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Visfatin is regulated by interleukin-6 and affected by the PPAR-γ pathway in BeWo cells

Visfatin, an adipocytokine and cytosolic enzyme with nicotinamide phosphoribosyltransferase (Nampt) activity, is involved in the pathogenesis of numerous metabolic disorders. In addition, the nuclear receptor peroxisome proliferator-activated receptor-γ (PPAR-γ) serves important roles in anti-inflammatory reactions and regulates glucose and lipid metabolism. The aim of the present study was to investigate the effect of interleukin-6 (IL-6) on the expression and secretion of visfatin in BeWo cells, and to determine whether the PPAR-γ pathway is involved in the regulation of visfatin by IL-6. Therefore, BeWo cells were stimulated with serial concentrations of IL-6 or pioglitazone, and the expression levels of visfatin and PPAR-γ were determined by reverse transcription-quantitative polymerase chain reaction and western blotting. The results of the present study demonstrated that IL-6 downregulated the mRNA levels of visfatin and PPAR-γ, which were strongly associated. Activation of PPAR-γ by pioglitazone resulted in significantly increased expression of visfatin, which abrogated the inhibitory effect of IL-6 on visfatin in BeWo cells. Furthermore, treatment using pioglitazone alone increased the expression and secretion of the visfatin protein, compared with the control or IL-6 alone group. In summary, the findings of the present study suggested that IL-6 inhibited the expression of visfatin and PPAR-γ at the transcriptional level; in addition, activation of PPAR-γ upregulated visfatin at the mRNA and protein expression levels. Therefore, the PPAR-γ signaling pathway may be involved in the regulation of visfatin by IL-6 in BeWo cells. These results may provide novel insight into the roles of visfatin in trophoblastic cells. Furthermore, thiazolidinedione pioglitazone, by upregulating visfatin expression, may promote the energy metabolism of trophoblastic cells, maintain the function of the placenta and improve the outcome of pregnancy.


Introduction
Visfatin, additionally termed pre-B-cell colony-enhancing factor or nicotinamide phosphoribosyltransferase (Nampt), is an adipocytokine that is predominantly produced in visceral adipose tissue (1)(2)(3); however, during pregnancy, visfatin is additionally expressed and secreted by the placental tissue (4,5). Visfatin is involved in the pathogenesis of various metabolic disorders; increased plasma concentrations of visfatin have been reported in individual with obesity, as well as in patients with gestational diabetes mellitus (GDM) or metabolic syndromes (6)(7)(8).
Visfatin functions as an immunomodulatory cytokine involved in the inflammatory responses, and is a factor associated with obesity, inflammation and insulin resistance (3,6). Upregulation of inflammatory cytokines, including tumor necrosis factor-α (TNF-α) and interleukin 6 (IL-6), serves important roles in the induction of insulin resistance (9). Numerous previous studies indicated that visfatin was associated with inflammation (10)(11)(12); however, the underlying molecular mechanisms remain unknown. Furthermore, visfatin functions as the rate-limiting enzyme of NAD + biosynthesis and converts nicotinamide into nicotinamide mononucleotide, a key NAD + intermediate (13). In addition, visfatin-mediated NAD + biosynthesis in adipocytes is an important physiological regulator of the metabolic function of adipose tissue and whole-body (13). Although still controversial, visfatin was reported to exert insulin mimetic or sensitizing effects; similar to insulin, visfatin increases lipogenesis, enhances glucose uptake of myocytes and adipocytes, and inhibits hepatocyte glucose release, consequently contributing to glucose and lipid metabolism (12,14,15). Previous studies identified the upregulation of IL-6 in the placenta of patients with GDM (16,17). In addition, it was indicated that the expression of visfatin in placenta was unchanged in patients with GDM (18). IL-6 (16,17) and visfatin (8,9) are associated with GDM. Kralisch et al (19) observed that IL-6 suppressed the mRNA synthesis of visfatin in 3T3-L1 adipocytes; however, in human adipocytes and amniotic epithelial cells, IL-6 significantly promoted visfatin protein expression (20,21). Therefore, the present study hypothesized that the modulation of visfatin expression by IL-6 may be tissue specific; however, how pro-inflammatory IL-6 regulates the expression of visfatin in placenta remains unknown.
