Effect of oxymatrine on liver gluconeogenesis is associated with the regulation of PEPCK and G6Pase expression and AKT phosphorylation

Abstract

An increase in liver gluconeogenesis is an important pathological phenomenon in type 2 diabetes mellitus (T2DM) and oxymatrine is an effective natural drug used for T2DM treatment. The present study aimed to explore the effect of oxymatrine on gluconeogenesis and elucidate the underlying mechanism. Male Sprague-Dawley rats were treated with a high-fat diet and streptozotocin for 4 weeks to induce T2DM, and HepG2 cells were treated with 55 mM glucose to simulate T2DM in vitro. T2DM rats were treated with oxymatrine (10 or 20 mg/kg weight) or metformin for 4 weeks, and HepG2 cells were treated with oxymatrine (0.1 or 1 µM), metformin (0.1 µM), or oxymatrine combined with MK-2206 (AKT inhibitor) for 24 h. Fasting blood glucose and insulin sensitivity of rats were measured to evaluate insulin resistance. Glucose production and uptake ability were measured to evaluate gluconeogenesis in HepG2 cells, and the expression of related genes was detected to explore the molecular mechanism. Additionally, the body weight, liver weight and liver index were measured and hematoxylin and eosin staining was performed to evaluate the effects of the disease. The fasting glucose levels of T2DM rats was 16.5 mmol/l, whereas in the control rats, it was 6.1 mmol/l. Decreased insulin sensitivity (K-value, 0.2), body weight loss (weight, 300 g), liver weight gain, liver index increase (value, 48) and morphological changes were observed in T2DM rats, accompanied by reduced AKT phosphorylation, and upregulated expression of phosphoenolpyruvate carboxykinase (PEPCK) and glucose-6-phosphatase (G6Pase). High-glucose treatment significantly increased glucose production and decreased glucose uptake in HepG2 cells, concomitant with a decrease in AKT phosphorylation and increase of PEPCK and G6Pase expression. In vivo, oxymatrine dose-dependently increased the sensitivity of T2DM rats to insulin, increased AKT phosphorylation and decreased PEPCK and G6Pase expression in the liver, and reversed the liver morphological changes. In vitro, oxymatrine dose-dependently increased AKT phosphorylation and glucose uptake of HepG2 cells subjected to high-glucose treatment, which was accompanied by inhibition of the expression of the gluconeogenesis-related genes, PEPCK and G6Pase. MK-2206 significantly inhibited the protective effects of oxymatrine in high-glucose-treated cells. These data indicated that oxymatrine can effectively prevent insulin resistance and gluconeogenesis, and its mechanism may be at least partly associated with the regulation of PEPCK and G6Pase expression and AKT phosphorylation in the liver.

Keywords: AKT; gluconeogenesis; glucose-6-phosphatase; oxymatrine; phosphoenolpyruvate carboxykinase; type 2 diabetes mellitus.

Figure 1

Schematic diagram of the time course of the experiment. Control group, normotolerant rats; model group, diabetic rats; treatment group, diabetic rats treated with oxymatrine (10 or 20 mg/kg body weight); and metformin group, diabetic rats treated with metformin (250 mg/kg body weight).

Figure 2

Effect of oxymatrine on fasting blood glucose in T2DM rats. The differences in the values between groups were analyzed using a one-way ANOVA. All experiments were repeated 3 times and all data are presented as the mean ± standard deviation. ^***P<0.001 vs. control; ^##P<0.01, ^###P<0.001 vs. T2DM. T2DM, type 2 diabetes mellitus; +oxymatrine (L), type 2 diabetic rats treated with 10 mg/kg body weight oxymatrine; +oxymatrine (H), type 2 diabetic rats treated 20 mg/kg body weight oxymatrine; +Metformin, type 2 diabetic rats treated with 250 mg/kg body weight metformin.

