabolic effects of nutraceutical compounds. In this study, human liposarcoma SW 872 cells, both non-differentiated and differentiated for 7 days with 100 mM oleic acid, have been used as the model system to (1) characterize these cells, concerning metabolic, pro-inflammatory, and morphologic features, and (2) begin to evaluate the “healthy” effects of some plant-derived nutraceutical compounds. Methods: In SW 872 cells, we evaluated the accumulation of triglycerides, glucose uptake, pro-inflammatory cytokine release, and the modulation of Akt protein phosphorylation (pAkt), highlighting the differences between differentiated and non-differentiated cells. Results: Oleic-acid-differentiated SW 872 cells have a higher triglyceride content (p < 0.001) than non-differentiated cells, as well as a lower glucose uptake (p < 0.001), but a higher insulin response (p < 0.05) and a specific activation of the Akt pathway. This cell model has thus been chosen for the preliminary evaluation of the effects of some phytochemical complexes on the modulation of these molecular parameters, observing, in some instances, promising effects on the reduction in triglyceride content and pro-inflammatory cytokine release, as well as on increased glucose uptake. Conclusions: Our study suggests that the SW 872 cell model could be useful for studies regarding human adipocyte function and dysfunction, as well as studies on the effects of bioactive compounds on dysfunctional adipose tissue, which will be addressed in future research.

1. Introduction

Dysfunctional adipose tissue can lead to a large spectrum of diseases, such as obesity, metabolic syndrome, and atherosclerosis, and can influence the molecular pathophysiology of cardio-metabolic diseases [1,2]. However, many pathogenetic mechanisms concerning metabolism, and, in particular, adipose tissue, are still unknown; for this reason, it is important to implement cellular models that can best represent human adipocytes and their biological and biochemical mechanisms. These models provide a more nuanced understanding of the development and causes of diseases related to adipose tissue dysfunction, such as obesity and diabetes, used to improve the diagnostic strategies for such diseases. Moreover, the availability of standardized and validated human adipocyte cell models can also facilitate many investigations aimed at testing novel compounds, nutrients, and plant-derived extracts with potential nutraceutical activity [3,4]. More specifically, in vitro models allow us to study the differentiation of adipocytes, and to understand the molecular and cellular events that lead to mature adipocytes, from preadipocytes, which are often more similar, to non-differentiated fibroblasts. Among the cell lines commonly proposed as adipocyte models, there is the 3T3-L1 preadipocyte murine cell line, which has been widely used in several studies, but shows the disadvantage of a low replicative capacity and consequently limited potential for in vitro differentiation [5]. Among the available adipocyte models of human origin, the human liposarcoma cell line, SW 872, has recently been developed. It represents an interesting human adipocyte model, which has been differentiated to more mature adipocytes through different protocols.

It is important to recall that the presence of dysfunctional adipose tissue, and therefore adipocytes, represents the pathophysiological driver of metabolic syndrome and associated complications. In this regard, dysfunctional adipocytes may show high oxidative stress, chronic low-grade inflammation, high pro-inflammatory cytokine release, insulin resistance, and triglyceride accumulation. Nonetheless, there is no pharmacological treatment that acts directly on the dysfunctional adipose tissue, and nutritional and nutraceutical approaches are still to be fully developed. In this paper, we have addressed some aspects related to the biomolecular characterization of SW 872 cells, concerning their metabolic and morphologic features, and started to validate this cell-based model as a potential tool to evaluate the effects of bioactive compounds with potential nutraceutical properties in more detail.

