Nutr Res Pract.  2015 Dec;9(6):592-598. 10.4162/nrp.2015.9.6.592.

The micosporine-like amino acids-rich aqueous methanol extract of laver (Porphyra yezoensis) inhibits adipogenesis and induces apoptosis in 3T3-L1 adipocytes

• Kim H
• Lee Y
• Han T
• Choi EM

• KMID: 2313882
• DOI: http://doi.org/10.4162/nrp.2015.9.6.592

Abstract

BACKGROUND/OBJECTIVES
Increased mass of adipose tissue in obese persons is caused by excessive adipogenesis, which is elaborately controlled by an array of transcription factors. Inhibition of adipogenesis by diverse plant-derived substances has been explored. The aim of the current study was to examine the effects of the aqueous methanol extract of laver (Porphyra yezoensis) on adipogenesis and apoptosis in 3T3-L1 adipocytes and to investigate the mechanism underlying the effect of the laver extract.
MATERIALS/METHODS
3T3-L1 cells were treated with various concentrations of laver extract in differentiation medium. Lipid accumulation, expression of adipogenic proteins, including CCAAT enhancer-binding protein alpha, peroxisome proliferator-activated receptor gamma, fatty acid binding protein 4, and fatty acid synthase, cell viability, apoptosis, and the total content and the ratio of reduced to oxidized forms of glutathione (GSH/GSSG) were analyzed.
RESULTS
Treatment with laver extract resulted in a significant decrease in lipid accumulation in 3T3-L1 adipocytes, which showed correlation with a reduction in expression of adipogenic proteins. Treatment with laver extract also resulted in a decrease in the viability of preadipocytes and an increase in the apoptosis of mature adipocytes. Treatment with laver extract led to exacerbated depletion of cellular glutathione and abolished the transient increase in GSH/GSSG ratio during adipogenesis in 3T3-L1 adipocytes.
CONCLUSION
Results of our study demonstrated that treatment with the laver extract caused inhibition of adipogenesis, a decrease in proliferation of preadipocytes, and an increase in the apoptosis of mature adipocytes. It appears that these effects were caused by increasing oxidative stress, as demonstrated by the depletion and oxidation of the cellular glutathione pool in the extract-treated adipocytes. Our results suggest that a prooxidant role of laver extract is associated with its antiadipogenic and proapoptotic effects.

Keyword

Laver extract; adipogenesis; apoptosis; oxidative stress; glutathione

MeSH Terms

3T3-L1 Cells
Adipocytes*
Adipogenesis*
Adipose Tissue
Apoptosis*
Carrier Proteins
Cell Survival
Glutathione
Humans
Methanol*
Oxidative Stress
PPAR gamma
Transcription Factors
Carrier Proteins
Glutathione
Methanol
PPAR gamma
Transcription Factors

Figure

• 

  Fig. 1 The effect of laver extract on lipid accumulation in 3T3-L1 adipocytes. Two-day postconfluent 3T3-L1 preadipocytes (day 0) were induced to differentiate. From day 0 to day 9, the cells were treated with various concentrations of laver extract in the differentiation medium. Accumulated lipids in cells were stained with Oil Red O on day 9 (A: representative images of stained cells). Lipid contents were determined by eluting the dye with isopropanol and measuring the absorbance at 510 nm (B). The lipid content was normalized to the protein content in each well. The results are expressed as the mean ± SD (n = 6). *** P < 0.001 vs. cells without extract treatment.

• 

  Fig. 2 The effect of laver extract on adipogenic protein levels in 3T3-L1 adipocytes. On day 9, the 3T3-L1 adipocytes which had been treated with various concentrations of laver extract were harvested and subjected to Western blot analysis using antibodies to PPARγ, C/EBPαs, FABP4, FAS, and β-actin (A). Then the blot was densitometrically analyzed and normalized to β-actin (B). The plots show the representative of three independent experiments.

• 

  Fig. 3 The effect of laver extract on viability and apoptosis in 3T3-L1 preadipocytes. 3T3-L1 preadipocytes were treated with various concentrations of laver extract for 48 h. Cell viability was determined by MTT assay (A). Cell death was analyzed by flow cytometry with annexin V-FITC and propidium iodide (PI) staining of the cells. The percentages of apoptotic cells, i.e., cells positive for annexin V and negative for PI, and necrotic cells, i.e., cells positive for both annexin V and PI, were determined. The results are expressed as the mean ± SD (n = 16 for MTT; n = 3 for flow cytometry). ** P < 0.01; *** P < 0.001 vs. cells without extract treatment.

• 

  Fig. 4 The effect of laver extract on apoptosis in 3T3-L1 adipocytes. Differentiated 3T3-L1 adipocytes were treated with various concentrations of laver extract for 48 h, i.e., during days 7-9. Cell death was analyzed by flow cytometry with annexin V-FITC and propidium iodide (PI) staining of the cells. The percentages of apoptotic cells, i.e., cells positive for annexin V and negative for PI, and necrotic cells, i.e., cells positive for both annexin V and PI, were determined. The results are expressed as the mean ± SD (n = 3). * P < 0.05 vs. cells without extract treatment.

• 

  Fig. 5 The effect of laver extract on the content and redox status of glutathione in 3T3-L1 adipocytes. From day 0 to day 9, the cells were treated with various concentrations of laver extract in the differentiation medium. On days 0, 3, 6, and 9, total glutathione content (A) and the ratio of GSH/GSSG (B) in cells were measured. The results are expressed as the mean ± SD (n = 6). *** P < 0.001 vs. cells without extract treatment on day 0.

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