Effect Of HMG-CoA Reductase Inhibitors On RaS/Raf/ERK/CREB Signal Transduction Pathway In INS-1Cells

Objective Insulin resistance and pancreatic β cell impaired insulin secretion are the most important pathophysiological mechanisms in type2diabetes. It has been the focus of attention of the medical profession that how to protect islet β cell function and prevent diabetic patients from avoiding further deterioration of the already impaired beta cell function.It has been confirmed that kinds of clinical drugs can affect the pancreatic β cell function, insulin secretion, and further influence glucose metabolism.As a3-hydroxy-3-methy1-glutaryl-coenzyme A (HMG-CoA) reductase inhibitor, atorvastatin is widely used in the treatment and prevention of diabetic patients because of clear lipid-lowering efficacy and tolerability, with the increasingly early time window, the longer application time, and the increasing dose. Non-lipid role of these drugs is also be concerned gradually, including the impact on diabetes. Recently, a number of clinical meta-analysis found that statins could increase the risk of diabetes with the dose-dependence. The mechanisms are unclear and need to be studied further. Results from in vitro and vivo experiments indicated that HMG-CoA reductase inhibitors not only reduce cholesterol levels, but also inhibit glucose-stimulated insulin secretion by inhibiting the activity of the pancreatic P cell ATP sensitive K+channel, voltage compliance calcium channel and inhibit insulin mRNA expression by inhibiting insulin promoter activity and ameliorate insulin resistance at the same time. However, the biological effect of HMG-CoA reductase inhibitors on insulin promoter in regulating the insulin synthesis is not clearly defined. The mechanism needs further evaluation in future prospective trials.In the cholesterol synthetic pathway, the inhibition of HMG-CoA reductase prevents the conversion of HMG-CoA to mevalonate, limiting the synthesis of cholesterol and its upstream intermediates such as isoprenoids, farnesyl, geranygeranyl pyrophosphate (FPP) and geranylgeranyl pyrophosphate(GGPP). Notably, FPP and GGPP are used as substrates for the prenylation of small GTP proteins, including Ras, Rho. A post-translational modification that is essential for the activation of these signaling effectors, thus enabling their critical roles in cell signal transduction pathway.The Ras/Raf/ERK/CREB signaling pathway is relatively clear insulin signaling pathway, mainly involved in the regulation of insulin gene transcription. Thus, the relationship between HMG-CoA reductase inhibitors and the Ras/Raf/ERK/CREB signal pathway are worthy of further exploration. In this study,we observed the Ras/Raf/ERK/CREB signaling pathway critical kinase activity and nuclear transcription factor CREB/DNA binding activity of pancreatic β cell line INS-1cells after the intervention of atorvastatin in high glucose, in order to identify the effect of atorvastatin on Ras signal transduction pathways at the molecular level. The aim of this study was to investigate how statins inhibit insulin promoter activity and insulin mRNA genes expression in pancreatic β cells.Methods INS-1cell, a pancreatic β cell line, was choosed as model. The impact of atorvastatin on the INS-1cells Ras signal transduction pathway (Ras/Raf/ERK/CREB) in vitro was investigated. Western blotting (WB), RT-PCR were used to detect Ras signaling pathway critical kinase (RasGTPase, p-Raf-1, p-CERB) expression. Chromatin immune-precipitation (ChIP) was used to detect the effect of atorvastatin on INS-1cells p-CREB/DNA binding activity.Results (1) High glucose induced the activation of Ras signaling in time-dependent manner in INS-1cells. Whether high glucose could alter protein expression in INS-1cells was examined. Cells were cultured in high glucose. The finding revealed high glucose rapidly increased Raf-1activation in six hours, but had no effect on total protein.(2) Whether atorvastatin could be linked to Raf-1activation was further investigated. Atorvastatin had inhibitory effect on the promotion of Raf-1activation with the concentration-dependence. Cells were cultured at different concentrations ofatorvastatin for24hours. With the increase in atorvastin concentration, the expression of active Raf-1protein gradually decreased. In comparison with the control group,1μM and1OμM atorvastatin were not detected the inhibition of the expression of active Raf-1protein (P>0.05).50μM and100μM atorvastatin had significantly decreased the expression of active Raf-1protein (P<0.01).(3) Atorvastatin can significantly reverse the promotion of RasGTPase at the concentration of50μM, p-Raf-1and p-CREB protein expression were significantly decreased compared with high glucose (25mmol/L) levels, and the inhibition ratio is61.8%,60.9%and55.4%, respectively (P<0.01).(4) Whether the high glucose the activation of Ras signal path protein expression was linked to promotion of Ras mRNA gene expression was studied finally.In comparison with the control group, real-time PCR showed that high glucose did not increase Ras mRNA gene expression. In comparison with the high glucose group, The Ras mRNA gene expression of the atorvastatin group and the RasGTPase inhibitor manumycin A group had no significant difference (P>0.05).(5) Atorvastatin could affect the combination of p-CREB and DNA in INS-1cells. In comparison with the control group, the p-CREB/DNA combining weight of INS-1cells of which were cultured in high glucose was significantly increased. Cells treated with atorvastatin or manumycin A had reduced the p-CREB/DNA combining weight(P<0.01).Conclusions(1) High glucose time-dependently increased the activity of critical enzyme of the Ras/Raf/ERK/CREB signaling pathway in pancreatic β cell line INS-1cells in vitro.(2) Atorvastatin statins could inhibit the activity of RasGTPase of Ras/Raf/ERK/CREB signaling pathway in pancreatic β cell lines. Moreover, atorvastatin inhibit the downstream molecules p-Raf-1, p-CERB protein expression. Atorvastatin statins could reduce the binding capacity of p-CREB and DNA in INS-1cells.(3) Atorvastatin inhibited post-translational modification of Ras proteins in the pancreatic β cells and RasGTPase activity, and futher inhibited the transduction of the Ras complex pathway of insulin signal transduction pathways (Ras/Raf/ERK/CREB), lead to the reducement of p-CREB/DNA binding capacity, suppression of insulin promoter activity and inhibition of insulin synthesis. Therefore, the impact of statins on diabetic islet function depended in part on the resistance of Ras/Raf/ERK/CREB signaling pathway.