Peroxisome proliferator-activated receptor-γ (PPAR-γ) is a nuclear receptor that is abundantly expressed in adipocytes, where it regulates adipogenesis and lipid homeostasis (22). PPAR-γ functions in other tissues by promoting anti-immune responses and lipid metabolism in macrophages, increasing lipid storage in the liver and enhancing glucose-stimulated insulin secretion in pancreatic β-cells (22,23). PPAR-γ is involved in the pathophysiology of GDM and serves key roles in the regulation of glucose metabolism, lipid homeostasis and anti-inflammatory processes (22,23). Previous studies identified the downregulation of PPAR-γ in the trophoblast cells of patients with GDM (24-26). Mayi et al (12) observed that synthetic PPAR-γ ligands upregulated the expression of visfatin gene in a PPAR-γ-dependent manner in resting primary human macrophages and adipose tissue macrophages, but not in adipocytes; however, whether PPAR-γ ligands induce the gene expression of visfatin in placenta remains unknown.
BeWo cells possess characteristics of the syncytiotrophoblast and extravillous trophoblast, and serve as an in vitro model to investigate trophoblast fusion (27,28). BeWo cells are frequently used to investigate the regulation of transplacental transport and cellular activities and uptake mechanisms (28). The aim of the present study was to determine the effect of IL-6 on the expression and secretion of visfatin in BeWo cells, and to determine whether the PPAR-γ pathway is involved in the regulation of visfatin by IL-6.
Statistical analysis. The experiments were repeated at least three times. Data were presented as the mean ± standard deviation and analyzed using SPSS 21.0 (IBM Corp., Armonk, NY, USA). Groups were compared by one-way analysis of variance and a Student-Newman-Keuls post-hoc test for normally distributed data. Correlation analysis was performed using Spearman's or Pearson's correlation coefficients. P<0.05 was considered to indicate a statistically significant difference.

IL-6 reduces the expression of visfatin gene in BeWo cells.
Treatment of BeWo cells with increasing concentrations of IL-6 (1, 5, and 10 ng/ml) resulted in significantly reduced levels of PPAR-γ and visfatin mRNA compared with the control; no further decrease was detected with 10 ng/ml IL-6 ( Fig. 1A and B). Time course experiments demonstrated that the inhibitory effects of IL-6 (5 ng/ml) on visfatin and PPAR-γ mRNA peaked at 48 h; no further reductions were observed at 72 h ( Fig. 1C and D). Pearson correlation analysis  Fig. 1E and F, respectively). In summary, the data of the present study demonstrated that IL-6 inhibited the expression of visfatin in BeWo cells, which was associated with PPAR-γ.

PPAR-γ agonist pioglitazone induces the expression of visfatin gene of BeWo cells.
Treatment with increasing concentrations of pioglitazone (0.1, 1 and 10 µM) upregulated the mRNA expression levels of visfatin in a dose-dependent manner, whereas, no significant differences were observed in the mRNA levels of PPAR-γ among the treatment groups (Fig. 2). Furthermore, activation of PPAR-γ by exposure to pioglitazone (10 µM) for 48 h resulted in significantly increased expression of visfatin compared with the control, which abrogated the inhibitory effects of IL-6 on visfatin in BeWo cells (Fig. 3A). In summary, the results of the present study demonstrated that visfatin may be a target gene of PPAR-γ in BeWo cells and that the PPAR-γ pathway may be involved in the regulation of visfatin by IL-6.

PPAR-γ activation regulates the expression and secretion of visfatin protein of BeWo cells.
