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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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Effects of the ERK inhibitor and activator on the inhibition of adipocyte differentiation by shikonin. (A) The protein level and mRNA expression were determined with ERK 1/2. Cells were treated with (A) Shikonin (1 μM) and the ERK inhibitor PD98059 (10 μM) during adipocyte differentiation. After 8 days, the cells were stained with Oil Red O to determine the degree of lipid accumulation. The inhibitory effects of shikonin and PD98059 on ERK 1/2 signaling pathway were observed during adipocyte differentiation. (B) Western blot analysis performed with the ERK pathway and adipogenic marker genes.
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Figure 4: Effects of the ERK inhibitor and activator on the inhibition of adipocyte differentiation by shikonin. (A) The protein level and mRNA expression were determined with ERK 1/2. Cells were treated with (A) Shikonin (1 μM) and the ERK inhibitor PD98059 (10 μM) during adipocyte differentiation. After 8 days, the cells were stained with Oil Red O to determine the degree of lipid accumulation. The inhibitory effects of shikonin and PD98059 on ERK 1/2 signaling pathway were observed during adipocyte differentiation. (B) Western blot analysis performed with the ERK pathway and adipogenic marker genes.

Mentions: ERK has been reported to promote differentiation in the early stages of adipogenesis in 3T3-L1 cells [9,25]. To confirm whether shikonin inhibits ERK phosphorylation in the early stages of adipogenesis, we examined time-course response of ERK 1/2 phosphorylation during the early differentiation period. Cells were pretreated with PD98059 (10 μM) or FGF-2 (1 nM) for 30 min prior to incubation with MDI in the presence or absence of 1 μM shikonin. ERK phosphorylation was determined at 5, 15, 30 min and 1 h after MDI treatment by Western blotting. As shown in Figure 4A, phosphorylation of ERK 1/2 was rapidly induced at 5 min after MDI treatment and maintained. FGF-2 also showed effects similar to MDI. ERK 1/2 phosphorylation was observed at 15 min after pretreatment of PD98059 and ceased after 1 h. ERK 1/2 phosphorylation was completely inhibited 30 min after treatment with shikonin. These results showed that shikonin suppressed at the early stage of adipogenesis after MDI treatment. To confirm the recovery effect of FGF-2 on ERK 1/2 phosphorylation inhibited by shikonin, cells were pretreated with 2 μM shikonin for 10 min and then various concentrations of FGF-2 for 30 min. As shown in Figure 4B, shikonin-mediated inhibition of ERK 1/2 phosphorylation was increased in a dose-dependent manner by FGF-2. These results showed that shikonin has an acute, direct effect on the ERK 1/2 signaling pathway through inhibition of ERK 1/2 phosphorylation.


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)

Effects of the ERK inhibitor and activator on the inhibition of adipocyte differentiation by shikonin. (A) The protein level and mRNA expression were determined with ERK 1/2. Cells were treated with (A) Shikonin (1 μM) and the ERK inhibitor PD98059 (10 μM) during adipocyte differentiation. After 8 days, the cells were stained with Oil Red O to determine the degree of lipid accumulation. The inhibitory effects of shikonin and PD98059 on ERK 1/2 signaling pathway were observed during adipocyte differentiation. (B) Western blot analysis performed with the ERK pathway and adipogenic marker genes.
© Copyright Policy - open-access
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Figure 4: Effects of the ERK inhibitor and activator on the inhibition of adipocyte differentiation by shikonin. (A) The protein level and mRNA expression were determined with ERK 1/2. Cells were treated with (A) Shikonin (1 μM) and the ERK inhibitor PD98059 (10 μM) during adipocyte differentiation. After 8 days, the cells were stained with Oil Red O to determine the degree of lipid accumulation. The inhibitory effects of shikonin and PD98059 on ERK 1/2 signaling pathway were observed during adipocyte differentiation. (B) Western blot analysis performed with the ERK pathway and adipogenic marker genes.
Mentions: ERK has been reported to promote differentiation in the early stages of adipogenesis in 3T3-L1 cells [9,25]. To confirm whether shikonin inhibits ERK phosphorylation in the early stages of adipogenesis, we examined time-course response of ERK 1/2 phosphorylation during the early differentiation period. Cells were pretreated with PD98059 (10 μM) or FGF-2 (1 nM) for 30 min prior to incubation with MDI in the presence or absence of 1 μM shikonin. ERK phosphorylation was determined at 5, 15, 30 min and 1 h after MDI treatment by Western blotting. As shown in Figure 4A, phosphorylation of ERK 1/2 was rapidly induced at 5 min after MDI treatment and maintained. FGF-2 also showed effects similar to MDI. ERK 1/2 phosphorylation was observed at 15 min after pretreatment of PD98059 and ceased after 1 h. ERK 1/2 phosphorylation was completely inhibited 30 min after treatment with shikonin. These results showed that shikonin suppressed at the early stage of adipogenesis after MDI treatment. To confirm the recovery effect of FGF-2 on ERK 1/2 phosphorylation inhibited by shikonin, cells were pretreated with 2 μM shikonin for 10 min and then various concentrations of FGF-2 for 30 min. As shown in Figure 4B, shikonin-mediated inhibition of ERK 1/2 phosphorylation was increased in a dose-dependent manner by FGF-2. These results showed that shikonin has an acute, direct effect on the ERK 1/2 signaling pathway through inhibition of ERK 1/2 phosphorylation.

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