Studies On The Mechanism Of Blood Glucose Reduction In T2DM Mice With Leaf Extracts Of Lithocarpus Polystachyus Rehd.

Lithocarpus polystachyus Rehd.is a bush tree,widely distributed in the south of the Yangtze River,which has a good hypoglycemic effect and has been get attention by scholars.There have been some preliminary studies on its hypoglycemic effect,but the specific active ingredients and their mechanism is not very clear.The germplasm resources of Guangxi,Hunan,Jiangxi,Fujian,Zhejiang and Chongqing were investigated,and the content of compounds in leaves was analyzed.The main purpose of this study was future to investigate the mechanism of blood glucose reduction of type 2 diabetes mellitus(T2DM)mice with leaf extract of Lithocarpus polystachyus Rehd.and the active constituents.In the present study,effect of leaf extract,phloridzin and trilobatin on blood glucose reduction were assessed in vivo(gavage)and in vitro(non-invasive micro-test technique,NMT)in experimental T2DM mice.The biochemical parameters measured include blood glucose and blood lipid level,liver biochemical indexes,and hepatic glycogen.The relative expression of glycometabolism-related genes was analysised.The effect of leaf extracts on physiological glucose flux in liver tissue from control and T2DM mice was also investigated.Serum samples and liver extract samples were studied by 1H NMR-based metabolomics method.Meanwhile,the liver weight and hepatic coefficient were analysised.To investigate the effects of leaf extract,trilobaloside and phloridzine on the secretion of insulin from isolated pancreatic islet ? cells,the transmembrane transport of K+,Ca2+,glucose and insulin in isolated pancreatic islet ? cells were studied.A fter treatments,the effects of leaf extract,trilobalide and phloridzine on intracellular Ca2+ accumulation were measured.The main conclusions are as following:(1)The distribution range of Lithocarpus polystuchyus Rehd.is about N 23°40'?32° and E 99°?123°,and the distribution altitude is 150?2200 m,mostly 350?1100 m.The adaptability is strong,and the growth conditions are not strict.From the middle part of hills to under the broad-leaved forest,the trees grow in the mountain yellow soil.When the altitude was more than 1400 m,the Lithocarpus polystachyus Rehd.was grown under the evergreen broad-leaved forest in the mountain yellow brown or mountain forest brown soil.At the same time,the effective content phloridzin and trilobatin in the extract was more than 12.05%and 34.87%respectively.(2)Body weight of T2DM mice increased significantly after the first week,but stabilized over the subsequent three weeks;body weight of all the other groups did not change during the four weeks' study.After four weeks,blood glucose of all the treatment groups decreased,and treatment with leaf extract had numerous positive effects:a)promotion in glucose uptake in liver,b)increasing in the synthesis of liver glycogen,c)reducing oxidative stress,d)up-regulation of Glucokinase(GK),Glucose Transporter 2(GLUT2),Insulin Receptor(IR)and Insulin Receptor Substrate(IRS)in liver,e)down-regulation of Glucose-6-Phosphatase(G-6-P)expression,and f)improving blood lipid levels.Treatment with either trilobatin or phloridzin accelerated liver glycogen synthesis,decreased oxidative stress and increased expression of GK.Both IRS and Phosphoenolpyruvate Carboxykinase(PEPCK)were up-regulated after treatment with trilobatin.Expression of GLUT2,PEPCK and G-6-P were also increased in liver tissue after treatment with phloridzin.Our data indicate that leaf extract from L.polystachyus Rehd.has a better hypoglycemic effects than trilobatin or phloridzin alone.Leaf extract significantly increased glucose uptake and hepatic glycogen synthesis,while,in the meantime,also caused a decline of hepatic gluconeogenesis and oxidative stress in T2DM mice.(3)Liver weight and hepatic coefficient were remarkably increased in T2DM mice than control,but had no decline after treatment.According to the metabonomic profiles and literatures,59 metabolites were identified in serum and liver extract samples.In the serum of T2DM,the amino acids including glutamine,valine,isoleucine,glycine and alanine were decreased,as well as pyruvate and creatine.However,the levels of glucose and lactate were increased in T2DM mice.After treatment of leaf extract,levels of pyruvate,creatine and glucose were improved.In liver extract,glutathione,glutamine and acetate declined,but fatty acids,cholesterol and TMAO were increased in T2DM.Leaf extracttreatment had a promoted dropping of glutamine,fatty acid and cholesterol.(4)Leaf extrqact improved the absorption of glucose in mouse pancreatic ? cells.Pancreatic ?cells respond to glucose stimulation to reduce K+and Ca2+ transmembrane outward transport.Leaf extract promoted the transmembrane transport of insulin.The mechanism of this action was similar to sulfonylurea glibenclamide,acting on KATP.Trilobatin was one of the active ingredients of the leaf extract to promote insulin transmembrane transport by acting on the KATP,but not the only one.In this work,L.polystachyus Rehd.was found to be widely distributed in the south,rich in resources and high in the active ingredients,which can be used for the development and utilization of the resources.The mechanism of blood glucose reduction by and target of leaf extract of L polystachyus Rehd.in T2DM mice were preliminarily revealed,proving the treatment had multiple targets,providing theoretical guidance for the clinical treatment of diabetes.Therefoe,this study was of great significance to the development and utilization L.polystachyus Rehd..