Glucogan-like Peptide-1 Potentiates Glucose-stimulated Insulin Secretion Via Transient Receptor Potential Melastatin 2

ObjectiveThe transient receptor potential(TRP)super family is a group of nonselective cation channel,which have seven sub-families including TRPM.There are eight members in TRPM subfamily,TRPM1 to 8.The transient receptor potential melastatin 2(TRPM2)channel is expressed on both excitable cells and non excitable cells including pancreatic beta cells.TRPM2 can be activated by H2O2,reactive oxygen species(ROS),temperature,and so on.As a Ca2+ permeable channel activated by c AMP it is responsible for the regulation of insulin secretion.Glucogan like peptide-1(GLP-1)is an important target for type 2 diabetes.It has the protection of islet beta cells,stimulate their proliferation and differentiation,inhibiting apoptosis to increase the number of islet beta cells,reduce patient weight,inhibition of gastrointestinal peristalsis and gastric secretion,suppress appetite,delayed gastric emptying,promoting synthesis and secretion of insulin,and so on.However,among them,we are more interested in its insulin regulatory effect.It is known that glucose-stimulated insulin secretion(GSIS)can be potentiated by GLP-1,and that the changes in the extracellular glucose concentration alter the level of intracellular ATP and c AMP.These findings prompted us to hypothesize that TRPM2 may mediate the modulatory effect of GLP-1 on insulin secretion.Our findings provide a new thinking for the prevention and treatment of diabetes and its complications.MethodRat pancreatic islets were isolated from healthy Sprague Dawley rats.Isolation and culture of primary pancreatic islets layout successfully.A patch clamp amplifier was used to measure the cytomembrane current at room temperature(22-25 oC).The PCLAMP v.10.2 software and Digidata-1440 A was adopted to give stimulatory commands and record data.Place primary ? cells into bath solution in the objective table.The flow velocity of HEPES buffer(containing 3.3 m M glucose)was 5 ml/min(electrical resistance was about 4.5 M?).After exposure to the low-glucose K-R buffer,the primary ? cells continued with low-glucose(control),or were exposed to high glucose,high potassium,TRPM2 stimulator,GLP-1,stimulator or inhibitors of PKA or Epac.The calcium current and the changes in membrane capacitance were recorded throughout continuous depolarization.The concentration of insulin in the supernatant was measured at 0,10 min and 60 min post-exposure by enzyme-linked immunosorbent assay(ELISA)using a commercial kit.In addition,10 nM GLP-1 was added at the end of the indicated interventions(high glucose for 10 min or 60 min,high potassium for 5 min,stimulator or inhibitor for 30 min,and)or to the TRPM2-silenced or overexpressed cells before assaying insulin levels.Use gene silence and overexpression technology to regulate expression level of TRPM2.By whole-cell current recording using patch clamp,we identified the activity of TRPM2 channel.In addition,insulin secretion assay was performed to confirm the involvement of TRPM2 in potentiation effect on GSIS of GLP-1.Result1.Different concentration of glucose stimulation effect on TRPM2: the primary ? cells were placed in bath solution at 37 oC under high glucose,normal glucose solution and low sugar environment in 15 minutes,there is no significant difference between the control group and low glucose group in the current of TRPM2(P>0.05),TRPM2 peak current intensity in high glucose group is higher than that of control group(P<0.05).2.Effects of high potassium and GLP-1 on TRPM2: using whole cell recording method to record the peak current of TRPM2 on primary ? cells after 10 nM GLP-1 treatment,compared with the control group GLP-1 stimulation can significantly enhance the strength of the TRPM2 current(P<0.05).30 m M KCl stimulation had no effect on TRPM2(P<0.05).3.TRPM2 gene silencing effects on insulin secretion: different concentration of glucose stimulation were given in control group and siTRPM2 group,insulin secretion level at 0-10 minutes(1st phase secretion)and 10-60 minutes(2nd phase secretion)were measured.SiTRPM2 group compared with the control group,the insulin secretion under low glucose and normal glucose condition had no significant difference(P>0.05),but in high glucose,insulin secretion in both 1st and 2nd phase decreased significantly compared with the control group(P<0.05).4.Effects of high potassium and GLP-1 on insulin secretion: under low glucose levels,insulin secretion in siTRPM2,GLP-1 and group GLP-1+siTRPM2 showed no obvious change compared with that in control group(P>0.05).The level of insulin secretion in KCl stimulated group was significantly higher than that in control group(P<0.05).The insulin secretion in group GLP-1+si TRPM2 was significantly higher than that in the control group(P<0.05),and the level of insulin secretion in GLP-1 group was not different from that in the control group(P>0.05).After treatment with KCl,there was no significant change in the level of insulin secretion between the two groups(P>0.05).5.Effects of 2-APB on GLP-1 mediated GSIS: 10 ?M 2-APB could significantly inhibit the overexpression group and control group TRPM2 current,GLP-1 can make the insulin secretion increased in high glucose level,and 2-APB can make GLP-1 group insulin secretion decreased to the level of control group,that is significantly lower than the GLP-1 group(P<0.05).6.Effects of agonists and inhibitors of PKA and Epac on GLP-1 mediated GSIS: PKA agonists and inhibitors had no significant effect on TRPM2 current(P>0.05).Compared with the control group,PKA agonists significantly increased GSIS.Compared with the GLP-1 group,the PKA inhibitor could not significant affect the insulintropic effect of GLP-1(P>0.05).Epac agonists significantly increased TRPM2 currents,in addition,the enhancement effect of GLP-1 on TRPM2 currents disappeared under the influence of Epac inhibitors.At the same time,Epac agonists can significantly enhance GSIS,and under these conditions,the addition of GLP-1 can not further promote insulin secretion,while Epac inhibitors can inhibit the secretion of insulin by GLP-1(P<0.05).ConclusionHigh concentration of glucose stimulated TRPM2 current intensity,but low sugar had no effect on TRPM2 current.TRPM2 was involved in the first and second phase insulin secretion of glucose stimulation.High potassium has no effect on the activity of TRPM2 channel.GLP-1 activates TRPM2 and regulates insulin secretion through this pathway.The KATP-Cav pathway is not involved in GLP-1 mediated GSIS.GLP-1 regulates GSIS through the Epac pathway rather than the PKA pathway,and the PKA pathway modulates GSIS through other mechanisms of action that are complementary to the regulation of GLP-1 by GSIS.