Nutr Res Pract.  2018 Dec;12(6):494-502. 10.4162/nrp.2018.12.6.494.

Red pepper seed water extract inhibits preadipocyte differentiation and induces mature adipocyte apoptosis in 3T3-L1 cells

• Kim HJ
• You MK
• Lee YH
• Kim HJ
• Adhikari D
• Kim HA

• KMID: 2448694
• DOI: http://doi.org/10.4162/nrp.2018.12.6.494

Abstract

BACKGROUND/OBJECTIVES
Reducing the number of adipocytes by inducing apoptosis of mature adipocytes as well as suppressing differentiation of preadipocytes plays an important role in preventing obesity. This study examines the anti-adipogenic and pro-apoptotic effect of red pepper seed water extract (RPS) prepared at 4℃ (RPS4) in 3T3-L1 cells.
MATERIALS/METHODS
Effect of RPS4 or its fractions on lipid accumulation was determined in 3T3-L1 cells using oil red O (ORO) staining. The expressions of AMP-activated protein kinase (AMPK) and adipogenic associated proteins [peroxisome proliferator-activated receptor-γ (PPAR-γ), CCAAT/enhancer-binding proteins α (C/EBP α), sterol regulatory element binding protein-1c (SREBP-1c), fatty acid synthase (FAS), and acetyl-CoA carboxylase (ACC)] were measured in 3T3-L1 cells treated with RPS4. Apoptosis and the expression of Akt and Bcl-2 family proteins [B-cell lymphoma 2 (Bcl-2), Bcl-2-associated death promoter (Bad), Bcl-2 like protein 4 (Bax), Bal-2 homologous antagonist/killer (Bak)] were measured in mature 3T3-L1 cells treated with RPS4.
RESULTS
Treatment of RPS4 (0-75 µg/mL) or its fractions (0-50 µg/mL) for 24 h did not have an apparent cytotoxicity on pre and mature 3T3-L1 cells. RPS4 significantly suppressed differentiation and cellular lipid accumulation by increasing the phosphorylation of AMPK and reducing the expression of PPAR-γ, C/EBP α, SREBP-1c, FAS, and ACC. In addition, all fractions except ethyl acetate fraction significantly suppressed cellular lipid accumulation. RPS4 induced the apoptosis of mature adipocytes by hypophosphorylating Akt, increasing the expression of the pro-apoptotic proteins, Bak, Bax, and Bad, and reducing the expression of the anti-apoptotic proteins, Bcl-2 and p-Bad.
CONCLUSIONS
These finding suggest that RPS4 can reduce the numbers as well as the size of adipocytes and might useful for preventing and treating obesity.

Keyword

Capsicum; adipocyte; adipogenesis; apoptosis; lipid metabolism

MeSH Terms

3T3-L1 Cells*
Acetyl-CoA Carboxylase
Adipocytes*
Adipogenesis
AMP-Activated Protein Kinases
Apoptosis Regulatory Proteins
Apoptosis*
Capsicum*
Humans
Lipid Metabolism
Lymphoma
Obesity
Phosphorylation
Sterol Regulatory Element Binding Protein 1
Water*
AMP-Activated Protein Kinases
Acetyl-CoA Carboxylase
Apoptosis Regulatory Proteins
Sterol Regulatory Element Binding Protein 1
Water

Figure

• 

  Fig. 1 Effect of red pepper seed water extract (RPS4) and its fractions on the viability in preadipocytes (A, B) and mature adipocytes (C). Cells were treated with various concentrations of RPS4 (A, C) or its fraction (B) for 24 h. C, PBS or DMSO; RPS4 25, red pepper seed water extract (4℃) 25 µg/mL; RPS4 50, red pepper seed water extract (4℃) 50 µg/mL; RPS4 75, red pepper seed water extract (4℃) 75 µg/mL; RPS4-H, n-hexane fractionate of red pepper seed water extract (4℃) ; RPS4-Cl, chloroform fractionate of red pepper seed water extract (4℃); RPS4-EA, ethylacetate fractionate of red pepper seed water extract (4℃); RPS4-B, butanol fractionate of red pepper seed water extract (4℃); RPS4-W, water fractionate of red pepper seed water extract (4℃).Values are Mean±SE. Stand error bars represent three independent experiments and each experiment was triplicated. Means with the same letter are not significantly different by Duncan's multiple range test (P < 0.05).

