Nutr Res Pract.  2021 Oct;15(5):555-567. 10.4162/nrp.2021.15.5.555.

Inhibitory effect of ethanolic extract of Abeliophyllum distichum leaf on 3T3–L1 adipocyte differentiation

• Thomas SS
• Eom J
• Sung NY
• Kim DS
• Cha YS
• Kim KA

• KMID: 2520658
• DOI: http://doi.org/10.4162/nrp.2021.15.5.555

Abstract

BACKGROUND/OBJECTIVES
Abeliophyllum distichum is a plant endemic to Korea, containing several beneficial natural compounds. This study investigated the effect of A. distichum leaf extract (ALE) on adipocyte differentiation.
MATERIALS/METHODS
The cytotoxic effect of ALE was analyzed using cell viability assay. 3T3-L1 preadipocytes were differentiated using induction media in the presence or absence of ALE. Lipid accumulation was confirmed using Oil Red O staining. The mRNA expression of adipogenic markers was measured using RT-PCR, and the protein expressions of mitogenactivated protein kinase (MAPK) and peroxisome proliferator-activated receptor gamma (PPARγ) were measured using western blot. Cell proliferation was measured by calculating the incorporation of Bromodeoxyuridine (BrdU) into DNA.
RESULTS
ALE reduced lipid accumulation in differentiated adipocytes, as indicated by Oil Red O staining and triglyceride assays. Treatment with ALE decreased the gene expression of adipogenic markers such as Pparγ, CCAAT/enhancer binding protein alpha (C/ebpα), lipoprotein lipase, adipocyte protein-2, acetyl-CoA carboxylase, and fatty acid synthase. Also, the protein expression of PPARγ was reduced by ALE. Treating the cells with ALE at different time points revealed that the inhibitory effect of ALE on adipogenesis is higher in the early period treatment than in the terminal period. Furthermore, ALE inhibited adipocyte differentiation by reducing the early phase of adipogenesis and mitotic clonal expansion. This was indicated by the lower number of cells in the Synthesis phase of the cell cycle (labeled using BrdU assay) and a decrease in the expression of early adipogenic transcription factors such as C/ebpβ and C/ebpδ. ALE suppressed the phosphorylation of MAPK, confirming that the effect of ALE was through the suppression of early phase of adipogenesis.
CONCLUSIONS
Altogether, the results of the present study revealed that ALE inhibits lipid accumulation and may be a potential agent for managing obesity.

Keyword

3T3-L1; adipocytes; adipogenesis; transcription factors; PPAR gamma

Figure

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  Fig. 1 Effect of ALE on cell viability. 3T3-L1 preadipocytes were treated with different concentrations of ALE for 24, 48, and 72 h, and cell viability was analyzed using MTT assay. Data expressed as mean ± SD (n = 8). Values with different superscripts; (a-d) for 24 h, (A-E) for 48 h, (w-z) for 72 h, indicate significant difference among groups by analysis of variance with Duncan's multiple range test at P < 0.05.ALE, Abeliophyllum distichum leaf extract; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide.

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  Fig. 2 Effect of ALE on adipocyte differentiation. 3T3-L1 preadipocytes were induced to differentiate with induction media in the presence or absence of ALE for 8 days and were analyzed for lipid accumulation. (A) Oil red O staining, (B) Oil Red O quantification, and (C) triglyceride quantification. Data expressed as mean ± SD (n = 6). Values with different superscripts indicate significant differences among groups by analysis of variance with Duncan's multiple range test at P < 0.05.ALE, Abeliophyllum distichum leaf extract; BS, non-differentiated cells; CON, differentiated adipocytes.

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  Fig. 3 Effect of ALE on adipocyte differentiation markers. 3T3-L1 preadipocytes were induced to differentiate with induction media in the presence or absence of ALE for 8 days and were analyzed for lipid accumulation. (A) mRNA expression levels of adipogenic and lipogenic genes and (B) Protein expression levels of PPARγ. Data expressed as mean ± SD (n = 6). Values with different superscripts indicate significant differences among groups by analysis of variance with Duncan's multiple range test at P < 0.05.ALE, Abeliophyllum distichum leaf extract; BS, non-differentiated cells; CON, differentiated adipocytes; Ppar, peroxisome proliferator-activated receptor; C/ebp, CCAAT/enhancer binding protein; Srebp, sterol regulatory element-binding protein; Fas, fatty acid synthase; Acc, acetyl CoA carboxylase; aP2, adipocyte protein-2; Lpl- lipoprotein lipase.

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  Fig. 4 Effect of ALE on adipocyte differentiation in cells treated at different time points. 3T3-L1 preadipocytes were induced to differentiate with induction media in the presence or absence of ALE for 8 days at different time points and were analyzed for lipid accumulation. (A) Oil red O staining, (B) Oil Red O quantification, and (C) Expression levels of genes associated with adipogenesis. Data expressed as mean ± SD (n = 6). Values with different superscripts indicate significant differences among groups by analysis of variance with Duncan's multiple range test at P < 0.05.ALE, Abeliophyllum distichum leaf extract; BS, non-differentiated cells; CON, differentiated adipocytes; Ep, early period; Tp, terminal period; Fp, full period; Ppar, peroxisome proliferator-activated receptor; C/ebp, CCAAT/enhancer binding protein; Fas, fatty acid synthase; Acc, acetyl CoA carboxylase.

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  Fig. 5 Effect of ALE on the MCE. 3T3-L1 preadipocytes were induced to differentiate with induction media in the presence or absence of ALE for 24 and 48 h. (A) Expression levels of genes associated with MCE and (B) BrdU incorporation. Data expressed as mean ± SE (n = 6). Values with different superscripts indicate significant differences among groups by analysis of variance with Duncan's multiple range test at P < 0.05.ALE, Abeliophyllum distichum leaf extract; BS, non-differentiated cells; CON, differentiated adipocytes; MCE, mitotic clonal expansion; BrdU, Bromodeoxyuridine; C/EBP, CCAAT/enhancer binding protein.

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  Fig. 6 Effect of ALE on MAPK signaling pathways. 3T3-L1 preadipocytes were induced to differentiate with induction media in the presence or absence of ALE for 2 h. (A) Western blot and (B) Quantification of protein levels. Data expressed as mean ± SD (n = 6). Values with different superscripts indicate significant differences among groups by analysis of variance with Duncan's multiple range test at P < 0.05.ALE, Abeliophyllum distichum leaf extract; BS, non-differentiated cells; CON, differentiated adipocytes; MAPK, mitogen-activated protein kinase; ERK, extracellular signal-regulated kinases; JNK, c-Jun amino-terminal kinase.

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