Basic Research Of Adiponectin And Insulin Resistance And Its Pathway Effect On Endometrial Cancer

THE EFFECT OF ADIPONECTIN ON AMPK PATHWAY IN ENDOMETRIAL CANCER CELLS AND INSULIN SENSITIVITYObjective:To investigate the effect of adiponectin on the AMPK/m TOR/S6K1 pathway proteins in endometrial cancer cells and the insulin receptor substrate 1(IRS1).Methods: The experiments groups is as follows:(1)Real-time quantitative PCR and western blot analysis was used to test the level of m RNA and protein of Adipo R1,Adipo R2.(2)Western blot analysis was used to detect phosphorylation of AMPK,m TOR,S6K1 or IRS1(tyr)protein expression with stimulation in different concentrations of Adiponectin(2.5,5,10,20 μg /m L),or following incubation with insulin 50 nmol/L for 5 minutes;or treated with 20μg /m L adiponectin for different times(15、30、45、60min),or following incubation with insulin 50 nmol/L for 5 minutes.(3)Ad group :HEC-1B cells treated with 20ug/m L Adiponectin for 30 min;inhibitor group :HEC-1B cellstreated with 10 umol/L compound C for 30 min;inhibitor + Ad group: HEC-1B cells were treated with compound C(10 umol/L,30min)followed by 20ug/m L adiponectin 30 min;control group:only adding the culture medium without serum DMEM.Results:(1)The relative expression level of Adipo R1 m RNA and protein is higher than Adipo R2 in HEC-1B 、RL95-2 cell(P<0.01).(2)HEC-1B and RL95-2 cell exhibited time-and dose-dependent increases in the p-AMPK、p-IRS1(Tyr895)levels after treatment with adiponectin,and which was more obvious at the concentration of 20 μg/m L adiponectin 30min(P<0.05).However,Adiponectin inhibited p-m TOR 、 p-S6K1 phosphorylation in time-and dose-dependent manner(P<0.05).(3)Adiponectin induced IRS1 tyrosine phosphorylation of endometrial cancer cell in time-and dose-dependent manner(P <0.05).(4)Comfound C could significantly block the activation of AMPK in cells and the inhibition of m TOR and S6K1(P<0.05).The IRS1 tyrosine activation induced by adiponectin was partially abolished by comfound C(P <0.05).Conclusions: Adiponectin inhibit proliferation of endometrial cancer cells through AMPK-m TOR/S6K1 signal pathway.Adiponectin sensitizes insulin signaling by regulating the AMPK-S6K1-IRS1 pathway.THE EFFECTS OF ADIPONECTIN AND HIGH-FAT DIET ON THE DUAL ANIMAL MODELS OF INSULIN RESISTANCE TO ENDOMETRIAL CARCER XENOGRAFT TUMORObjective: To establish a dual animal model of high-fat diet feeding insulin resistance and endometrial cancer transplantation tumor,and to explore the mechanism of adiponectin and insulin resistance on occurrence and progression of endometrial cancer.Method:(1)Feeding high-fat diet induced insulin resistance model of nude mouse: choose 40 BABL/C nude mice were randomly divided into model group and the control group,20 respectively with high fat feed(high-fat diet,HFD)and normal feed(common diet,CD)feeding.(2)After feeding 10 weeks,Fasting plasma glucose(FBG),fasting blood insulin(FINS),intraperitoneal glucose tolerance test(IPGTT)and intraperitoneal insulin tolerance testing(IPITT)were tested,confirm the high-fat group IR model is successfully established.(3)Subcutaneous inoculation of endometrial carcinoma in the inguinal region after the high-fat group successfully induced IR model.(4)After 0.5cm in tumors diameter,according to the different diet tumor-burdened nude mice were randomly divided into 4 groups: CD+Ad group and HFD+Ad group(25μ g/kg· d rat adiponectin),CD group and HFD group(0.9%Na Cl sodium chloride injection)intraperitoneal injection for 2 weeks(QOD).(5)Record and analyze the growth metabolic indicators: The volume change of subcutaneous transplantation tumor was recorded with vernier caliper every 3 days.After the experiment,take the eyeball for bloodand separate the serum,fasting blood glucose(FBG),fasting blood insulin(FINS),serum triglyceride(TG),serum total cholesterol(TC)and serum adiponectin(Ad)were also detected.Meanwhile,Separation of tumor,visceral fat and liver,weighing.The liver were taken HE staining compared with the group of fat varying degrees.Qrt-PCR was used to detect the expression of adiponectin receptor and PPAR alpha-gene m RNA.Western blot was used to detect changes in Adipo R1,Adipo R2,PPARα protein and phosphorylation of AMPK,m TOR,S6K1,4EBP1,IRS1 and p38 MAPK in different groups.Results:(1)The FBG,FINS and HOMR were significantly higher in HFD group than in CD group for 10 weeks of pure high-fat diet(P < 0.05).IPGTT and IPITT tests showed that the HFD group rats showed impaired glucose tolerance and insulin resistance,and successfully constructed the insulin resistance model.(2)At the end of the experiment,the level of TG,TC,FBG,FINS,HOMR and visceral fat in HFD group was higher than that in the CD group(P < 0.05),while the serum adiponectin was lower than that in the CD group,CD + Ad group serum adiponectin is highest(P< 0.05).In the whole experiment,The average daily intake of nude mice in the HFD group was lower than that in the CD group,but the average daily energy intake of the HFD group was significantly higher than that of the CD group(P < 0.05).HE staining showed liver fat change in HFD group,while the CD group did not show hepatic fat lesion.There were no statistically significant differences in liver weight.(3)In the four groups,the tumor volume and weight were ranked : CD +Adgroup < CD group< HFD+Ad group< HFD group(P < 0.05).Compared with non-adiponectin injection group,the inhibition rate of CD +Ad group and HFD+Ad group was 38.06% and 52.31% respectively.The results of q RT-PCR showed no difference in the expression of Adipo R m RNA in 4 groups,and PPARα m RNA was the highest expression in CD +Ad group.Western Blot results showed that there was no significant difference in the expression of Adipo R in the four groups.In the four groups,In the CD +Ad group,p38MAPK-PPAR α pathway,AMPK-m TOR-S6K1-IRS1(Tyr)pathway expression level was the highest(P< 0.05),while the expression level of m TOR-S6K1/4EBP1 pathway was the lowest(P < 0.05).Conclusion:In this study,the model of insulin resistance was successfully induced by feeding the nude mice with high fat diet.High fat diet and insulin resistance can promote the growth of endometrial cancer transplantation tumor.Adiponectin can inhibit the growth of endometrial cancer transplantation tumor through AMPK-m TOR-S6K1/4EBP1 pathway.Adiponectin increased insulin sensitivity by activating AMPK-m TOR-S6K1-IRS1 pathway.Adiponectin can improve insulin resistance in nude mice and increase the expression of p38MAPK-PPARα protein pathway.