Geniposide Regulates Insulin Secretion And It's Molecular Mechanisms In INS-1 Cells

Type 2 diabetes mellitus (T2DM) is a metabolic disorder that is characterized by high blood glucose in the context of insulin resistance and relative insulin deficiency caused by decrease of pancreaticβcells mass or the increase ofβcells apoptosis. Current drugs have limited efficacy and do not address the most important problems, the decliningβcells function and the associated obesity.GLP-1 is an incretin, which is released in response to carbohydrates and fat intake and stimulates glucose-stimulated insulin secretion (GSIS). Increasing studies have demonstrated the benefits of GLP-1 on pancreaticβcell mass and function. As GLP-1 exerts its beneficial effects through binding and activating its receptor, GLP-1R is a valuable target for the treatment of diabetes. However, GLP-1 has an extremely short half-life due to the rapid degradation by dipeptidyl peptidase-IV (DPP-IV), which limits the therapeutic value of exogenous GLP-1. It is, therefore, important to develop some small molecules, which are able to activate GLP-1R.As we reported in the past that, geniposide, a natural compound from Gardenia jasminoides, was a novel agonist for GLP-1 receptor. Geniposide showed neurotrophic and neuroprotective characteristics via the activation of GLP-1R. In streptozocin-induced diabetic rat models, we have further found that geniposide lowered blood glucose levels, improved insulin secretion and insulin resistance. All the results suggest that geniposide might be a promising lead compound for the treatment of diabetes. However, the molecular mechanism of geniposide regulating pancreaticβcells keeps unknown. The aim of this study is to investigate the effect of geniposide on glucose-stimulating insulin secretion and its signal pathway in pancreaticβcell line INS-1 cells.Firstly, we explored the effect of geniposide on insulin secretion in INS-1 cells, after the cells were incubated with geniposide in the presence or absence of glucose, the level of insulin was determined with commercial ELISA kits. The results demonstrated that geniposide directly induced insulin secretion in a dose-dependent manner in INS-1 cells. Moreover, accompanied with the increase of glucose uptake and ATP level, geniposide also enhanced GSIS in the presence of low and moderately high concentrations of glucose in INS-1 cells.Secondly, to investigate the role of GLP-1R in geniposide regulating GSIS, we determined the effect of geniposide on insulin secretion in the presence or absence of a GLP-1R antagonist, exendin (9-39) amide. The data demonstrated that pretreatment with 200 nM exendin (9-39) for 1 hour prevented the increase of insulin secretion by geniposide in INS-1 cells. At the same time, we also checked the effect of GLP-1R interfere on geniposide-inducing insulin secretion in INS-1 cells, the results showed that, in GLP-1R interfered INS-1 cells, the level of insulin secretion induced by 5.5 mM glucose did not differ from the control cells, hinted that glucose-stimulated insulin secretion was no relative to GLP-1R. However, not as in normal cultured INS-1 cells, geniposide failed to enhance glucose stimulated insulin secretion in GLP-1R interfered INS-1 cells. Together with the effect of exendin (9-39) on geniposide regulating glucose stimulated insulin secretion, it is suggestive that GLP-1R plays an important role in the regulation of geniposide on insulin secretion in INS-1 cells.And then, we tried to probe the possible signal pathway of geniposide- regulating GSIS in INS-1 cells. The stimulatory effect of geniposide on insulin secretion was further mechanistically addressed by application of specific inhibitors of PI3K and EGFR. After pretreatment with 10μM LY294002 (a specific inhibitor for PI3K) or 500 nM AG1478 (a specific inhibitor of EGFR) for 1 hour, the insulin secretion induced by geniposide was partly abolished in INS-1 cells. These results implicate that PI3K and EGFR signal are involved in geniposide-inducing insulin secretion in INS-1 cells. In addition, we determined the effects of geniposide on the phosphorylation of Akt473 and Akt308. The results showed that, after treatment with geniposide, the phsophorylation of Akt308 and Akt473 increased gradually to the maximum at 30 minutes or 120 minutes respectively. Furthermore, geniposide also enhanced the phosphorylation of PDK1, the key signal factor of PI3K signal pathway, and preincubated with LY294002, a selective inhibitor of PI3K, inhibited the phosphorylation of PDK1, suggesting that geniposide regulating GSIS might be involved in its influence on the phosphorylation of PDK1 and Akt.FoxO1 transcription factors emerges as a key factor for the maintenance of a functional pancreasβcells and represents an extraordinary element in the development of therapeutic approaches to treat diabetes. At present, the influence of geniposide on the phosphorylation of FoxO1 was explored in INS-1 cells. The results demonstrated that geniposide enhanced the phosphorylation of FoxO1, with a maximal effect observed at 60 minutes. And, the phosphorylation of FoxO1 induced by geniposide was partially blocked by pretreatment with the inhibitors of PI3K (LY294002, 10μM) and EGFR (AG1478, 500 nM) in INS-1 cells. By contrast, geniposide inhibited GSK3βphosphorylation, and this effect was counteracted by pre-incubation of INS-1 cells with LY294002, suggesting that geniposide might regulate FoxO1 and GSK3βphosphorylation in INS-1 cells via PI3K or EGFR signaling pathway.The homeodomain transcription factor PDX1 is also known as insulin promoter factor 1, which is essential for pancreas development,βcell maturation, insulin production. Finally, the results showed that geniposide activated PDX1 and accelerated the translocation of PDX1 from the cytoplasm to the nucleus to enhance the expression of GLUT2. And this effect was prevented by a pre-incubation with PI3K inhibitor (LY294002) or EGFR inhibitor (AG1478). These results suggested that geniposide might regulate PDX1 translocation in INS-1 cells via PI3K or EGFR signaling pathway, enhance the binding of PDX-1 with DNA, accelerate the expression of regulating insulin factors, and induce the production of insulin.It is concluded that, with the activation of GLP-1R, geniposide activate PI3K/Akt signaling pathway directly or through inducing EGFR transactivation, to inhibit the activity of FoxO1 through phosphorylation-dependent nuclear exclusion and induce the translocation of PDX1 protein from cytoplasm to nucleus in INS-1 cells, which ultimately contribute to regulate glucose-stimulating insulin secretion in INS-1 cells.