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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.


Expression of GLUT4 in baicalein- or Akt inhibitor-treated 3T3-L1 cells.A, Expression of the recombinant mouse GLUT4 protein in 3T3-L1 cells. 3T3-L1 cells were transfected with GLUT4 (pcDNA-mGLUT4) or the empty (pcDNA) vector. GLUT4 levels were detected by Western blot analysis using cell extracts (15 μg/lane). B, Intracellular triglyceride level in GLUT4-transfected cells. 3T3-L1 cells were transfected with GLUT4 vector by electroporation. After 24 h, 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<0.05, **p<0.01, as indicated by the brackets. C, Change in glucose uptake in GLUT4-transfected cells. 3T3-L1 cells were cultured as described in the legend of Fig 9B. Cells were then incubated with fluorescent 2-NBDG, and then observed under a fluorescence microscope. The results are representative from three experiments. D, Measurement of glucose uptake in GLUT4-transfected cells. 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.g009: Expression of GLUT4 in baicalein- or Akt inhibitor-treated 3T3-L1 cells.A, Expression of the recombinant mouse GLUT4 protein in 3T3-L1 cells. 3T3-L1 cells were transfected with GLUT4 (pcDNA-mGLUT4) or the empty (pcDNA) vector. GLUT4 levels were detected by Western blot analysis using cell extracts (15 μg/lane). B, Intracellular triglyceride level in GLUT4-transfected cells. 3T3-L1 cells were transfected with GLUT4 vector by electroporation. After 24 h, 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<0.05, **p<0.01, as indicated by the brackets. C, Change in glucose uptake in GLUT4-transfected cells. 3T3-L1 cells were cultured as described in the legend of Fig 9B. Cells were then incubated with fluorescent 2-NBDG, and then observed under a fluorescence microscope. The results are representative from three experiments. D, Measurement of glucose uptake in GLUT4-transfected cells. 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 obtain the further evidence that baicalein or Akt inhibitor suppress the very early stage of adipogenesis by decreased intracellular triglyceride level through reduced Akt-C/EBPα-GLUT4-mediated glucose uptake in adipocytes, we performed the rescue study by expressing GLUT4 (Fig 9A). 3T3-L1 cells were transfected with the GLUT4 expression vector. After that, the cells were caused to differentiate into adipocytes in medium with MDI and baicalein or Akt inhibitor for 1.5 h after the initiation of adipogenesis. Baicalein- or Akt inhibitor-mediated reduction of intracellular triglyceride levels were mostly cleared by the expression of GLUT4 (Fig 9B). Moreover, the suppression of glucose uptake into the cells by either treatment was negated by expressing GLUT4 (Fig 9C and 9D). These results, taken together, indicate that suppression of the very early stage of adipogenesis by baicalein or Akt inhibitor occurred by decreased intracellular triglyceride levels through reduced Akt-C/EBPα-GLUT4-mediated glucose uptake in adipocytes.


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
Expression of GLUT4 in baicalein- or Akt inhibitor-treated 3T3-L1 cells.A, Expression of the recombinant mouse GLUT4 protein in 3T3-L1 cells. 3T3-L1 cells were transfected with GLUT4 (pcDNA-mGLUT4) or the empty (pcDNA) vector. GLUT4 levels were detected by Western blot analysis using cell extracts (15 μg/lane). B, Intracellular triglyceride level in GLUT4-transfected cells. 3T3-L1 cells were transfected with GLUT4 vector by electroporation. After 24 h, 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<0.05, **p<0.01, as indicated by the brackets. C, Change in glucose uptake in GLUT4-transfected cells. 3T3-L1 cells were cultured as described in the legend of Fig 9B. Cells were then incubated with fluorescent 2-NBDG, and then observed under a fluorescence microscope. The results are representative from three experiments. D, Measurement of glucose uptake in GLUT4-transfected cells. 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.g009: Expression of GLUT4 in baicalein- or Akt inhibitor-treated 3T3-L1 cells.A, Expression of the recombinant mouse GLUT4 protein in 3T3-L1 cells. 3T3-L1 cells were transfected with GLUT4 (pcDNA-mGLUT4) or the empty (pcDNA) vector. GLUT4 levels were detected by Western blot analysis using cell extracts (15 μg/lane). B, Intracellular triglyceride level in GLUT4-transfected cells. 3T3-L1 cells were transfected with GLUT4 vector by electroporation. After 24 h, 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<0.05, **p<0.01, as indicated by the brackets. C, Change in glucose uptake in GLUT4-transfected cells. 3T3-L1 cells were cultured as described in the legend of Fig 9B. Cells were then incubated with fluorescent 2-NBDG, and then observed under a fluorescence microscope. The results are representative from three experiments. D, Measurement of glucose uptake in GLUT4-transfected cells. 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 obtain the further evidence that baicalein or Akt inhibitor suppress the very early stage of adipogenesis by decreased intracellular triglyceride level through reduced Akt-C/EBPα-GLUT4-mediated glucose uptake in adipocytes, we performed the rescue study by expressing GLUT4 (Fig 9A). 3T3-L1 cells were transfected with the GLUT4 expression vector. After that, the cells were caused to differentiate into adipocytes in medium with MDI and baicalein or Akt inhibitor for 1.5 h after the initiation of adipogenesis. Baicalein- or Akt inhibitor-mediated reduction of intracellular triglyceride levels were mostly cleared by the expression of GLUT4 (Fig 9B). Moreover, the suppression of glucose uptake into the cells by either treatment was negated by expressing GLUT4 (Fig 9C and 9D). These results, taken together, indicate that suppression of the very early stage of adipogenesis by baicalein or Akt inhibitor occurred by decreased intracellular triglyceride levels through reduced Akt-C/EBPα-GLUT4-mediated glucose uptake in 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&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.