The Effect Of Metformin On AMPK, PPARγand Insulin Resistance

Objective:1. To observe the expression levels of AMP-activated protein kinase (AMPK) and peroxisome proliferator-activated receptor γ (PPARγ) in adipose tissue of type2diabetes with metformin treatment, and the effect on glucose and lipid metabolism to improve insulin sensitivity2. Established the insulin resistance model in vitro,utilized metformin to interfere in the adipocyte,observed the expression of AMPK、PPARγ、GLUT4,at the same time observed the effect that metformin contribute to AMPK、PPARγ、analysis if the effect that metformin improve insulin resistance has something to do with the effect that it increased the expression of AMPK、PPARγ.Methods:1. High fat diet with intraperitoneal injection of STZ (40mg/kg) was prepared with type2diabetes model, after the success of modeling, diabetic rat model were randomly divided into two group:diabetic group (DM-C) and treatment group (DM-T). DM-T group was given metformin hydrochloride orally for4weeks. Weight of rats was measured before and after treatment, at the end of the experiment the rats were measured in adipose perirenal and testis weight. Then to Calculate of total body weight ratio of fat.Their serum were used to detect of Fasting plasma glucose (FPG), Fasting insulin (FINS), and Fasting sensitive index (ISI) were Calculated, serum total cholesterol (TC), triglycerides (TG), high density lipoprotein(HDL),Low density lipoprotein(LDL) levels also were detect; real time-polymerase chain reaction (RT-PCR) to detect the AMPK and PPARy mRNA adipose tissue expression.2. Chose the American Type Culture Collection,3T3-L1adipocyte as our experimental object than culture and derivation. Refer the differentiated method of Anti Kumar KL differentiated the adipocyte. Cultured3T3-L1adipocyte in the DMEM high glucose medium that contain15%calf serum, and the condition of the evidence is37℃,5%C02when the cell Was in good condition,put it to cultivated plates, when the cell conjugation after two days, culture the cells for48hours use the DMEM medium that contain0.5mmol/L IBMX10mg/L insulin lumo/L Dexamethasone15%calf semm. and than change the DMEM medium that contain lOmg/L insulin1/Mmol/L dexamethasone15%calf serum,cultivate for48hours,after that culture with DMEM medium cantina15%calf serum for8-12days. When there are90%3T3-L1cells become mature adipocyte,it can use in study. Use oil red O to improve3T3cells had yet induced to mature adipocyte:Remove the old medium; wash the cells by PBS, use Ca-formalin fixation for30min; and move oil red O’dyeing for30min,remove oil red O,avantin clean the rest oil red O, than campeachy dyeing the cell nucleus, observe the cell appearance and get the photos.3T3-L1adipocyte insulin resistance model established. After the cells differentiation, use DMEM medium that contain lumoI/L dexamethasone to induce insulin resistance model, change the medium every two days,and than use the old medium to exam the consumption of glucose, depend on the consumption we can judge the level of resistance. We use the GOD-POD kit to exam the level of glucose in the old medium. When the resistance model has established,set up the adipocyte as blank group,dexamethasone model group, metformin treatment group. After metformin has intervention for48hours, exam the level of glucose in the medium, than exam the mRNA level of AMPK,PPARγ and GLUT4.Results:In diabetic rats fed with high fat diet and injected with a small dose of STZ:The level of height,Fasting plasma glucose,blood lipid of the diabetic rats were markely increased as compared with the NC group.ISI of the DM-C group was reduced as compared with the NC group.And the DM-T group compare with DM-C group, AMPK and PPARy mRNA in adipose tissue was significantly increased (P <0.05), serum high density lipoprotein level was significantly higher (P<0.05); serum total cholesterol, triglycerides LDL decreased significantly (P<0.05). In3T3-L1adipocyte:1. the absorbance level OD of dexamethasone model group is significant higher than the blank group(P<0.05).2. the absorbance level OD of metformin group is lower than dexamethasone model group (P<0.05).3. The expression of AMPK,PPARy,GLUT4mRNA in dexamethasone model group is significant lower than in the blank group(P<0.05).4. The expression of AMPK,PPARy,GLUT4mRNA in metformin group is significant higher than the dexamethasone model group(P<0.05).Conclusion:In diabetic rats fed with high fat diet and injected with a small dose of STZ:(1) AMPK and PPARy mRNA were low in diabetic rats adipose tissue (2) Metformin increased adipose tissue expression of AMPK and PPARy mRNA.(2)(3) Metformin regulated glucose,lipid metabolism and insulin resistance.(4) Metformin in type2diabetic rats may regulate glucose,lipid metabolism and insulin resistance by increased adipose tissue AMPK and PPARy mRNA. In3T3-L1adipocyte:(1) The glucose intake in3T3-L1adipocyte that used dexamethasone induced into insulin resistance model is low.(2)Metformin can increase the glucose intake in3T3-L1adipocyte.(3)The mRNA of AMPK,PPARr,GLUT4in3T3-L1adipocyte that used dexamethasone induced into insulin resistance model growth lower.(4)Metformin can increase the AMPK,PPARr,GLUT4mRNA in3T3-L1adipocyte that used dexamethasone induced into insulin resistance model.(5) Metformin may regulate insulin resistance by increased expression of AMPK,PPARy and GLUT4mRNA.