2. SW 872 Adipocyte Differentiation

The SW 872 cell line has an immature adipocyte phenotype, constitutively expressing certain genes involved in the metabolism of fatty acids, such as lipoprotein lipase (LPL), the cholesterol ester transfer protein (CETP), CD36, peroxisome proliferator-activated receptors-α (PPAR-α), peroxisome proliferator-activated receptors-γ (PPAR-γ), and LDL receptor-related protein 1 (LRP1) [6]. The differentiation of these cells to mature adipocytes can be achieved with oleic acid (OA), a PPAR-γ agonist, which in previous studies had shown to increase triglycerides within SW 872 cells [6,7,8]. OA stimulates the PPAR-γ transcription factor, which is crucial for the regulation of specific adipocyte genes and has a fundamental role in adipocyte differentiation. There are some studies that mention additional methods of differentiation of these cells to mature adipocyte-like cells, e.g., by adding dexamethasone (DEX), insulin, and 3-isobutyl-1-methylxanthine (IBMX) [9]. Furthermore, it has been confirmed that these cells are capable of triglyceride accumulation, such as mature adipocytes, simply by adding 100 μM of OA in the culture medium [4]. In this case, SW 872 cells are grown in DMEM-F12 medium containing HEPES buffer, supplemented with 10% FBS, 1% penicillin, and streptomycin (100 μg/mL). The cells are kept in culture in Petri dishes at 37 °C in an atmosphere containing 5% CO₂. When a confluence of 80–100% is reached, a 100 μM solution of OA is added to initiate cell differentiation [7,10]. Additionally, in order to accelerate the induction of the differentiation process in only 3 days, a higher concentration of OA is also used (0.5 mmol/L [11] or 0.6 mol/L [12]). Nonetheless, differentiated cells do not express the uncoupling protein 1 (UCP-1) and there is no change in the number of mitochondria present in the cell [10]. In this study, fatty acid accumulation was evaluated by oil red O (ORO) lipid staining, in order to observe the impact of differentiation on triglyceride accumulation (Figure 1A). The triglyceride content in undifferentiated cells slightly increased by 49.63% after 24 h, while the triglyceride content increased by 63.96% after 48 h. In contrast, cells differentiated with OA showed an almost double increase only after 24 h, compared to untreated cells (Figure 1B). Despite the considerable variation in the accumulation of triglycerides between differentiated and undifferentiated cells, no clear morphological cellular changes were induced by the treatment with OA. Resveratrol (10 µM), used as a positive control on differentiated cells, led to a 30.4% reduction in triglyceride cell content, while metformin (1 µM) and epigallocatechin (40 µM) showed no changes in the SW 872 cells lipid content (data not shown). Among all differentiation options, the OA treatment seems the best and simplest method to acquire mature adipocyte-like cells.

3. Glucometabolic Studies in SW 872 Cells

It is well known that dysfunctional adipocytes display a certain degree of insulin-resistance, with reduced glucose uptake, due to different molecular changes. Using SW 872 cells, glucose uptake was evaluated by observing the ability of these cells to internalize glucose (2-deoxy-2-[(7-nitro-2,1,3-benzoxadiazol-4-yl)amino]-D-glucose; 2-NBDG) after treatment with OA [3]. Basal glucose uptake in SW 872 cells was reduced by 54.81% after treatment with OA, compared to non-differentiated cells (Figure 2A). The modulation of glucose uptake following insulin administration in differentiated and non-differentiated cells was evaluated as a function of time (15 min, 30 min, and 1 h) and dose (10 nM, 100 nM, and 1 µM). Although differentiated cells have lower glucose uptake than non-differentiated cells, it has been observed that these cells respond better to insulin. In particular, it has been shown that after an incubation of 60 min with 10 nM insulin, glucose uptake increased by 16.8%, and, at the same time, 100 nM insulin led to an 18.8% increase in glucose absorption (Figure 2D). For all other conditions, insulin did not promote a significant increase in glucose uptake, as it did in non-differentiated SW 872 cells (Figure 2C). We then explored the role of the Akt pathway in these events. The phosphorylation of Akt protein is responsible for GLUT4 translocation on the cell membrane, and its activation is induced upon insulin stimuli; therefore, it has an impact on cell glucose uptake. We used the Western blot technique to evaluate the presence and quantity of the Akt protein in the SW 872 cell model, followed by the treatment of cells with 10 µM insulin [3]. In differentiated cells, Akt activation was undetectable at T0. Yet, since the Akt phosphorylation is an immediate event, after 5, 15, and 30 min, the pAkt amount increased, and it started leveling down after 1 h. In contrast, pAkt quantity was reduced in undifferentiated cells, even after insulin administration (Figure 2B). To summarize, SW 872 differentiated cells showed lower glucose uptake, resembling that of dysfunctional adipocytes. Furthermore, cells treated with OA, a PPAR-γ agonist, showed an increased response to insulin, as expected.

4. Inflammation

Dysfunctional adipocytes release pro-inflammatory cytokines, such as interleukin 6 (IL-6) and interleukin 8 (IL-8), leading to a high level of inflammation, which leads to worse health conditions. In our study, SW 872 cells showed a time-dependent modulation of the basal secretion of these pro-inflammatory cytokines, with an increase in IL-6 and a reduction in IL-8, after treatment with OA (Figure 3). Other studies show contradictory results on IL-6 and IL-8 release, depending on the PPAR-γ agonist and the adipocyte cell model used [13,14,15]. Considering these features, pro-inflammatory cytokine release and modulation must be further investigated in order to validate and better understand the best dysfunctional adipocytes in the cell model.

