Intervention of Gastrodin in Type 2 Diabetes Mellitus and Its Mechanism

Abstract

As a severe metabolic disease, type 2 diabetes mellitus (T2DM) has become a serious threat to human health in recent years. Gastrodin, as a primary chemical constituent in Gastrodia elata Blume, has antidiabetic effects. However, the possible mechanisms are unclear. The aim of the present study was to investigate the effects and possible mechanisms of gastrodin on the treatment of T2DM. In vivo, after treatment with gastrodin for 6 weeks, fasting blood glucose levels, blood lipid metabolism, and insulin sensitivity index values were remarkably reduced compared with those of the diabetic control group. The values of aspartate aminotransferase and alanine aminotransferase also showed that gastrodin alleviates liver toxicity caused by diabetes. Moreover, gastrodin relieved pathological damage to the pancreas in T2DM rats. In vitro, gastrodin alleviated insulin resistance by increasing glucose consumption, glucose uptake, and glycogen content in dexamethasone-induced HepG2 cells. The Western blotting results showed that gastrodin upregulated the expression of insulin receptors and ubiquitin-specific protease 4 (USP4) and increased the phosphorylation of GATA binding protein 1 (GATA1) and protein kinase B (AKT) in vivo and in vitro. Furthermore, gastrodin decreased the ubiquitin level of the insulin receptor via UPS4 and increased the binding of GATA1 to the USP4 promoter. Additionally, administration of the phosphatidylinositol 3-kinase (PI3K)/AKT signaling pathway inhibitors MK-2206 and LY294002 abolished the beneficial effects of gastrodin. Our results indicate that gastrodin promotes the phosphorylation of GATA1 via the PI3K/AKT pathway, enhances the transcriptional activity of GATA1, and then increases the expression level of USP4, thereby reducing the ubiquitination and degradation of insulin receptors and ultimately improving insulin resistance. Our study provides scientific evidence for the beneficial actions and underlying mechanism of gastrodin in the treatment of T2DM.

Keywords: AKT pathway; USP4; gastrodin; insulin resistance; type 2 diabetes.

FIGURE 1

The effects of Gastrodin on body weight, OGTT and ITT in T2DM rats. Body weight (A), oral glucose tolerance test curve of each group (B) and insulin tolerance test curve of each group of each group (C). The results were presented as mean ± SD (n = 6). **p < 0.01, vs. NC group, #p < 0.05, ##p < 0.01, vs. DC group.

FIGURE 2

The effects of Gastrodin on blood glucose levels, FINS and insulin sensitivity index in T2DM rats. Fasting blood glucose level (A), Serum insulin concentration (B) and insulin sensitivity index (ISI) of each group (C). The results were presented as mean ± SD (n = 6). **p < 0.01, vs. NC group, #p < 0.05, ##p < 0.01, vs. DC group.

FIGURE 3

The effects of Gastrodin on serum lipid profiles. Effects of Gastrodin on the TG (A), TC (B), LDL cholesterol (C), HDLcholesterol (D), ALT and AST levels in T2DM rats. The results were presented as mean ± SD (n = 6). **p < 0.01, vs. NC group, #p < 0.05, ##p < 0.01, vs. DC group.

FIGURE 4

HE staining of the pancreas in rat. (n = 6).

FIGURE 5

Glucagon and insulin staining of the pancreas tissues in rat. The results were presented as mean ± SD (n = 6). **p < 0.01, vs. NC group, #p < 0.05, ##p < 0.01, vs. DC group.

FIGURE 6

The phosphorylation levels of AKT, GATA1 and the levels of USP4, insulin receptor expressions in liver from different groups were detected by western blot assay, and representative bands were shown in (A). The phosphorylation levels of GATA1 (C), AKT (D) and the levels of USP4 (B), insulin receptor (E) were normalized to NC. The results were presented as mean ± SD (n = 3). **p < 0.01, vs. NC group, ##p < 0.01, vs. DC group.

FIGURE 7

Inhibitory effect of Gastrodin in HepG2 cells. Results were expressed as mean ± SD (n = 3). *p < 0.05, **p < 0.01 vs Control group.

FIGURE 8

Effects of Gastrodin on Glucose uptake assay and Glycogen content assay in HepG2 cells. The fluorescence intensity of 2-NBDG into HepG2 cells (A). The glycogen content in HepG2 cells (B). The results were presented as mean ± SD (n = 3). **p < 0.01, vs. insulin group, #p < 0.05, ##p < 0.01, vs. Insulin + Dex group.

FIGURE 9

The levels of insulin receptor expressions and mRNA expressions of insulin receptor in HepG2 from different groups. The representative bands were shown in (A). The levels of insulin receptor (B) were normalized to control. The mRNA levels of insulin receptor (C) was normalized to control. The results were presented as mean ± SD (n = 3). **p < 0.01, vs. control group, ##p < 0.01, vs. Model group.

FIGURE 10

The ubiquitin level of insulin receptor and levels of MARCH1, USP4 and insulin receptor were measured with Co-IP (n = 3). MG132 was a proteasome inhibitor. The concentration of gastrodin was 100 μM. *p < 0.05, **p < 0.01, vs. control+MG132 group, ##p < 0.01, vs. Dex+MG132 group.

FIGURE 11

The levels of USP4 expressions and mRNA expressions of USP4 in HepG2 from different groups. The representative bands were shown in (A). The levels of USP4 (B) were normalized to Dex. The mRNA levels of USP4 (C) was normalized to Dex. The concentration of gastrodin was 100 μM. The results were presented as mean ± SD (n = 3). **p < 0.01, vs. Control group, ##p < 0.01, vs. Dex group.

FIGURE 12

Chromatin immunoprecipitation (ChIP) assay on the promoter of GATA1. The results were presented as mean ± SD (n = 3). **p < 0.01, vs. Control, ##p < 0.01, vs. Dex. The concentration of gastrodin was 100 μM.

FIGURE 13

The phosphorylation levels of AKT, GATA1 in HepG2 from different groups were detected by western blot assay, and representative bands were shown in (A). The phosphorylation levels of GATA1 (B) and AKT (C) were normalized to Dex. The results were presented as mean ± SD (n = 3). **p < 0.01, vs. Control, ##p < 0.01, vs. Dex.

FIGURE 14

The phosphorylation levels of GATA1 and the levels of USP4 expressions in HepG2 from different groups were detected by western blot assay, and representative bands were shown in (A). The phosphorylation levels of GATA1 (C), the levels of USP4 (B) and the levels of insulin receptor (D) were normalized to Dex. The results were presented as mean ± SD (n = 3). **p < 0.01, vs. Control, ##p < 0.01, vs. Dex, ^ap < 0.05, ^aap < 0.01, vs. Dex + Gastrodin group.