To investigate whether pioglitazone-induced expression of visfatin resulted in increased protein levels, BeWo cells treated with IL-6 (5 ng/ml) and/or pioglitazone (10 µM) for 48 h were subjected to western blot analysis. IL-6 exhibited no significant effect on the protein expression of visfatin, while activation of PPAR-γ resulted in significantly increased protein expression levels of visfatin compared with the control, IL-6 treatment alone or in combination with pioglitazone (Fig. 3B); however, the expression levels of PPAR-γ protein were not affected by IL-6 or pioglitazone (data not shown). To examine whether induction by pioglitazone promoted the secretion of visfatin secretion, the ability of PPAR-γ to stimulate the release of visfatin was evaluated. As presented in Fig. 3C, treatment with pioglitazone (10 µM) significantly increased the levels of visfatin in BeWo cell compared with IL-6 alone (5 ng/ml) or in combination with pioglitazone. The findings suggested that pioglitazone upregulated the expression level of visfatin by activating the PPAR-γ pathway and the PPAR-γ pathway may be involved in the regulation of visfatin by IL-6.

Discussion
In the present study, the expression of visfatin in BeWo cells was evaluated in the presence of IL-6. Downregulation of visfatin was observed in BeWo cells treated with IL-6, which was consistent with the findings in 3T3-L1 adipocytes by Kralisch et al (19). The present study additionally identified that IL-6 reduced the mRNA expression level of PPAR-γ in BeWo cells and was associated with the expression of visfatin. The PPAR-γ agonist pioglitazone induced the expression of visfatin in BeWo cells, possibly in a PPAR-γ-dependent manner, which was consistent with the findings by Mayi et al (12) in resting primary human macrophages and adipose tissue macrophages. To the best of our knowledge, the present study is the first to demonstrate that visfatin may be a target gene of PPAR-γ in BeWo cells. In addition, the PPAR-γ pathway could be involved in the regulation of visfatin by IL-6; however, Lv et al (15) reported that pioglitazone ameliorates insulin resistance by downregulating visfatin expression in visceral adipose tissue and reducing circulating visfatin in high-fat-fed rats, whereas, no effect of pioglitazone was observed during the differentiation of 3T3-L1 adipocytes. This discrepancy may be due to the different sources of cells and tissues, which indicates that the effects of pioglitazone on visfatin expression could be cell-or tissue-specific.
PPARs are a member of the nuclear hormone receptor superfamily consisting of three isotypes: PPAR-α, PPAR-δ and PPAR-γ. All three PPAR isotypes are expressed in the placenta (30). The expression levels of PPAR-γ were downregulated in the placenta of females with GDM; however, that of PPAR-δ remained unchanged. A previous study demonstrated that PPAR-α agonist fenofibrate stimulated the expression of PPAR-α and induced the mRNA expression of visfatin in visceral fat deposits of Otsuka Long-Evans Tokushima fatty rats (31). The protein expression levels of PPAR-α in placenta were reduced, whereas, the mRNA levels were unchanged in females with GDM (24). Therefore, PPAR-γ was selected as the potential target in the present study. A chemical agonist of PPARs was easier to obtain compared with overexpression vectors and/or PPAR small interfering RNAs, thus, pioglitazone was used in the present study.
The results of the present study demonstrated that IL-6 downregulated PPAR-γ and visfatin at the transcriptional level; the expression levels of visfatin and PPAR-γ were strongly correlated in BeWo cells. In addition, visfatin and PPAR-γ expression was reduced as the concentration of IL-6 increased between 0 and 5 ng/ml in BeWo cells, but their expression levels increased as the concentration of IL-6 reached 10 ng/ml; however, the expression levels of visfatin and PPAR-γ were significantly decreased compared with control group. The results of the present study suggested that the inhibitory effect of IL-6 on visfatin and PPAR-γ did not occur in a dose-dependent manner. The underlying mechanisms require further investigation.
