Effects And Mechanisms Of The Ethyl Acetate Extract Of Acorus Calamus On The Expression And Secretion Of GLP-1

ObjectivesThe purpose of this study is to investigate the anti-diabetic effect of ethyl acetate extract of Acorus calamus (ACE), and its effect on the expression and secretion of intestinal glucagon like peptide-1 (GLP-1) as well as its molecular mechanism. It is hoped that this research can provide basis and direction for the further development and application of ACE and provide ideas and methods for exploring anti-diabetic effects and molecular mechanism based on GLP-1.Methods1. The anti-diabetic effect and mechannism of ACE based on GLP-1 in vivo STZ-induced hyperglycemic mice, genetical obese diabetic mice, and diet-induced obesity mice were used to evaluate the anti-diabetic effect of ACE. The mice were randomly divided into three groups:model control group, positive control group receiving metformin (100 mg/kg), experimental group receiving ACE (100 mg/kg), and the mice at the same age were chosen as normal control. The mice were sacrificed after 4-5 weeks treatment, and blood samples, pancreas, intestine were preserved and stored at-80℃ for GLP-1 and ralated mechanism analysis. During the experiment, blood glucose levels and oral glucose tolerance test (OGTT) were used to determine the anti-diabetic effect of ACE in mice.ELISA was used for plasma insulin levels determination, HE staining and immunofluorescence staining were used to investigate the effect of ACE on the secretion of insulin and the function of pancreatic islet. Plasma GLP-1 levels, intestinal GLP-1 related gene expression (gcg, pc3), and in situ GLP-1 expression were analysed to demonstrate the effect of ACE on the secretion and expression of GLP-1.2. The effect of ACE on the expression and secretion of GLP-1, and the mechanism invloved.The effect and molecular mechanism of ACE on the expression and secretion of GLP-1 in different culture conditions were detected by using intestinal L cell line NCI-H716. Basic condition:NCI-H716 cells were treated with KRB buffer with 0.2% BSA plus DMSO, metformin (1 mM) or ACE (12.5μg/ml) after two days’differentiation. Then cells were incubated at 37℃ for 2 h, cell supernatant was stored at-80℃ for GLP-1 analysis and cells were preserved for further machanism study. Pathological condition:all procedure was the same with that in basic condition except the treatment with palmitic acid before drug administration.(1) ELISA was used to determine GLP-1 levels in supernatant and cellcular cAMP levels.(2) Immunofluorescence staining was used to determine the intestinal GLP-1 expression.(3) Lowery assay was used to measure the protein concentration.(4) Nucleocytoplasmic separation was used to separate the unclear and endochylema protein.(5) Western blotting was used to explore the key protein in insulin signaling pathway, PKA and key protein in Wnt signaling pathway.Results1. The anti-diabetic effect of ACE in vivo(1) The anti-diabetic effect of ACE in STZ-induced hyperglycemic mice Blood glucose was decreased by ACE in STZ-induced hyperglycemic mice after 4-weeks treatment with the reduction of 27.9%(P<0.05) while metformin showed a 32.9%(P<0.01) reduction compared with model control.(2) The anti-diabetic effect of ACE in db/db diabetic mice Blood glucose was decreased by ACE in db/db diabetic mice after 5-weeks treatment, with the reduction of 65.8%(P<0.05) while metformin showed a 60.3%(P<0.05) reduction compared with model control.(3) The anti-diabetic effect of ACE in DIO miceBlood glucose was decreased by ACE in DIO mice after 4-weeks treatment, with the reduction of 15.0%(P<0.05) in fasting blood glucose and 12.7%(P<0.01) in random blood glucose compared with model control. OGTT was improved with a reduction of 19.4%(P<0.05) for ACE and 21.1%(P<0.05) for metformin, respectively.2. The effect of ACE on plasma insulin levels and islet morphologyAfter ACE administration, plasma insulin levels were increased in STZ-induced hyperglycemic mice, db/db mice and DIO mice by 43.7%(P<0.01),53.1%(P<0.001) and 77.1%(P<0.001), respectivley. And in situ insulin expression was increased along with augmented islet tissue volume and clearer boundary compared with model control in STZ-induced hyperglycemic mice; pathological morphology of pancreatic islet was improved compared with model control in db/db diabetic mice; and the co-expression of insulin and proliferation related marker Ki67 of pancreatic islet were improved in DIO mice, suggesting the promotion of ACE on the proliferation of pancreatic islet β cells.3. The effect of ACE on the secretion and expression of GLP-1 in vivoPlasma GLP-1 levels were raised by ACE in STZ-induced hyperglycemic mice, db/db mice and DIO mice. And in situ GLP-1 expression were increased by ACE in db/db diabetic mice and DIO mice. Intesinal GLP-1 related gene expression (gcg and pc3) were increased by ACE in STZ-induced hyperglycemic mice and DIO mice, pc3 gene expression was augmented in db/db diabetic mice.4. The effect and mechanism of ACE on the expression and secretion of GLP-1 in intestinal L cells under basic culture conditionGLP-1 levels in supernatant and GLP-1 related gene expression (gcg,pc3) of intestinal cells NCI-H716 were increased by ACE. The key protein insulin receptor substrate-1 (IRS-1) and protein kinase B (PKB/Akt) were phosphorylated at Tyr989 and Ser473, Thr308, respectively. In addition, the cAMP levels, the phosphorylation of downstream protein PKA, as well as the translocation of the key protein β-catenin and increased levels of target gene expression cyclin-D in wnt signaling pathways were elevated.5. The effect and mechanism of ACE on the expression and secretion of GLP-1 in intestinal L cells under pathological culture conditionThe expression and secretion of GLP-1 in supernatant of NCI-H716 cells were reduced by palmitic acid along with lowered cellcular cAMP levels, decreased phosphoated PKA levels and reduced nuclear β-catenin levels. These effects were restored by ACE, turning out the elevated cAMP levels, the phosphorylation of downstream protein PKA, as well as the rise of nuclear β-catenin levels in wnt signaling pathways. But the key protein in insulin signaling pathway remained unchanged as that after the palmitic acid treatment.ConclusionThe expression and secretion of GLP-1 were increased by ACE in vivo and in vitro, further research showed the involvement of cAMP/PKA and Wnt signaling pathways. 1. Blood glucose was decreased by ACE in STZ-induced hyperglycemic mice, db/db diabetic mice and DIO mice, and random glucose levels were reduced as well as OGTT was improved in DIO mice. Plasma insulin levels were elevated and the potential islet protective effect of ACE was observed in all kinds of model mice, which might be associated with increased intestinal gene expression of gcg and pc3 levels which related to GLP-1 synthesis, as well as elevated plasma GLP-1 levels.2. ACE promoted expression and secretion of GLP-1 in basic culture condition of intestinal NCI-H716 cells, and the mechanism may relate to the activation of the insulin receptor, cAMP/PKA and wnt signaling pathway. And in pathological condition induced by palmitic acid in NCI-H716 cells, ACE restored the inhibited GLP-1 expression and secretion mainly by the enhancement of cAMP/PKA and wnt signaling pathway.