• 

  Fig. 2 Effect of red pepper seed water extract (RPS4) and its fractions on lipid accumulation in 3T3-L1 cells. The 3T3-L1 cells treated with PBS, DMSO, 25, 50, or 75 µg/mL RPS4 or 50 µg/mL its fractions during the differentiation. After 8 days, 3T3-L1 cells were stained using Oil Red O (A) and measured lipid droplet by ELISA (B). C, PBS or DMSO; RPS4 25, red pepper seed water extract (4℃) 25 µg/mL; RPS4 50, red pepper seed water extract (4℃) 50 µg/mL; RPS4 75, red pepper seed water extract (4℃) 75 µg/mL; RPS4-H, n-hexane fractionate of red pepper seed water extract (4℃) ; RPS4-Cl, chloroform fractionate of red pepper seed water extract (4℃); RPS4-EA, ethylacetate fractionate of red pepper seed water extract (4℃); RPS4-B, butanol fractionate of red pepper seed water extract (4℃); RPS4-W, water fractionate of red pepper seed water extract (4℃). Values are Mean±SE. Stand error bars represent three independent experiments and each experiment was triplicated. Means with the same letter are not significantly different by Duncan's multiple range test (P < 0.05).

• 

  Fig. 3 Effect of red pepper seed water extract (RPS4) on the expression of AMPK (A) and adipogenic associated proteins (B) in 3T3-L1 adipocytes. C, PBS; RPS4 25, red pepper seed water extract (4℃) 25 µg/mL; RPS4 50, red pepper seed water extract (4℃) 50 µg/mL; RPS4 75, red pepper seed water extract (4℃). Values are Mean±SE. Stand error bars represent three independent experiments and each experiment was triplicated. Means with the same letter are not significantly different by Duncan's multiple range test (P < 0.05).

• 

  Fig. 4 Effect of red pepper seed water extract (RPS4) on apoptosis in mature adipocytes. Mature adipocytes were incubated with various concentration of PBS, 25, 50, 75 µg/mL of RPS4 for 24 h and then cell apoptosis was evaluated by Muse Cell Analyzer. C, PBS; RPS4 25, red pepper seed water extract (4℃) 25 µg/mL; RPS4 50, red pepper seed water extract (4℃) 50 µg/mL; RPS4 75, red pepper seed water extract (4℃). Values are Mean±SE. Stand error bars represent three independent experiments and each experiment was triplicated. Means with the same letter are not significantly different by Duncan's multiple range test (P < 0.05).

• 

  Fig. 5 Effect of red pepper seed water extract (RPS4) on the expression of Akt and p-Akt in mature adipocytes. (A) Mature adipocytes were treated with RPS4 for 2, 6, or 24 h; (B) Mature adipocytes were treated with various concentration RPS4 for 6 h. C, PBS; RPS4 25, red pepper seed water extract (4℃) 25 µg/mL; RPS4 50, red pepper seed water extract (4℃) 50 µg/mL; RPS4 75, red pepper seed water extract (4℃) 75 µg/mL. Values are Mean±SE. Stand error bars represent three independent experiments and each experiment was triplicated. Means with the same letter are not significantly different by Duncan's multiple range test (P < 0.05).

• 

  Fig. 6 Effect of red pepper seed water extract (RPS4) on the expression of Bcl-2 family proteins in mature adipocytes. (A) Bcl-2 family protein, (B) Bcl-2/Bax and (C) p-Bad/Bad ratio. After differentiation, cells treated with PBS, 25, 50, 75 µg/mL of RPS4 for 24 h to measure the expression of Bcl-2 family proteins using Western blotting. C, PBS; RPS4 25, red pepper seed water extract (4℃) 25 µg/mL; RPS4 50, red pepper seed water extract (4℃) 50 µg/mL; RPS4 75, red pepper seed water extract (4℃) 75 µg/mL. Values are Mean±SE. Stand error bars represent three independent experiments and each experiment was triplicated. Means with the same letter are not significantly different by Duncan's multiple range test (P < 0.05).

• 

  Fig. 7 Proposed working model: PRS4 suppresses adipogenisis and induces apoptosis through AMPK pathways

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