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Suppression of Very Early Stage Of Adipogenesis by Baicalein, a Plant-Derived Flavonoid through Reduced Akt-C/EBP α -GLUT4 Signaling-Mediated Glucose Uptake in 3T3-L1 Adipocytes

View Article: PubMed Central - PubMed

ABSTRACT

Baicalein has been used as a Chinese medicine, and is an abundant plant flavonoid present in fruits and vegetables. Here, we examined the effects of baicalein in adipogenesis and investigated its molecular mechanism in adipocytes. Baicalein lowered the intracellular lipid accumulation and decreased the transcription levels of the adipocyte-specific genes in mouse 3T3-L1 adipocytes. Glucose uptake mediated by glucose transporter 4 (GLUT4) was reduced, causing down-regulation of the intracellular lipid accumulation. These reductions were also observed even when baicalein was added in only early stage of adipogenesis (0–2 days) of 6-day-adipogenesis. Chromatin immunoprecipitation assay showed that baicalein decreased the binding level of C/EBPα protein to the promoter region of the GLUT4 gene. Phosphorylation of Akt at 1 h after the initiation of adipogenesis was inhibited by the treatment with baicalein. Inhibition during only the first 1.5 h after the initiation of adipogenesis by baicalein or an Akt inhibitor was enough to decrease the lipid contents in the cells undergoing adipocyte differentiation for 6 days. These results indicate that baicalein decreased the intracellular lipid accumulation by down-regulation of glucose uptake via repression of Akt-C/EBPα-GLUT4 signaling in the very early stage of adipogenesis of 3T3-L1 adipocytes.

No MeSH data available.


Inhibition of very early stage of adipogenesis in 3T3-L1 cells by baicalein or Akt inhibitor.A, Intracellular triglyceride level. 3T3-L1 cells (undifferentiated cells: U; white column) were differentiated into adipocytes (differentiated cells: D; gray column) for 6 days and treated with baicalein or an Akt inhibitor (Akt Inh.; black columns) for the initial 1.5 h of adipogenesis. Data are presented as the means ± S.D. from three experiments. **p<0.01, as indicated by the brackets. B, Expression levels of adipogenic genes. Cells were cultured as described in the legend of Fig 8A. Data are shown as the means ± S.D. from three experiments. **p<0.01, as indicated by the brackets. C, ChIP assay. 3T3-L1 cells were cultured as described in the legend of Fig 8A. The results are representative from three experiments. Band intensity was measured with MultiGauge software. D, Change in glucose uptake. 3T3-L1 cells were cultured as described in the legend of Fig 8A. Cells were then incubated with fluorescent 2-NBDG, and then observed under a fluorescence microscope. The results are representative from three experiments. E, Quantification of glucose uptake in baicalein- or Akt inhibitor (Akt Inh.)-treated 3T3-L1 cells. The data are presented as the value relative to that of the undifferentiated cells and are shown as the means ± S.D. from three experiments **p<0.01, as indicated by the brackets.
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pone.0163640.g008: Inhibition of very early stage of adipogenesis in 3T3-L1 cells by baicalein or Akt inhibitor.A, Intracellular triglyceride level. 3T3-L1 cells (undifferentiated cells: U; white column) were differentiated into adipocytes (differentiated cells: D; gray column) for 6 days and treated with baicalein or an Akt inhibitor (Akt Inh.; black columns) for the initial 1.5 h of adipogenesis. Data are presented as the means ± S.D. from three experiments. **p<0.01, as indicated by the brackets. B, Expression levels of adipogenic genes. Cells were cultured as described in the legend of Fig 8A. Data are shown as the means ± S.D. from three experiments. **p<0.01, as indicated by the brackets. C, ChIP assay. 3T3-L1 cells were cultured as described in the legend of Fig 8A. The results are representative from three experiments. Band intensity was measured with MultiGauge software. D, Change in glucose uptake. 3T3-L1 cells were cultured as described in the legend of Fig 8A. Cells were then incubated with fluorescent 2-NBDG, and then observed under a fluorescence microscope. The results are representative from three experiments. E, Quantification of glucose uptake in baicalein- or Akt inhibitor (Akt Inh.)-treated 3T3-L1 cells. The data are presented as the value relative to that of the undifferentiated cells and are shown as the means ± S.D. from three experiments **p<0.01, as indicated by the brackets.

Mentions: To further confirm the importance of the suppression of Akt activation in the very early stage of adipogenesis, we treated the cells with baicalein or an Akt inhibitor (Akt Inh.) for 1.5 h after the initiation of adipogenesis during 6-day-adipocyte differentiation process. To completely inhibit Akt activation at around 1 h after the initiation of adipogenesis, we treated the cells with baicalein or an Akt inhibitor for 1.5 h. 3T3-L1 cells were differentiated into adipose cells in medium with MDI and baicalein or Akt inhibitor for 1.5 h after the initiation of adipogenesis, and the cells were continued to differentiate into adipose cells for 6 days in medium with MDI or insulin alone. The triglyceride level in the cells was decreased by the treatment with baicalein or Akt inhibitor for 1.5 h after the initiation of adipogenesis during 6-day-adipocyte differentiation process (Fig 8A). In addition, the expression levels of the PPARγ, C/EBPα, and GLUT4 genes were reduced by either treatment (Fig 8B). Moreover, these treatments reduced the binding of C/EBPα to the GLUT4 gene promoter after 6 days of adipocyte differentiation (Fig 8C). In addition, the treatment of Akt inhibitor did not affect the expression of C/EBPβ and C/EBPδ genes (data not shown). Furthermore, the glucose uptake into the cells were also repressed by either treatment (Fig 8D and 8E).


