Brown Algae Dictyopteris divaricata Attenuates Adipogenesis by Modulating Adipocyte Differentiation and Promoting Lipolysis through Heme Oxygenase-1 Activation in 3T3-L1 Cells

Abstract

The present study aims to explore the probable anti-adipogenesis effect of Dictyopteris divaricata (D. divaricata) in 3T3-L1 preadipocytes by regulating heme oxygenase-1 (HO-1). The extract of D. divaricata retarded lipid accretion and decreased triglyceride (TG) content in 3T3-L1 adipocytes but increased free glycerol levels. Treatment with the extract inhibited lipogenesis by inhibiting protein expressions of fatty acid synthase (FAS) and lipoprotein lipase (LPL), whereas lipolysis increased by activating phosphorylation of hormone-sensitive lipase (p-HSL) and AMP-activated protein kinase (p-AMPK). The extract inhibited adipocyte differentiation of 3T3-L1 preadipocytes through down-regulating adipogenic transcription factors, including peroxisome proliferator-activated receptor gamma (PPARγ) and CCAAT/enhancer-binding protein α (C/EBPα), and sterol regulatory element-binding protein 1 (SREBP1). This is attributed to the triggering of Wnt/β-catenin signaling. In addition, this study found that treatment with the extract activated HO-1 expression. Pharmacological approaches revealed that treatment with Zinc Protoporphyrin (ZnPP), an HO-1 inhibitor, resulted in an increase in lipid accumulation and a decrease in free glycerol levels. Finally, three adipogenic transcription factors, such as PPARγ, C/EBPα, and SREBP1, restored their expression in the presence of ZnPP. Analysis of chemical constituents revealed that the extract of D. divaricata is rich in 1,4-benzenediol, 7-tetradecenal, fucosterol, and n-hexadecanoic acid, which are known to have multiple pharmacological properties.

Keywords: Dictyopteris divaricata; HO-1; adipogenesis; lipid accumulation; lipolysis.

Figure 1

GC-MS chromatogram of D. divaricata extract.

Figure 2

Effect of Dictyopteris divaricata (DD) on viability of 3T3-L1 cell. (A) Differentiation procedure and assay schedule; (B) Effect of DD on 3T3-L1 cell viability. The cells were incubated with 10–100 µg/mL of DD for 48 h, and cell viability was assessed by MTT assay. All data given are means ± SD (n = 3) and error bars with different letters are significantly different at p < 0.05. DMEM, Dulbecco’s modified Eagle’s medium; DEX, dexamathanose; FBS, fetal bovine serum; IBMX, 3-isobutyl-1-methylxanthine; INS, insulin; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; ORO, Oil red O; TG, triglyceride; WB; Western blot.

Figure 3

Effect of Dictyopteris divaricata (DD) on adipogenesis of 3T3-L1 cells. (A) Illustrative images and quantitative analysis of lipid accumulation; (B) intra-cellular TG accumulation; (C) free glycerol release. Cells were differentiated in the presence and absence of DD for 6 days as described in material and methods followed by staining with Oil Red O reagent, colorimetric determination of TG content, and free glycerol release assay. All data given are means ± SD (n = 3) and error bars with different letters are significantly different at p < 0.05. MDI, differentiation initiation media; TG, triglyceride.

Figure 4

Effect of Dictyopteris divaricata (DD) on protein levels of PPARγ, C/EBPα, SREBP-1, and β-catenin by 3T3-L1 cells. (A) Representative Western blots of PPARγ, C/EBPα, and SREBP-1; relative intensities were normalized to the band of beta-actin; (B) Representative Western blots of β-catenin; relative intensities of nucleus and cytosol fractions were normalized to the band of Lamin B and β-actin, respectively. Cells were differentiated in the presence and absence of DD for 6 days as described in materials and methods, followed by Western blot analysis. All data given are means ± SD (n = 3) and error bars with different letters are significantly different at p < 0.05. MDI, differentiation initiation media; PPARγ, peroxisome proliferator-activated receptor gamma; C-EBPα, CCAAT/enhancer-binding protein alfa; SREBP-1, sterol regulatory element-binding protein-1.

Figure 5

Effect of Dictyopteris divaricata (DD) on protein levels of AMPK, HSL, FAS, and LPL by 3T3-L1 cells. (A) Representative Western blots of p-HSL and p-AMPK; relative intensities were normalized to the band of HSL and AMPK, respectively; (B) Representative Western blots of FAS and LPL; relative intensities were normalized to the band of β-actin. Cells were differentiated in the presence and absence of DD for 6 days as described in the materials and methods section, followed by Western blot analysis. All data given are means ± S. D (n = 3) and error bars with different letters are significantly different at p < 0.05. MDI, differentiation initiation media; p-HSL, phosphorylation of hormone-sensitive lipase; p-AMPK, phosphorylation of AMP-activated protein kinase; FAS, fatty acid synthase; LPL, lipoprotein lipase.

Figure 6

Effect of Dictyopteris divaricata (DD) on HO-1 activation. Representative Western blot of HO-1. The relative intensity of HO-1 was normalized to the band of β-actin. Cells were differentiated in the presence and absence of DD for 6 days as described in materials and methods followed by Western blot analysis. All data given are means ± SD (n = 3) and error bars with different letters are significantly different at p < 0.05. MDI, differentiation initiation media; HO-1, heme oxygenase 1.

Figure 7

Dictyopteris divaricata (DD) induced HO-1 activation and HO-1 inhibitor attenuates adipogenesis inhibition by DD. (A) Representative Western blot of HO-1. (B) Illustrative images and quantitative analysis of lipid accumulation; (C) Free glycerol release; (D) Representative Western blots C/EBPα, PPARγ, and SREBP-1; relative intensities were normalized to the band of β-actin. Cells were differentiated and treated with DD as described in materials and methods with 2 h pretreatment of ZnPP (10 µM) followed by Western blot analysis, Oil Red O staining, and free glycerol release. All data given are means ± SD (n = 3) and error bars with different letters are significantly different at p < 0.05. MDI, differentiation initiation media; HO-1, heme oxygenase 1. PPARγ, peroxisome proliferator-activated receptor gamma; C-EBPα, CCAAT/enhancer-binding protein alfa; SREBP-1, sterol regulatory element-binding protein-1.

Figure 8

Proposed model of the anti-adipogenesis effects of the extract. D. divaricata inhibits adipogenesis by reducing adipocyte differentiation, and lipogenesis and promoting lipolysis in 3T3-L1 adipocytes.