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Shikonin suppresses ERK 1/2 phosphorylation during the early stages of adipocyte differentiation in 3T3-L1 cells.

Gwon SY, Ahn JY, Jung CH, Moon BK, Ha TY - BMC Complement Altern Med (2013)

Bottom Line: To investigate the effects of shikonin on adipocyte differentiation, 3T3-L1 cells were induced to differentiate using 3-isobutyl-1-methylzanthine, dexamethasone, and insulin (MDI) for 8 days in the presence of 0-2 μM shikonin.Shikonin effectively suppressed adipogenesis and downregulated the protein levels of 2 major transcription factors, PPARγ and C/EBPα, as well as the adipocyte specific gene aP2 in a dose-dependent manner. qRT-PCR analysis revealed that shikonin inhibited mRNA expression of adipogenesis-related genes, such as PPARγ, C/EBPα, and aP2.We also confirmed that FGF-2-stimulated ERK 1/2 activity was attenuated by shikonin.

View Article: PubMed Central - HTML - PubMed

Affiliation: Functional Food Technology Research Group, Korea Food Research Institute, 516, Baekhyn-dong, Seongnam-si, Gyeonggi-do 463-746, Republic of Korea.

ABSTRACT

Background: The naphthoquinone pigment, shikonin, is a major component of Lithospermum erythrorhizon and has been shown to have various biological functions, including antimicrobial, anti-inflammatory, and antitumor effects. In this study, we investigated the effect of shikonin on adipocyte differentiation and its mechanism of action in 3T3-L1 cells.

Methods: To investigate the effects of shikonin on adipocyte differentiation, 3T3-L1 cells were induced to differentiate using 3-isobutyl-1-methylzanthine, dexamethasone, and insulin (MDI) for 8 days in the presence of 0-2 μM shikonin. Oil Red O staining was performed to determine the lipid accumulation in 3T3-L1 cells. To elucidate the anti-adipogenic mechanism of shikonin, adipogenic transcription factors, the phosphorylation levels of ERK, and adipogenic gene expression were analyzed by Western blotting and quantitative real-time PCR. To further confirm that shikonin inhibits adipogenic differentiation through downregulation of ERK 1/2 activity, 3T3-L1 cells were treated with shikonin in the presence of FGF-2, an activator, or PD98059, an inhibitor, of the ERK1/2 signaling pathway.

Results: Shikonin effectively suppressed adipogenesis and downregulated the protein levels of 2 major transcription factors, PPARγ and C/EBPα, as well as the adipocyte specific gene aP2 in a dose-dependent manner. qRT-PCR analysis revealed that shikonin inhibited mRNA expression of adipogenesis-related genes, such as PPARγ, C/EBPα, and aP2. Adipocyte differentiation was mediated by ERK 1/2 phosphorylation, which was confirmed by pretreatment with PD98059 (an ERK 1/2 inhibitor) or FGF-2 (an ERK 1/2 activator). The phosphorylation of ERK1/2 during the early stages of adipogenesis in 3T3-L1 cells was inhibited by shikonin. We also confirmed that FGF-2-stimulated ERK 1/2 activity was attenuated by shikonin.

Conclusions: These results demonstrate that shikonin inhibits adipogenic differentiation via suppression of the ERK signaling pathway during the early stages of adipogenesis.

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Inhibitory effect of shikonin on ERK 1/2 phosphorylation in 3T3-L1 adipocytes. (A) Protein level and (B) mRNA expression were determined with ERK 1/2. (C) Time-course of ERK expression as measured by qRT-PCR.
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Figure 3: Inhibitory effect of shikonin on ERK 1/2 phosphorylation in 3T3-L1 adipocytes. (A) Protein level and (B) mRNA expression were determined with ERK 1/2. (C) Time-course of ERK expression as measured by qRT-PCR.

Mentions: Many studies have suggested that MAPKs promote early-stage adipocyte differentiation by activating transcription factors [11,19-21]. The ERK1/2 signaling pathways has been reported to play a critical role for controlling adipogenesis [22]. To elucidate possible mechanisms underlying the inhibition of adipocyte differentiation by shikonin, we further examined whether regulation of ERK 1/2 phosphorylation is associated with the inhibition of adipocyte differentiation by shikonin (Figure 3A). Interestingly, shikonin markedly decreased the phosphorylation of ERK 1/2 in a dose-dependent manner. Additionally, mRNA expression of ERK 1/2 was inhibited by shikonin (Figure 3B). We also evaluated the effect of shikonin on ERK 1/2 mRNA expression at various time points during adipocyte differentiation. MDI-treated control cell showed significantly elevated ERK 1/2 phosphorylation between day 0 and day 2 compared with shikonin-treated cells. However, shikonin significantly downregulated ERK mRNA levels from day 4 to 6. These results suggest that shikonin inhibited adipocyte differentiation in the early stages.