5. Modulation of Dysfunctional Aspects of SW 872 Cell Line by Plant Extracts

The SW 872 cell model was then utilized as a potential adipocyte model to test the effects of Cameroonian spice extracts in order to modify the above-reported cellular mechanisms [3]. Interestingly, these plant extracts were found active in improving some of these functions in HepG2 human hepatocyte [16].

Table 1. Specific effects of the Cameroonian spice extracts tested on differentiated SW 872 cells. Adapted with permission from [3].

	Triglyceride Reduction	Glucose Uptake Stimulation	ROS Production	IL-6 Reduction	IL-8 Reduction
Xylopia aethiopica	−14.5%		+55.8%		−21.1%
Xylopia parviflora	−13.8%		−50.5%		−36.8%
Scorodophloeus zenkeri	−18.5%
Monodora myristica	−15.3%		−40%		−24.3%
Tetrapleura tetraptera	−13.8%	+40.8%	−27.4%	−29.7%
Echinops giganteus	−11.3%		−43.6%	−29%
Afrostyrax lepidophyllus	−16.5%		−24.6%
Dichrostachys glomerata	−17.4%			−40%
Aframomum melegueta	−13%	+41.7%		−43.1%
Aframomum citratum	−16%				−58.6%
Zanthoxylum leprieurii	−13.4%	+56.6%			−32.7%

reports the effects of a set of 11 Cameroonian on triglyceride content, glucose uptake stimulation, ROS production, and cytokines release. Plant extracts were used only on differentiated cells, at different concentrations (1 µg/mL, 10 µg/mL, and 20 µg/mL). The results showed that all plants reduced the triglyceride contents, while the other parameters were modulated by only some of them.

6. Conclusions and Discussion

The global prevalence of metabolic diseases, such as obesity, type 2 diabetes mellitus (T2DM), metabolic syndrome, and the consequent atherosclerotic cardiovascular diseases, is increasing. In order to improve living conditions, the biochemical and molecular pathways of dysfunctional adipocytes require further investigation. To reach this aim, different dysfunctional adipocyte models are used. The SW 872 human liposarcoma model is mainly used for in vitro studies in oncological studies, but there is also evidence that support its use for in vitro studies of dysfunctional adipose tissue and related diseases. Literature shows different methods to differentiate SW 872 cells to mature-like adipocytes; however, to the best of our knowledge, there are no studies that fully characterize these cells as part of a dysfunctional adipocyte model. It has been shown that this cell model can spontaneously differentiate into a mature adipocyte model when placed in high density culture for 10 days [8]. This can be due to both the presence of triglycerides within the cell, and also the number of other parameters. However, we decided to promote differentiation with 100 µM OA, as it induces a faster differentiation [3].

In this study, the accumulation of intracellular lipids was evaluated using ORO staining, which shows a higher accumulation of triglycerides in differentiated cells than non-differentiated cells. This accumulation is similar to the one induced in adipocytes following activation of glitazone-mediated PPAR-γ—a side effect that frequently occurs in patients on glitazonic therapy for the treatment of T2DM [17].

In addition, we evaluated the glucose uptake by cytofluorometric analysis, showing that non-differentiated SW 872 cells allow twice as much glucose input than differentiated cells, despite the fact that non-differentiated cells do not respond to insulin. In addition, the results obtained by Western blotting analysis support this; in fact, the phosphorylated Akt fraction in differentiated cells increases in a time-dependent manner, more markedly than in non-differentiated cells. Other studies on the secretion of pro-inflammatory cytokines showed an increase in IL-6 associated to differentiation. It has also been observed that secretion of IL-6 increases from T0 to T7 in non-differentiated cells. On the contrary, differentiated cells showed a lower release of IL-8 than non-differentiated cells. This can be due to the inhibition of IL-8 by PPAR-γ activation by OA, which is an agonist of the receptor [13]. Plant extracts of Cameroonian origin, used as nutritional spices and in traditional medicine, were found to modulate the glucometabolic and inflammatory aspects of SW 872 cells; thus, these cells could be used for the screening of functional compounds or extracts of natural origin (nutraceutical and plant extracts). Based on the highlighted characteristics, this model could be used in the future to study nutraceuticals or drugs used for the treatment of cardiometabolic pathologies related to a dysfunction of adipose tissue.