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. 2018 Dec 19;23(12):3382.
doi: 10.3390/molecules23123382.

Theaflavins Improve Insulin Sensitivity through Regulating Mitochondrial Biosynthesis in Palmitic Acid-Induced HepG2 Cells

Tuantuan Tong  1   2 , Ning Ren  3   4 , Park Soomi  5   6 , Jiafan Wu  7   8 , Na Guo  9   10 , Hyunuk Kang  11   12 , Eunhye Kim  13   14 , Yuanyuan Wu  15   16 , Puming He  17   18 , Youying Tu  19   20 , Bo Li  21   22
Affiliations

Theaflavins Improve Insulin Sensitivity through Regulating Mitochondrial Biosynthesis in Palmitic Acid-Induced HepG2 Cells

Tuantuan Tong et al. Molecules. .

Abstract

Theaflavins, the characteristic and bioactive polyphenols in black tea, possess the potential improving effects on insulin resistance-associated metabolic abnormalities, including obesity and type 2 diabetes mellitus. However, the related molecular mechanisms are still unclear. In this research, we investigated the protective effects of theaflavins against insulin resistance in HepG2 cells induced by palmitic acid. Theaflavins significantly increased glucose uptake of insulin-resistant cells at noncytotoxic doses. This activity was mediated by upregulating the total and membrane bound glucose transporter 4 protein expressions, increasing the phosphor-Akt (Ser473) level, and decreasing the phosphorylation of IRS-1 at Ser307. Moreover, theaflavins were found to enhance the mitochondrial DNA copy number, down-regulate the PGC-1β mRNA level and increase the PRC mRNA expression. Mdivi-1, a selective mitochondrial division inhibitor, could attenuate TFs-induced promotion of glucose uptake in insulin-resistant HepG2 cells. Taken together, these results suggested that theaflavins could improve hepatocellular insulin resistance induced by free fatty acids, at least partly through promoting mitochondrial biogenesis. Theaflavins are promising functional food ingredients and medicines for improving insulin resistance-related disorders.

Keywords: hepatocyte; insulin resistance; insulin signaling pathway; mitochondrial biogenesis; peroxisome proliferator-activated receptor coactivator-1 (PGC-1); theaflavins.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
HPLC chromatogram of theaflavins (TFs). 1, Theaflavin (TF): R1=R2=H; 2, Theaflavin-3-gallate (TF-3-G): R1=H, R2=galloyl; 3, Theaflavin-3′-gallate (TF-3′-G): R1=galloyl, R2=H; 4. Theaflavins-3, 3′-digallate (TFDG): R1=R2=galloyl.
Figure 2
Figure 2
Effect of theaflavins (TFs) on HepG2 cell growth at 24 h. Cell viability was determined by MTT assay. Data are represented as means ± SD from five replicates. Significant differences between different treatments are showed by different letters (p < 0.05).
Figure 3
Figure 3
Palmitic acid (PA) induces IR in HepG2 cells. (A) Effect of PA on HepG2 cell growth at 24 h. Cell viability was determined by MTT assay. (B) PA reduces 2-NBDG uptake of HepG2 cells with or without insulin (500 nM). Data are represented as means ± SD from five replicates. Significant differences between different treatments are showed by different letters (p < 0.05).
Figure 4
Figure 4
Effects of theaflavins (TFs) on 2-NBDG uptake of insulin-resistant HepG2 cells induced by PA. Metformin is used as a positive control. Data are represented as means ± SD from five replicates. Significant differences between different treatments are showed by different letters (p < 0.05).
Figure 5
Figure 5
Effects of theaflavins (TFs, 2.5–10 µg/mL) on insulin signaling pathway in insulin-resistant HepG2 cells induced by PA after 24h treatment. (A) Protein expression of total GLUT4 and membrane bound GLUT4. (B) Protein expressions of Akt and phosphor-Akt (Ser473). (C) Protein expressions of IRS-1 and phosphor-IRS-1 (Ser307). The protein levels were determined by western blot assay and quantified by Image J. Metformin (5 µg/mL) was used as a positive control. Data were represented as means ± SD from three replicates. Significant differences between different treatments were showed by different letters (p < 0.05).
Figure 5
Figure 5
Effects of theaflavins (TFs, 2.5–10 µg/mL) on insulin signaling pathway in insulin-resistant HepG2 cells induced by PA after 24h treatment. (A) Protein expression of total GLUT4 and membrane bound GLUT4. (B) Protein expressions of Akt and phosphor-Akt (Ser473). (C) Protein expressions of IRS-1 and phosphor-IRS-1 (Ser307). The protein levels were determined by western blot assay and quantified by Image J. Metformin (5 µg/mL) was used as a positive control. Data were represented as means ± SD from three replicates. Significant differences between different treatments were showed by different letters (p < 0.05).
Figure 6
Figure 6
Effect of theaflavins (TFs) on the mtDNA copy number of insulin-resistant HepG2 cells induced by PA after 24 h treatment. Data are represented as means ± SD from three replicates. Significant differences between different treatments are showed by different letters (p < 0.05).
Figure 7
Figure 7
Effects of mdivi-1 on 2-NBDG uptake of insulin-resistant HepG2 cells with theaflavins (TFs) treatment. All six groups were treated with mdivi-1 (10 μM). Metformin was used as a positive control. Data are represented by means ± SD from five replicates. Significant differences between different treatments are showed by different letters (p < 0.05).
Figure 8
Figure 8
Effects of theaflavins (TFs) on the PGC-1β (A) and PRC (B) mRNA expressions of insulin-resistant HepG2 cells induced by PA after 24h treatment. The relative mRNA levels (PGC-1β and PRC) were determined by quantitative real-time PCR assay and calculated by the mean value with the comparative Ct method (ΔΔCt). Data were represented as means ± SD from three replicates. Significant differences between different treatments are showed by different letters (p < 0.05).
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