Effects Of Ecdysterone On Protein (GLUT4, PPARr) And Gene (PPARr MRNA) In The Insulin Resistance Cellular Model Of3T3-L1

Objective: Through the mouse3T3-L1preadipocytes’(mouseembryonic fibroblast cells) induction and differentiation，select growthexperiment conditions cell with free fatty acid (FFA) incubationestablished a cellular model of insulin resistance (IR).On the basis ofusing ecdysterone (ECR) intervention on the proliferation, andcomparison with pioglitazone, in order to investigate ecdysterone in vitroeffect of cells to insulin resistance and its intensity. The priority ofglucose transporter4(GLUT4) protein, peroxisome proliferator activatedreceptor γ (PPARγ) protein and the effects of PPARγ mRNA expressionof transcription factors, and preliminary discussion on ecdysteroneaffected part of the mechanism of the above factor expression.Methods:Regular training in DMEM (high glucose) cultures of fat cells before3T3-L1, requires every48hours in a fluid, when the cell fusion after48hours, when the cell fusion after48h, containing3-isobutyl-1--methylxanthine (IBMX，0.5mmol/L), insulin（10μg/ml）, dexterity（1μmmol/L） in DMEM (H) for two days.Then change including10g/ml INSULIN culture to cultivate6-8days; To continue to develop fullythe culture medium,2days in a fluid, differentiation12to14days, tofully differentiated into3T3-L1fat cells; And then change to contain fetal bovine serum (BSA,2g/L) of DMEM (high glucose) nutrientsolution culture3T3-L1fat cells after12h, change become has added aPA (0.5tendency/L), FAF BSA (10g/L) in DMEM culture for the night(24h), IR cell model modeling success. Adding different concentrationsin the cultures of ecdysterone and concentration of1×10-5mol/Lpioglitazone incubation24hours at the same time. Extract relevantmaterial, after appropriate treatment using Western blot determineproteins and protein expression level, using RT-PCR detection of PPARγgene expression levels. The experiment is divided into six groups:(1)blank control group:3T3-L1adipocytes;(2) model group: FFA inducedwith insulin resistance of3T3-L1adipocytes;(3,4,5) ECR groups: toECR1×10-7mol/L low concentration group, ECR1×10-6mol/L middleconcentration group, ECR1×10-5mol/L high concentration group，calledECR low, medium and high concentration groups;(6): Pioglitazoneconcentration of1×10-5mol/L, called the pioglitazone group. Results:Cell culture, building and cells grow well in the process of intervention,the concentration in1×10-7～1×10-5mol/L ECR, experimental cellsshowed good adaptability; Compared with model group, at1×10-7mol/L～1×10-5mol/L concentration range of ecdysterone and1×10-5mol/L pioglitazone, can make the insulin resistance cellular modelof the mouse3T3-L1adipocytes’ glucose transporter4(GLUT4) protein,peroxisome proliferator activated receptor γ (PPARγ) protein expression and transcription factor PPARγ quantity increase. Conclusions:(1)Ecdysterone in1×10-7mol/L～1×10-5mol/L concentration, andconcentration of1×10-5mol/L pioglitazone，all can increase expressionof the insulin resistance cellular model of the mouse3T3-L1adipocytes’glucose transporter4(GLUT4) protein; Ecdysterone and pioglitazone in1×10-5mol/L concentration increase expression of GLUT4protein in3T3-L1adipocytes is equivalent（P=0.234）.（2）Ecdysterone in1×10-7mol/L～1×10-5mol/L concentration, and concentration of1×10-5mol/L pioglitazone，all can increase expression of the insulin resistancecellular model of the mouse3T3-L1adipocytes’ protein of PPARγ;Statistically significant effects on increase expression of PPAR γ proteinin3T3-L1adipocytes by ecdysterone and pioglitazone in1×10-5mol/Lconcentration was differences (P=0.009).(3) Concentration in1×10-7mol/L～1×10-5mol/L ECR, all can increase the IR model cellPPARγ mRNA expression, concentration of1×10-5mol/L pioglitazoneincrease IR model cell PPARγ mRNA expression，all can increaseexpression of the insulin resistance cellular model of the mouse3T3-L1adipocytes’ PPARγ mRNA; Ecdysterone and pioglitazone in1×10-5mol/L concentration increase expression of PPARγ mRNA in3T3-L1adipocytes is equivalent（P=0.486）.Display of ecdysterone can improvethe free fatty acid-induced insulin resistance, the molecular mechanismmight be related to ecdysterone activated PPAR γ gene, and increase of protein GLUT4and PPAR γ expression.