Following treatment with pioglitazone, the mRNA and protein expression levels of visfatin were significantly increased in a dose-dependent manner in the present study. Therefore, visfatin may serve within trophoblastic cells as a protective factor, and may be involved in the regulation of energy metabolism and inflammatory reactions. It is well-established that visfatin/Nampt promotes intracellular NAD + synthesis and induces the activation of sirtuin1 (12,(32)(33)(34). Furthermore, placental visfatin/Nampt may additionally serve as the Nampt enzyme within trophoblasts (34). The overexpression of visfatin/Nampt may increase intracellular NAD + and stimulate sirtuin1, consequently affecting DNA repair, protecting telomeres and reducing inflammation (34). NAD + is an important coenzyme in numerous oxidation-reduction reactions (34). NAD + accepts and donates electrons in reactions, thus leading to the production of ATP, which is required by cells for most energy-consuming processes (34). Sirtuin1, additionally termed NAD + -dependent deacetylase, is a negative regulator of pro-inflammatory cytokines (35)(36)(37). In summary, visfatin/Nampt may be the regulatory factor underlying the survival of trophoblastic cells and maintenance of placental function in a hostile environment, including the inflammation response or hyperglycemia. Upon inflammation, reduced expression of visfatin may lead to disrupted placental function and increased risk of abortion, preterm birth, and/or fetal distress (33).
However, treatment with the PPAR-γ agonist pioglitazone induced the mRNA and protein expression of visfatin by increasing the secretion of visfatin and offsetting the inhibitory effect of IL-6 at the mRNA level. The findings of the present study suggested that visfatin was a potential target gene of PPAR-γ in BeWo cells, and pioglitazone upregulated the expression level of visfatin by activating the PPAR-γ pathway. Additionally, the mRNA expression levels of PPAR-γ remained unchanged following the treatment of pioglitazone. Subsequent to being activated by its ligands, PPAR-γ forms a heterodimer with the retinoic X receptor, which transactivates the PPAR-response elements of target genes involved in insulin sensitivity, glucose metabolism and immune responses (12,38,39). Therefore, upon activation, PPAR-γ upregulates its target genes, including visfatin, rather than promoting its own transcription. Furthermore, treatment with pioglitazone alone for 48 h resulted in increased secretion of visfatin compared with IL-6 alone or in combination with pioglitazone; however, the expression levels of visfatin in all groups remained low, indicating that visfatin may serve as an autocrine/paracrine factor rather than a typical endocrine agent in trophoblastic cells. Therefore, the present study hypothesized that visfatin was able to exert local effects in the placenta. Additionally, the activation of PPAR-γ suppressed the activity of nuclear factor-κB, consequently regulating the balance between cytokine production and lipid metabolism (40); however, the present study indicated that treatment with visfatin for 48 h did not alter the secretion levels of inflammatory cytokines, including IL-6 and TNF-α (data not shown). In summary, the results of the present study provide novel insight into the roles of visfatin in trophoblastic cells. Placental visfatin may serve as the Nampt enzyme by increasing NAD + biosynthesis and activating sirtuin1, consequently functioning as an anti-inflammatory factor rather than a pro-inflammatory factor, and serving an important role in maintaining the energy metabolism of trophoblastic cells and the function of the placenta.
In conclusion, the findings of the present study suggested that IL-6 downregulated visfatin and PPAR-γ at the transcriptional level; in addition, activation of PPAR-γ induced the expression of visfatin in BeWo cells. The thiazolidinedione pioglitazone may promote the energy metabolism of trophoblastic cells, maintain the function of the placenta and improve the outcome of pregnancy; however, further study is required to confirm whether visfatin may be a target gene of PPAR-γ in BeWo cells. The roles of visfatin as a Nampt enzyme, as well as its downstream signaling pathway and underlying mechanisms in trophoblastic cells or placenta, require further investigation.