The suppressive effect of Gelidium amansi- EtOH extracts on the adipogenesis with MAPK signals in adipocytes with or without macrophages

Abstract

To elucidate the anti-inflammatory and anti-adipogenetic effects of Gelidium amansii (GA) ethanol extracts and their mechanisms, we performed two culture systems, adipocytes cultured with or without macrophages. Purified GA-3 fraction (GAE) contains high flavonoids and phenolics, reduced the mRNA levels of PPARγ and C/EBPα with GLUT4 expression in adipocyte with or without macrophages. GAE also increased the protein expression of HSL and ATGL enzymes, lipolysis biomarkers in fat cells. In co-culture system, GAE suppressed not only the transcription factors for adipogenesis, but also the production of pro-inflammatory cytokines, TNF-α. Compared to MAPK pathways such as JNK and p-38, the phosphorylation of both ERK1/2 (Thr²⁰²/Tyr²⁰⁴) was strongly suppressed by GAE with dose-dependent manner in both culture system. Otherwise, an increased JNK expression caused by GAE treatments blocked an insulin-induced GLUT4 translocation in adipocytes culture. In conclusion, GAE depressed the expression of adipogenetic genes, corresponding to a reduction in fat accumulation while preadipocytes developed into adipocytes with the modulation of MAPK pathways and inflammatory cytokines.

Keywords: Adipocytes; ERK1/2; Gelidium amansii; MAPK; Macrophages; TNF-α.

Fig. 1

Effect of GAE on the cell viability, lipid accumulation and TG concentration in adipocytes in adipocytes or co-cultured with RAW264.7 macrophages. After various concentrations of GAE (0–200 µg/mL) were treated into adipocyte or co-cultured with macrophages, cell viability using the MTT or CCK-8 assay (A), lipid accumulation (B, D) and triglyceride content (B, D) were measured by ORO staining or a triglyceride assay, respectively. A reduction in TG concentration sustained until 16 days’ incubation with GAE treatment of 100 µg/mL (C; BODIPY 493/503 and Hoechst 33342 dyes). Each value represents the mean ± SE (n = 5) with significance against the control (*p < 0.05, **p < 0.01, ***p < 0.001)

Fig. 2

Effect of GAE on the expression of transcription factors related to adipogenesis in adipocytes (A) or co-cultured with RAW264.7 macrophages (B). After various concentrations of GAE (0–200 µg/mL) were treated for 8 days, the expression of protein levels of PPARγ, C/EBPα, and GLUT4 were measured. Each value represents the mean ± SE (n = 5) with significance compared to the control (*p < 0.05, **p < 0.01, ***p < 0.001)

Fig. 3

Effects of GAE on the TG mobilization in matured adipocytes. Matured adipocytes were incubated with various concentrations of GAE (0–200 µg/mL) for 24 h. The expression of protein levels of HSL, ATGL, AMPK (A) and production of glycerol were measured in adipocytes. (B) Each value represents the mean ± SE (n = 5) with significance compared to the control (*p < 0.05, **p < 0.01, ***p < 0.001)

Fig. 4

Effect of GAE on the changes of MAPK signaling in adipocytes (A) or co-cultured with RAW264.7 macrophages (B). After various concentrations of GAE (10–200 µg/mL) treated for 8 days in adipocytes alone (A) or co-cultured with macrophages (B), the expression of protein or phosphorylation for MAPK were detected. Each value represents the mean ± SE (n = 5) with significance compared to the control (*p < 0.05, **p < 0.01, ***p < 0.001)

Fig. 5

The changes of TNF-α production after various concentration of GAE (0–200 µg/mL) were treated in adipocytes or co-cultured with RAW264.7 macrophages using ELISA (A) and the flow cytometry (B). After various concentrations of GAE (10–100 µg/mL) treated every 2 days for 8 days in both adipocytes with or without macrophages, TNF-α producing inflammatory monocytes (F4/80⁺ and CD11b⁺) were detected by FACS, respectively. Means of fluorescence intensity (MFI) described as changes in percentages of positive cells (the upper right and corner of each panel). Each value represents the mean ± SE (n = 5) with significance compared to the control (*p < 0.05, **p < 0.01, ***p < 0.001)