Screening The Anti-hyperglycemic Agents By An Insulin Resistant Cell Model And Research Of Anti-hyperglycemic Mechanism Of Iridoid Glucosides

Type 2 diabetes mellitus (T2DM) is a multifactorial chronic disease characterized by hyperglycemia that results from insulin resistance (IR.) and defects in insulin secretion. With the improvement of living standards, the incidence of T2DM increased significantly, which worsens the health of the patients and reduces their living quality. Therefore, it is essential to develop new drugs to deal with T2DM.Hepatic glucose metabolism plays a significant role in controlling plasma glucose levels. Early research has shown that the rate of gluconeogenesis is increased after an overnight fasting in T2DM patients, which raises the level of fasting plasma-glucose directly. Therefore, inhibition of hepatic gluconeogenesis has been considered an effective therapeutic strategy for treating hyperglycemia. In fact, both metformin and thiazolidinediones can reduce blood glucose levels by inhibiting hepatic gluconeogenesis. In this research, three different hepatic cells including human liver carcinoma cell line HepG2, human liver cell line L02, and primary mouse hepatocytes, were selected to construct the insulin resistant (IR) model induced by treatment with high concentration of insulin. Afterwards, the glucose consumptions of the cells were measured to screen the anti-hyperglycemic agents. By comparison of the three types of cells, the L02 cell was finally selected to be the screening model, and 16 natural compounds including geniposide, loganin, aucubin, gentiopicroside, oloeside dimethly ester, ligstroside, nuzhenide, salidroside, nuzhenide G13, ECG, EGC, GCG, TF, TF-3-G, TF,-3'-G, and TF-3,3'-diG were tested by the IR L02 cell model. The results show that the two iridoid glucosides, namely geniposide and loganin, and one seco-iridoid glucoside, namely gentiopicroside, display excellent anti-hyperglycemic effects. Meanwhile, the compounds salidroside and aucubin also indicate fairly good effect. The other compounds such as ligstroside, oloeside dimethly ester, ECG, EGC, GCG, TF, TF-3-G, TF,-3'-G, and TF-3,3'-diG only represent weaker effects. The compounds nuzhenide and nuzhenide G13, however, exhibit no anti-hyperglycemic effect at all.Geniposide and gentiopicroside were then chosen for the further mechanism research. We found geniposide and gentiopicroside suppress gluconeogenesis by down regulating the transcription and expression of PEPCK and G6Pase, which are the key emzymes in the gluconeogenesis. Furthermore, luciferase reporter assay and immunofluorescence assay were performed to investigate the inhibition of transcription factor FOXO1 by geniposide and gentiopicroside. After that we demonstrated geniposide and gentiopicroside were able to activate the phosphorylation of both AKT and AMPK, which sequentially inhibit the transcriptional activity of FOXO1.In addition, we found that geniposide and gentiopicroside could attenuate the hepatocyte injury induced by H2O2.100?M geniposide/gentiopicroside could partially reverse the apoptosis induced by H2O2, and 100 ?M gentiopicroside could significantly reverse the S-phase arrest induced by H2O2. The results indicate geniposide and gentiopicroside could attenuate the the hepatocyte injury induced by oxidative stress, and protect liver from the IR induced by oxidative stress.Our research would provide an explanation for the anti-diabetic effect of G. jasminoides, G. macrophylla, and G. scabra Bunge, and the potential use of geniposide and gentiopicroside as anti-diabetic agents merits further in-depth exploration.