Limits...
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.

Show MeSH

Related in: MedlinePlus

Effect of shikonin on the ERK pathway during the early stages of adipogenesis. (A) The time-dependent effect of PD98059 (10 μM), FGF-2 (1 nM), and shikonin (1 μM) on ERK 1/2 activity was analyzed by Western blotting. (B) The cells were treated with shikonin (2 μM) plus different doses of FGF-2. Cells were harvested at 10 min and examined with Western blotting as described in the Figure 2 legend.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC3750626&req=5

Figure 5: Effect of shikonin on the ERK pathway during the early stages of adipogenesis. (A) The time-dependent effect of PD98059 (10 μM), FGF-2 (1 nM), and shikonin (1 μM) on ERK 1/2 activity was analyzed by Western blotting. (B) The cells were treated with shikonin (2 μM) plus different doses of FGF-2. Cells were harvested at 10 min and examined with Western blotting as described in the Figure 2 legend.

Mentions: Shikonin treatment inhibited ERK 1/2 phosphorylation in a time-dependent manner, which suggests that shikonin inhibits adipocyte differentiation by regulating ERK 1/2 phosphorylation in the early stages of adipogenesis (Figure 5A). To further confirm the inhibition of ERK 1/2 phosphorylation by shikonin, we investigated whether shikonin has a direct effect on ERK 1/2 phosphorylation. As expected, FGF-2 treatment inhibited shikonin-induced ERK 1/2 phosphorylation (Figure 5B). Taken together, these findings suggest that shikonin is able to block ERK phosphorylation at an early stage and inhibit the expression of adipogenic transcription factors by modulating the ERK-mediated signaling pathway during adipocyte differentiation. Further in vivo studies are necessary to determine the molecular mechanisms of shikonin-induced ERK 1/2 phosphorylation inhibition.


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)

Effect of shikonin on the ERK pathway during the early stages of adipogenesis. (A) The time-dependent effect of PD98059 (10 μM), FGF-2 (1 nM), and shikonin (1 μM) on ERK 1/2 activity was analyzed by Western blotting. (B) The cells were treated with shikonin (2 μM) plus different doses of FGF-2. Cells were harvested at 10 min and examined with Western blotting as described in the Figure 2 legend.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 5: Effect of shikonin on the ERK pathway during the early stages of adipogenesis. (A) The time-dependent effect of PD98059 (10 μM), FGF-2 (1 nM), and shikonin (1 μM) on ERK 1/2 activity was analyzed by Western blotting. (B) The cells were treated with shikonin (2 μM) plus different doses of FGF-2. Cells were harvested at 10 min and examined with Western blotting as described in the Figure 2 legend.
Mentions: Shikonin treatment inhibited ERK 1/2 phosphorylation in a time-dependent manner, which suggests that shikonin inhibits adipocyte differentiation by regulating ERK 1/2 phosphorylation in the early stages of adipogenesis (Figure 5A). To further confirm the inhibition of ERK 1/2 phosphorylation by shikonin, we investigated whether shikonin has a direct effect on ERK 1/2 phosphorylation. As expected, FGF-2 treatment inhibited shikonin-induced ERK 1/2 phosphorylation (Figure 5B). Taken together, these findings suggest that shikonin is able to block ERK phosphorylation at an early stage and inhibit the expression of adipogenic transcription factors by modulating the ERK-mediated signaling pathway during adipocyte differentiation. Further in vivo studies are necessary to determine the molecular mechanisms of shikonin-induced ERK 1/2 phosphorylation inhibition.

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