Carpachromene Ameliorates Insulin Resistance in HepG2 Cells via Modulating IR/IRS1/PI3k/Akt/GSK3/FoxO1 Pathway

Abstract

Insulin resistance contributes to several disorders including type 2 diabetes and cardiovascular diseases. Carpachromene is a natural active compound that inhibits α-glucosidase enzyme. The aim of the present study is to investigate the potential activity of carpachromene on glucose consumption, metabolism and insulin signalling in a HepG2 cells insulin resistant model. A HepG2 insulin resistant cell model (HepG2/IRM) was established. Cell viability assay of HepG2/IRM cells was performed after carpachromene/metformin treatment. Glucose concentration and glycogen content were determined. Western blot analysis of insulin receptor, IRS1, IRS2, PI3k, Akt, GSK3, FoxO1 proteins after carpachromene treatment was performed. Phosphoenolpyruvate carboxykinase (PEPCK) and hexokinase (HK) enzymes activity was also estimated. Viability of HepG2/IRM cells was over 90% after carpachromene treatment at concentrations 6.3, 10, and 20 µg/mL. Treatment of HepG2/IRM cells with carpachromene decreased glucose concentration in a concentration- and time-dependant manner. In addition, carpachromene increased glycogen content of HepG2/IRM cells. Moreover, carpachromene treatment of HepG2/IRM cells significantly increased the expression of phosphorylated/total ratios of IR, IRS1, PI3K, Akt, GSK3, and FoxO1 proteins. Furthermore, PEPCK enzyme activity was significantly decreased, and HK enzyme activity was significantly increased after carpachromene treatment. The present study examined, for the first time, the potential antidiabetic activity of carpachromene on a biochemical and molecular basis. It increased the expression ratio of insulin receptor and IRS1 which further phosphorylated/activated PI3K/Akt pathway and phosphorylated/inhibited GSK3 and FoxO1 proteins. Our findings revealed that carpachromene showed central molecular regulation of glucose metabolism and insulin signalling via IR/IRS1/ PI3K/Akt/GSK3/FoxO1 pathway.

Keywords: HepG2 cells; PI3K/Akt/GSK3/FoxO1 pathway; carpachromene; insulin receptor; insulin resistance.

Figure 1

Establishment of insulin resistant model on HepG2 cells through examining the effect of different concentrations of insulin (0.005, 0.05, 0.5, 5, 50 µM) on glucose consumption after 12, 24, 36, 48 h, when compared to control non-insulin treated HepG2 cells. Bars represent mean ± SD.

Figure 2

Cell viability after treatment with different concentrations (0.4, 1.6, 6.3, 10, 20, 25, 100 µg/mL) of carpachromene or metformin on HepG2/IRM cells for 48 h. Bars represent mean ± SD. Significant difference was analyzed by two-way ANOVA test followed by post hoc Dunnett test, where ** p < 0.01, *** p < 0.001, compared to untreated HepG2/IRM cells.

Figure 3

Glucose concentration in the media after treatment of HepG2/IRM cells with carpachromene or metformin at the concentrations of 5, 10, and 20 µg/mL for 12, 24, 36, and 48 h. Bars represent mean ± SD. Significant difference was analyzed by two-way ANOVA test followed by post hoc Dunnett test, where # p < 0.001, compared to untreated HepG2/IRM cells.

Figure 4

Glycogen content before and after treatment of HepG2/IRM cells with carpachromene or metformin (20 µg/mL), where untreated HepG2/IRM cells were considered 100%. Bars represent mean ± SD. Significant difference was analyzed by one-way ANOVA test followed by post hoc Dunnett test, where ** p < 0.01, *** p < 0.001, compared to control untreated HepG2/IRM cells.

Figure 5

Effect of Carpachromene on the expression of phosphorylated and total proteins of IR, IRS1, IRS2, PI3K, Akt, GSK3, and FoxO1 proteins in HepG2/IRM cells. (A) Representative Western blots of phosphorylated and total proteins of IR, IRS1, IRS2, PI3K, Akt, GSK3, and FoxO1 in HepG2/IRM cells before (left lane) and after (right lane) treatment with 20 µg/mL carpachromene. β-actin was used as internal loading control. (B) Phosphorylated/total protein expression ratio in HepG2/IRM cells relative to untreated HepG2/IRM cells, after normalization to the corresponding β-actin protein expression. Bars represent mean ± SD. Significant difference was analyzed by student t test, where ** p < 0.01, compared to untreated HepG2/IRM cells.

Figure 6

Effect of carpachromene (20 µg/mL) on the activity of (A) PEPCK and (B) HK enzymes on HepG2/IRM cells. Metformin was used as positive control. Bars represent mean ± SD (n= 3), significant difference was analyzed by one-way ANOVA followed by Dunnett test, *** p < 0.001, when compared to untreated HepG/IRM cells.

Figure 7

Chemical structure of carpachromene extracted from the ethyl acetate fraction of fresh leaves of Ficus benghalensis.