Suppression of Very Early Stage Of Adipogenesis by Baicalein, a Plant-Derived Flavonoid through Reduced Akt-C/EBP α -GLUT4 Signaling-Mediated Glucose Uptake in 3T3-L1 Adipocytes
Inhibition of very early stage of adipogenesis in 3T3-L1 cells by baicalein or Akt inhibitor.A, Intracellular triglyceride level. 3T3-L1 cells (undifferentiated cells: U; white column) were differentiated into adipocytes (differentiated cells: D; gray column) for 6 days and treated with baicalein or an Akt inhibitor (Akt Inh.; black columns) for the initial 1.5 h of adipogenesis. Data are presented as the means ± S.D. from three experiments. **p<0.01, as indicated by the brackets. B, Expression levels of adipogenic genes. Cells were cultured as described in the legend of Fig 8A. Data are shown as the means ± S.D. from three experiments. **p<0.01, as indicated by the brackets. C, ChIP assay. 3T3-L1 cells were cultured as described in the legend of Fig 8A. The results are representative from three experiments. Band intensity was measured with MultiGauge software. D, Change in glucose uptake. 3T3-L1 cells were cultured as described in the legend of Fig 8A. Cells were then incubated with fluorescent 2-NBDG, and then observed under a fluorescence microscope. The results are representative from three experiments. E, Quantification of glucose uptake in baicalein- or Akt inhibitor (Akt Inh.)-treated 3T3-L1 cells. The data are presented as the value relative to that of the undifferentiated cells and are shown as the means ± S.D. from three experiments **p<0.01, as indicated by the brackets.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC5036867&req=5

pone.0163640.g008: Inhibition of very early stage of adipogenesis in 3T3-L1 cells by baicalein or Akt inhibitor.A, Intracellular triglyceride level. 3T3-L1 cells (undifferentiated cells: U; white column) were differentiated into adipocytes (differentiated cells: D; gray column) for 6 days and treated with baicalein or an Akt inhibitor (Akt Inh.; black columns) for the initial 1.5 h of adipogenesis. Data are presented as the means ± S.D. from three experiments. **p<0.01, as indicated by the brackets. B, Expression levels of adipogenic genes. Cells were cultured as described in the legend of Fig 8A. Data are shown as the means ± S.D. from three experiments. **p<0.01, as indicated by the brackets. C, ChIP assay. 3T3-L1 cells were cultured as described in the legend of Fig 8A. The results are representative from three experiments. Band intensity was measured with MultiGauge software. D, Change in glucose uptake. 3T3-L1 cells were cultured as described in the legend of Fig 8A. Cells were then incubated with fluorescent 2-NBDG, and then observed under a fluorescence microscope. The results are representative from three experiments. E, Quantification of glucose uptake in baicalein- or Akt inhibitor (Akt Inh.)-treated 3T3-L1 cells. The data are presented as the value relative to that of the undifferentiated cells and are shown as the means ± S.D. from three experiments **p<0.01, as indicated by the brackets.
Mentions: To further confirm the importance of the suppression of Akt activation in the very early stage of adipogenesis, we treated the cells with baicalein or an Akt inhibitor (Akt Inh.) for 1.5 h after the initiation of adipogenesis during 6-day-adipocyte differentiation process. To completely inhibit Akt activation at around 1 h after the initiation of adipogenesis, we treated the cells with baicalein or an Akt inhibitor for 1.5 h. 3T3-L1 cells were differentiated into adipose cells in medium with MDI and baicalein or Akt inhibitor for 1.5 h after the initiation of adipogenesis, and the cells were continued to differentiate into adipose cells for 6 days in medium with MDI or insulin alone. The triglyceride level in the cells was decreased by the treatment with baicalein or Akt inhibitor for 1.5 h after the initiation of adipogenesis during 6-day-adipocyte differentiation process (Fig 8A). In addition, the expression levels of the PPARγ, C/EBPα, and GLUT4 genes were reduced by either treatment (Fig 8B). Moreover, these treatments reduced the binding of C/EBPα to the GLUT4 gene promoter after 6 days of adipocyte differentiation (Fig 8C). In addition, the treatment of Akt inhibitor did not affect the expression of C/EBPβ and C/EBPδ genes (data not shown). Furthermore, the glucose uptake into the cells were also repressed by either treatment (Fig 8D and 8E).

View Article: PubMed Central - PubMed

ABSTRACT

Baicalein has been used as a Chinese medicine, and is an abundant plant flavonoid present in fruits and vegetables. Here, we examined the effects of baicalein in adipogenesis and investigated its molecular mechanism in adipocytes. Baicalein lowered the intracellular lipid accumulation and decreased the transcription levels of the adipocyte-specific genes in mouse 3T3-L1 adipocytes. Glucose uptake mediated by glucose transporter 4 (GLUT4) was reduced, causing down-regulation of the intracellular lipid accumulation. These reductions were also observed even when baicalein was added in only early stage of adipogenesis (0&ndash;2 days) of 6-day-adipogenesis. Chromatin immunoprecipitation assay showed that baicalein decreased the binding level of C/EBP&alpha; protein to the promoter region of the GLUT4 gene. Phosphorylation of Akt at 1 h after the initiation of adipogenesis was inhibited by the treatment with baicalein. Inhibition during only the first 1.5 h after the initiation of adipogenesis by baicalein or an Akt inhibitor was enough to decrease the lipid contents in the cells undergoing adipocyte differentiation for 6 days. These results indicate that baicalein decreased the intracellular lipid accumulation by down-regulation of glucose uptake via repression of Akt-C/EBP&alpha;-GLUT4 signaling in the very early stage of adipogenesis of 3T3-L1 adipocytes.

No MeSH data available.