Shikonin suppresses ERK 1/2 phosphorylation during the early stages of adipocyte differentiation in 3T3-L1 cells.

Gwon SY, Ahn JY, Jung CH, Moon BK, Ha TY - BMC Complement Altern Med (2013)

Inhibitory effect of shikonin on ERK 1/2 phosphorylation in 3T3-L1 adipocytes. (A) Protein level and (B) mRNA expression were determined with ERK 1/2. (C) Time-course of ERK expression as measured by qRT-PCR.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3750626&req=5

Figure 3: Inhibitory effect of shikonin on ERK 1/2 phosphorylation in 3T3-L1 adipocytes. (A) Protein level and (B) mRNA expression were determined with ERK 1/2. (C) Time-course of ERK expression as measured by qRT-PCR.
Mentions: Many studies have suggested that MAPKs promote early-stage adipocyte differentiation by activating transcription factors [11,19-21]. The ERK1/2 signaling pathways has been reported to play a critical role for controlling adipogenesis [22]. To elucidate possible mechanisms underlying the inhibition of adipocyte differentiation by shikonin, we further examined whether regulation of ERK 1/2 phosphorylation is associated with the inhibition of adipocyte differentiation by shikonin (Figure 3A). Interestingly, shikonin markedly decreased the phosphorylation of ERK 1/2 in a dose-dependent manner. Additionally, mRNA expression of ERK 1/2 was inhibited by shikonin (Figure 3B). We also evaluated the effect of shikonin on ERK 1/2 mRNA expression at various time points during adipocyte differentiation. MDI-treated control cell showed significantly elevated ERK 1/2 phosphorylation between day 0 and day 2 compared with shikonin-treated cells. However, shikonin significantly downregulated ERK mRNA levels from day 4 to 6. These results suggest that shikonin inhibited adipocyte differentiation in the early stages.

Bottom Line: To investigate the effects of shikonin on adipocyte differentiation, 3T3-L1 cells were induced to differentiate using 3-isobutyl-1-methylzanthine, dexamethasone, and insulin (MDI) for 8 days in the presence of 0-2 μM shikonin.Shikonin effectively suppressed adipogenesis and downregulated the protein levels of 2 major transcription factors, PPARγ and C/EBPα, as well as the adipocyte specific gene aP2 in a dose-dependent manner. qRT-PCR analysis revealed that shikonin inhibited mRNA expression of adipogenesis-related genes, such as PPARγ, C/EBPα, and aP2.We also confirmed that FGF-2-stimulated ERK 1/2 activity was attenuated by shikonin.

View Article: PubMed Central - HTML - PubMed

Affiliation: Functional Food Technology Research Group, Korea Food Research Institute, 516, Baekhyn-dong, Seongnam-si, Gyeonggi-do 463-746, Republic of Korea.

ABSTRACT

Background: The naphthoquinone pigment, shikonin, is a major component of Lithospermum erythrorhizon and has been shown to have various biological functions, including antimicrobial, anti-inflammatory, and antitumor effects. In this study, we investigated the effect of shikonin on adipocyte differentiation and its mechanism of action in 3T3-L1 cells.

Methods: To investigate the effects of shikonin on adipocyte differentiation, 3T3-L1 cells were induced to differentiate using 3-isobutyl-1-methylzanthine, dexamethasone, and insulin (MDI) for 8 days in the presence of 0-2 μM shikonin. Oil Red O staining was performed to determine the lipid accumulation in 3T3-L1 cells. To elucidate the anti-adipogenic mechanism of shikonin, adipogenic transcription factors, the phosphorylation levels of ERK, and adipogenic gene expression were analyzed by Western blotting and quantitative real-time PCR. To further confirm that shikonin inhibits adipogenic differentiation through downregulation of ERK 1/2 activity, 3T3-L1 cells were treated with shikonin in the presence of FGF-2, an activator, or PD98059, an inhibitor, of the ERK1/2 signaling pathway.

Results: Shikonin effectively suppressed adipogenesis and downregulated the protein levels of 2 major transcription factors, PPARγ and C/EBPα, as well as the adipocyte specific gene aP2 in a dose-dependent manner. qRT-PCR analysis revealed that shikonin inhibited mRNA expression of adipogenesis-related genes, such as PPARγ, C/EBPα, and aP2. Adipocyte differentiation was mediated by ERK 1/2 phosphorylation, which was confirmed by pretreatment with PD98059 (an ERK 1/2 inhibitor) or FGF-2 (an ERK 1/2 activator). The phosphorylation of ERK1/2 during the early stages of adipogenesis in 3T3-L1 cells was inhibited by shikonin. We also confirmed that FGF-2-stimulated ERK 1/2 activity was attenuated by shikonin.

Conclusions: These results demonstrate that shikonin inhibits adipogenic differentiation via suppression of the ERK signaling pathway during the early stages of adipogenesis.

Show MeSH
Related in: MedlinePlus