Study On The Regulation And Molecular Mechanism Of Carrot Peptide DCHP On Glucose Metabolism

Type 2 diabetes mellitus(T2DM)is a metabolic disease characterized by insulin resistance(IR).It is mainly characterized by hyperglycemia and usually leads to the disturbance of glucose,lipid and protein metabolism.In recent years,with the surge in the number of obese and overweight people,the incidence of T2 DM is increasing year by year.Therefore,the development of safe and efficient hypoglycemic drugs has become the focus of worldwide attention.Among them,plant hypoglycemic peptides are one of the most widely studied potential therapeutic agents,trypsin inhibitors being one of them.Trypsin inhibitors were generally considered as antinutritional factors,which required high temperature to inactivate them in animal culture and human food applications to facilitate nutrient absorption and utilization.But every coin has two sides,trypsin inhibitors derived from natural plants have advantages such as wide range,good stability,less toxic and side effects compared with general small molecule drugs,and natural resistance to protease in the digestive tract,etc.,which have great potential utility in the development of hypoglycemic functional foods and in the prevention and treatment of diabetes as oral drugs.Therefore,it is particularly important to study the hypoglycemic activity and hypoglycemic mechanism of trypsin inhibitors.DCHP is a kind of active peptide that belongs to the trypsin inhibitor family in terms of protein classification,which was obtained from carrot in our laboratory in the early stage.DCHP does not have trypsin inhibitor activity,but has good resistance to trypsin hydrolysis,high α-glucosidase inhibitory activity and good p H and temperature stability in vitro,but lack of studies on its hypoglycemic effect and mechanism in vivo.In this paper,DCHP was used as the research object,HepG2 cells of human liver cancer and Caenorhabditis elegans model were used to explore the regulatory effect of DCHP on glucose metabolism and further explore its potential mechanism of action.The main research contents and conclusions are as follows:(1)Study on the regulation of glucose metabolism in HepG2 cells by DHCP.DCHP dosing was performed on untreated HepG2 cells and on HepG2 cells that were induced to produce IR by glucose and palmitic acid.The results indicate that DCHP has a positive effect on the regulation of glucose homeostasis in liver.100 μg/m L DCHP can promote glucose consumption,transport and uptake of glucose in HepG2 cells without inhibiting cell proliferation.DCHP inhibited gluconeogenesis by down-regulating the expression level and protein activity of phosphoenolpyruvate carboxy kinase(PEPCK)and inhibiting the expression of glucose-6 phosphatase(G6P)in HepG2 cells.Meanwhile,DCHP can also promotes glycogen synthesis by regulating the expression levels of glycogen synthesis kinase 3β(GSK3β)and glycogen synthase(GS)in HepG2 cells.In IR-HepG2 cells,50 μg/m L DCHP significantly increased glucose consumption in IR-HepG2 cells and promoted glucose uptake in model cells.DCHP could promote the phosphorylation of GSK3β by activating Phosphoinositide 3-kinase(PI3K),release the inhibition of GS,and promote liver glycogen synthesis.Meanwhile,the activation of PI3 K by DCHP also increased the phosphorylation level of forkhead box protein o1(Fox O1),inhibited the activity of Fox O1,and inhibited the activity of gluconeogenic ring-limiting enzyme,which weakened the process of gluconeogenesis.In addition,DCHP can also reduce the malondialdehyde(MDA)level in HepG2 cells,restore the activity of superoxide dismutase(SOD)and increase the intracellular glutathione(GSH)content,so as to improve the cellular oxidative stress damage caused by IR.(2)Protective effect of DCHP on Caenorhabditis elegans under high concentration of glucose(HCG)condition.50μg/m L DCHP treatment of Caenorhabditis elegans with oxidative stress induced by walnut quinone was found to shorten the lifespan of Caenorhabditis elegans.DCHP can reduce the damage of walnut quinone to Caenorhabditis elegans and prolong its lifespan.At the same time,under HCG stimulation,DCHP can reduce MDA content and increase GSH content in Caenorhabditis elegans.It is suggested that the enhancement of antioxidant capacity is one of the reasons for the protective effect of DCHP on Caenorhabditis elegans under the HCG condition.In addition,HCG also shortened the lifespan of wild type nematode N2 and daf-2 mutants,but extended the lifespan of daf-16 mutants,suggesting that the effect of HCG on the lifespan of nematode is regulated by DAF-16.DCHP treatment can inhibit the above damage caused by HCG to a certain extent,suggesting that the inhibition of DCHP on the damage effect is partly through the activation of DAF-16 expression.In addition,DCHP treatment further extended the lifespan of daf-16 mutants,suggesting that the protective effect of DCHP against HCG stimulation of Caenorhabditis elegans may be mediated by other unknown factors.Transcriptome sequencing results showed that,compared with the HCG treatment group,DCHP intervention affected many biological processes,mainly involving cell growth,proliferation regulation,protein metabolism,endocrine regulation,etc.