Berberine reduces endoplasmic reticulum stress and improves insulin signal transduction in Hep G2 cells

Abstract

Aim: Endoplasmic reticulum (ER) stress plays an important role in the pathogenesis of insulin resistance and pancreatic beta-cell dysfunction. The aim of this study is to investigate whether the insulin-sensitizing action of berberine is related to reducing ER stress.

Methods: ER stress in cultured Hep G2 cells was induced with tunicamycin. Cells were pretreated with berberine in combination with or without insulin. The concentration of glucose was measured by glucose oxidase method. The molecular markers of ER stress, including ORP150, PERK, and eIF2 alpha were analyzed by Western blot or real time PCR. The activity of JNK was also evaluated. Moreover, the insulin signaling proteins such as IRS-1 and AKT were determined by Western blot.

Results: The production of glucose stimulated with insulin was reduced. The expressions of ORP150 was decreased both in gene and protein levels when cells were pretreated with berberine, while the activation of JNK was blocked. The levels of phosphorylation both on PERK and eIF2 alpha were inhibited in cells pretreated with berberine. The level of IRS-1 ser(307) phosphorylation was decreased, whereas IRS-1 tyr phosphorylation was increased notablely. AKT ser(473) phosphorylation was also enhanced significantly in the presence of berberine.

Conclusion: The antidiabetic effect of berberine in Hep G2 cells maybe related to attenuation of ER stress and improvement of insulin signal transduction.

Figure 1

Dose-dependent cytotoxicity of berberine on Hep G2 cells. Cells were incubated with different concentrations of berberine for 24 h. Data were means±SEM. n=6 in each group. ^cP<0.01 vs Nor.

Figure 2

Effect of berberine on basal and insulin-stimulated glucose production in HepG2 cells. Tests were performed in the absence or presence of insulin (100 nmol/L) after cells were exposed to Tu and pretreated with berberine. Data are means±SEM. n=6 in each condition. ^cP<0.01 vs Nor. ^fP<0.01 vs Tu (0.5 μg/mL). ⁱP<0.01 vs Tu (2.0 μg/mL).

Figure 3

Effect of berberine on the expressions of markers of ER stress in HepG2 cells. Cells were pretreated with or without berberine in the absence or presence of different concentrations of Tu for 4 h. The mRNA expression of ORP150 (A) was analyzed using quantitative real-time RT-PCR whereas the protein level of ORP150 was analyzed with Western blot analysis (B). Total JNK, p-c-Jun, PERK, and eIF2α were examined by Western blot analysis (C, D, and E). Data are means±SEM of three independent experiments in each condition. ^cP<0.01 vs Nor. ^fP<0.01 vs Mod.

Figure 4

Effect of berberine on insulin signaling transduction of HepG2 cells. Cells were pretreated with berberine and exposed to different concentrations of Tu, total proteins were detected by Western blot analysis, cells were stimulated with insulin insulin (100 nmol/L) for 20 min to measure the levels of phosphorylation. Total protein concentration and phosphorylation level of IRS-1 (A) and AKT (B) were shown respectively. Data are means±SEM of three independent experiments in each condition.

Figure 5

The effects of the berberine on ER stress and insulin resistance. The ER stress in the hepatocyte cells was induced with Tu. The activity of JNK was elevated and expression ORP150 were up regulated, at the same time, phosphorylation of PERK and eIF2α were enhanced, serine phosphorylation of IRS-1 was increased while tyrosine phosphorylation of IRS-1 was inhibited, phosphorylation of AKT was decreased too. All these changes lead to an increase in insulin resistance and glucose production was enhanced. Treated with berberine reversed these changes and enhances insulin signaling which leads to a decrease of glucose production in cells stimulated with insulin.