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Strongylocentrotus intermedius Extract Suppresses Adiposity by Inhibiting Adipogenesis and Promoting Adipocyte Browning via AMPK Activation in 3T3-L1 Cells.
Dayarathne LA
,
Jasmadi
,
Ko SC
,
Yim MJ
,
Lee JM
,
Kim JY
,
Oh GW
,
Lee DS
,
Jung WK
,
Lee SJ
,
Je JY
.
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The current study aimed to determine whether Strongylocentrotus intermedius (S. intermedius) extract (SIE) exerts anti-obesity potentials employing 3T3-L1 cells as in vitro model. Herein we reported that treatment of SIE for 6 days reduced lipid accretion and TG (triglyceride) content whereas it increased the release of free glycerol. The inhibited lipid accumulation and induced lipolysis were evidenced by the downregulation of lipogenesis proteins, such as fatty acid synthase and lipoprotein lipase, and the upregulation of hormone-sensitive lipase expression. Furthermore, the downregulation of adipogenic transcription factors, including peroxisome proliferator-activated receptor gamma, CCAAT/enhancer-binding protein α, and sterol regulatory element-binding protein 1, highlights that reduced lipid accumulation is supported by lowering adipocyte differentiation. Additionally, treatment activates brown adipocyte phenotype in 3T3-L1 cells by inducing expression of brown adipose tissue-specific proteins, such as uncoupling protein 1 (UCP-1) and peroxisome proliferator-activated receptor-γ coactivator 1α (PGC-1α). Moreover, SIE induced the phosphorylation of AMP-activated protein kinase (AMPK). The pharmacological approach using AMPK inhibitor revealed that the restraining effect of SIE on adipogenesis and promotion of adipocyte browning were blocked. In GC-MS analysis, SIE was mainly composed of cholest-5-en-3-ol (36.71%) along with saturated and unsaturated fatty acids which have favorable anti-obesity potentials. These results reveal that SIE has the possibility as a lipid-lowering agent for the intervention of obesity.
Fig. 1. Cell viability of SIE in 3T3-L1.Differentiation procedure and assay schedule (A) 3T3-L1 cell viability (B) The cells were treated with 10-100 μg/ml of SIE for 48 h, and cell viability was measured by MTT assay. All values given are means ± SD (n = 3).
Fig. 2. Lipid accumulation, TG content, and free glycerol release by SIE in 3T3-L1 cells.(A) Oil Red O images and quantitative analysis of lipid accumulation; (B) TG accumulation; (C) free glycerol release. Cells were differentiated in the presence and absence of extract 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). **p < 0.01 and ***p < 0.001.
Fig. 3. Expressions of adipogenesis and lipogenesis proteins by SIE in 3T3-L1 cells.(A) Protein expressions of PPARγ, C/EBPα, and SREBP-1; results are expressed as fold changes compared to the band of β-actin; (B) Protein expressions of FAS and LPL; results are expressed as fold changes to the band β-actin. Cells were differentiated in the presence and absence of extract for 6 days as described in materials and methods followed by Western blot analysis. All data given are means ± SD (n = 3). *p < 0.05, **p < 0.01, and ***p < 0.001.
Fig. 4. Expression of AMPK, HSL, PGC-1α, and UCP-1 by SIE in 3T3-L1 cells.(A) Protein expressions of p-HSL, PGC-1α, and UCP-1; results are expressed as fold changes compared to the band of HSL and β-actin. (B) Protein expressions of p-AMPK; results are expressed as fold changes compared to the band of AMPK. Cells were differentiated in the presence and absence of extract for 6 days as described in materials and methods followed by Western blot analysis. All data given are means ± SD (n = 3). *p < 0.05 and ***p < 0.001.
Fig. 5. AMPK inhibitor attenuates adipogenesis inhibition by SIE.(A) Protein expression of p-AMPK. (B) Representative images and quantitative analysis of lipid accumulation; (C) TG accumulation; (D) Free glycerol release; (E) Protein expression of p-HSL, PGC-1α, and UCP-1; results are expressed as fold changes compared to the band of HSL and β- actin respectively. Cells were differentiated and treated with extract as described in materials and methods with 2 h pretreatment of Compound C (CC) (10 μM) followed by Western blot analysis, Oil Red O staining, TG assay, and free glycerol release. All data given are means ± SD (n = 3). *p < 0.05, **p < 0.01, and ***p < 0.001.