Figure 3

Effect of oxymatrine on insulin sensitivity in T2DM rats. (A) Blood glucose levels and (B) K value in the treated rats. Differences between groups were compared using a one-way ANOVA. All experiments were repeated 3 times and all data are presented as the mean ± standard deviation. ^***P<0.001 vs. control; ^#P<0.05, ^##P<0.01, ^###P<0.001 vs. T2DM. T2DM, type 2 diabetes mellitus; +oxymatrine (L), type 2 diabetic rats treated with 10 mg/kg body weight oxymatrine; +oxymatrine (H) type 2 diabetic rats treated 20 mg/kg body weight oxymatrine; +Metformin, type 2 diabetic rats treated with 250 mg/kg body weight metformin

Figure 4

Effect of oxymatrine on the expression of AKT, PEPCK and G6Pase in the liver of type 2 diabetic rats. (A) mRNA expression levels of PEPCK and (B) G6Pase. (C) Protein expression levels of PEPCK, G6Pase, AKT and pAKT. (D) Ratio of PEPCK to β-actin. (E) Ratio of G6Pase to β-actin. (F) Ratio of pAKT to β-actin. Differences between groups were compared using a one-way ANOVA. All experiments were repeated 3 times and all data are presented as the mean ± standard deviation. ^***P<0.05 vs. control; ^#P<0.05, ^##P<0.01 vs. T2DM. T2DM, type 2 diabetes mellitus; +oxymatrine (L) type 2 diabetic rats treated with 10 mg/kg body weight oxymatrine; +oxymatrine (H) type 2 diabetic rats treated 20 mg/kg body weight oxymatrine; +Metformin, type 2 diabetic rats treated with 250 mg/kg body weight metformin; p, phospho; PEPCK, phosphoenolpyruvate carboxykinase; glucose-6-phosphatase.

Figure 5

Effects of oxymatrine on liver index and liver tissue morphology of the T2DM rats. (A) Body weight, (B) liver weight and (C) liver index of the treated rats. (D) Representative images of HE staining of the liver. Differences between groups were compared using a one-way ANOVA. All experiments were repeated 3 times and all data are presented as the mean ± standard deviation. ^***P<0.001 vs. control; ^#P<0.05, ^##P<0.01, ^###P<0.001 vs. T2DM. T2DM. T2DM, type 2 diabetes mellitus; +oxymatrine (L), type 2 diabetic rats treated with 10 mg/kg body weight oxymatrine; +oxymatrine (H) type 2 diabetic rats treated 20 mg/kg body weight oxymatrine; +Metformin, type 2 diabetic rats treated with 250 mg/kg body weight metformin; HE, hematoxylin and eosin.

Figure 6

Effect of oxymatrine on glucose production and uptake in HepG2 cells. (A) glucose production assay, and (B) 2-NBDG uptake assay in the treated cells. Differences between groups were compared using a one-way ANOVA. All experiments were repeated 3 times and all data are presented as the mean ± standard deviation. ^***P<0.001 vs. control; ^#P<0.05, ^##P<0.01 vs. High Glucose. +oxymatrine (L) cells treated with 0.1 µM oxymatrine; +oxymatrine (H) cells treated with 1 µM oxymatrine; +Metformin, cells treated with 0.1 µM metformin.

Figure 7

Effect of oxymatrine on the expression of AKT, PEPCK and G6Pase in HepG2 cells. (A) mRNA expression levels of PEPCK and (B) G6Pase in the treated cells. (C) Protein expression of PEPCK, G6Pase, AKT and pAKT. (D) Ratio of PEPCK, (E) G6Pase and (F) pAKT to β-actin. Differences between groups were compared using a one-way ANOVA. All experiments were repeated 3 times and all data are presented as the mean ± standard deviation. ^***P<0.001 vs. control; ^#P<0.05, ^##P<0.01 vs. High Glucose. +oxymatrine (L) cells treated with 0.1 µM oxymatrine; +oxymatrine (H) cells treated with 1 µM oxymatrine; +Metformin, cells treated with 0.1 µM metformin; PEPCK, phosphoenolpyruvate carboxykinase; glucose-6-phosphatase; p, phospho.

Figure 8

Effect of inhibition of AKT on the expression of AKT, PEPCK and G6Pase in HepG2 cells. (A) Protein expression of AKT and pAKT. (B) Ratio of pAKT to AKT. mRNA expression levels of (C) PEPCK and (D) G6Pase. (E) Protein expression of PEPCK and G6Pase. Ratio of (F) G6Pase to β-actin and (G) PEPCK to β-actin. Differences between groups were compared using a one-way ANOVA. All experiments were repeated 3 times and all data are presented as the mean ± standard deviation. ^**P<0.01 vs. control; ^#P<0.05 vs. High Glucose; ⁺⁺P<0.01, ⁺⁺⁺P<0.001 vs. +oxymatrine. PEPCK, phosphoenolpyruvate carboxykinase; G6Pase, glucose-6-phosphatase.