The Effects Of Diethylhexyl Phthalate On The Fat And Glucose Metabolism In Mice

ObejectObesity is an excessive accumulation of fat caused by a metabolic abnormality,which is manifested by an abnormal increase and enlargement of the number and size of fat cells.Studies in recent years have shown that the incidence of obesity worldwide has increased year by year,especially in children and adolescents.The main cause of obesity is the imbalance of energy intake and consumption,and excess calories are stored in the form of fat.Genetic factors,unhealthy diets,and lifestyles have long been associated with obesity.However,with the rapid development of modern chemical industry,some environmental endocrine disrupting chemicals(EDCs)are appearing more and more in our living environment,and the correlation between EDCs and obesity has gradually attracted widespread attention.A large number of studies at home and abroad have confirmed that EDCs exposure is significantly positively correlated with obesity.These materials are widely used in industrial,agricultural production,as well as in daily chemical,food packaging,pharmaceutical,and cosmetic production as plasticizers,rust removers,and flame retardants.These substances are currently known to enter the human body through various exposure routes such as air,food,and water.Because these substances are lipophilic and non-degradable,they can remain in the environment and the body for a long time,which can cause environmental pollution and affect the health of the body.It has become an important public health problem.2-ethylhexyl phthalate(DEHP),one of the EDCs,is a plasticizer widely used in the production of household products,food packaging,cosmetics and medical devices to enhance plastics and extensibility.DEHP easily escapes from plastic materials and pollutes the environment.The oral route is the most important form of exposure for the human body.Epidemiological studies suggest that DEHP exposure is closely related to obesity.Labrotory data have supported that DEHP can promote fat accumulation in the body.Whether lipolysis mechanism is involved in the obesity induced by DEHP has not been reported.In the present study,we oral administrated the male C57 mice with the 0.05mg/kg.bw(human daily exposure doses)and 500 mg/kg.bw DEHP,(liver damaged doses)under normal diet and high fat diet(HFD),which mimic human normal diet and high-fat diet patterns.The effects of DEHP on body weight,body fat content,fat metabolism,glucose metabolism and blood lipids of the experimental animals were observed respectively.Followed by the changes of lipolysis pathway of experimental animals,as well as the analysis the interaction between DEHP and high-fat diet.At the same time,the effects of DEHP on the adipogenic differentiation and lypolysis of 3T3-L1 cells were observed in vitro,and the mechanism of fat accumulation caused by DEHP was explored in order to provide basic data for effective intervention of obesity.Method1.Animal rearing and treatment:72 SPF C57 male mice were acclimated for 1 week,and randomly divided into 6 groups of 12 animals each.They were control group,0.05 mg/kg.bw DEHP group,500 mg/kg.bw DEHP group,high fat diet(HFD)group,0.05 mg/kg.bw DEHP+HFD group,and 500 mg/kg.Bw DEHP+HFD group.All the mice in HFD groups were fed with HFD,others were fed conventional pellet feed.DEHP diluted in corn oil was administered to mice by gavage according to the set doses.The body weight were recorded during the experimental period,and the animals were sacrificed at 13 weeks,and the tissue samples were taken for determination.2.Glucose tolerance test and insulin resistance test:Glucose tolerance test(GTT)and insulin tolerance test(ITT)were performed at the 11th and 12th of the experiment respectively.After fasting for 6 hours,the blood glucose were detected with the blood of tail vein,which was recorded as 0 min blood glucose.Then the animals were given 1.5g/kg.bw glucose for GTT or 0.4u insulin/kg.bw for ITT by intraperitoneal injection,then the the blood glucose were measured at 15min,30min,45min,60min,90min,120min respectively.The blood glucose curve was drawn and the area under the curve(AUC)was calculated3.In vitro cell culture and treatment:3T3-L1 mouse embryonic fibroblasts were cultured in high glucose DMEM containing 10%fetal bovine serum,1%penicillin-streptomycin at 37 ?,5%C02,and saturated humidity.The cells in logarithmic growth phase were seeded into 6-well plates and cultured in a 37 ? for 2 to 3 days.After the cell fusion degree reached 90%or more,the final concentration of 3.125 ?M/L and 6.25 ?M/L,12.5 ?M/L,25 ?M/L,and 50 ?M/L DEHP were added into the DMEM.Then,the adipose differentiation was induction by the differentiation solution 1 containing 4,3-isobutyl-1-methylxanthine solution(IBMX),insulin and dexamethasone and differentiation solution 2 containing insulin respectively.After the induction was completed,the fat in the cells were detected by oil red O staining,and the triglyceride level in the cell culture was examined.The cells were collected for target proteins determination.4.Western Blot:Western Blot was used to detect the changes of proteins expressions related to lipolysis pathway in 3T3-L1 cells and animal fat tissues,which includes protein kinase A(PKA),hormone-dependent lipolytic enzyme(HSL),phosphorylated HSL(Ser563 and Ser660),and uncoupling protein 1(UCP1),and upstream triacylglyceride lipase(ATGL).5.Biochemical detection:The high-density lipoprotein cholesterol(HDL-C),low density lipoprotein cholesterol(LDL-C),total cholesterol(total cholesterol,CHOL),triglyceride(TG),total bilirubin(TBIL),urea nitrogen(BUN),albumin(albmin,ALB),total protein(total protein,TP),aspartate aminotransferase(AST)and alanine aminotransferase(ALT)were detected using automatic biochemistry analyzer.6.Morphological examination:Cryosection of mouse liver tissue and 3T3-L1 cells were stained with oil red O to detect the fat contents.The paraffin sections of mouse adipose tissue were stained with HE.Result1.Effects of DEHP on body weight,fat content,organ coefficient of mice:Under normal feeding and high-fat diet feeding,mice were orally administered with different concentrations of DEHP for 12 weeks.Compared with the control group,the weight gain of 0.05 mg/kg.bw DEHP group was significantly higher(P<0.01);the weight gain of HFD group and 0.05 mg/kg.bw DEHP+HFD group was also significantly higher than that of the control group(P<0.01),but the difference between the two groups was not statistically significant(P>0.05).Compared with the control group,the weight of epididymal fat and abdominal wall fat in the 0.05 mg/kg.bw DEHP group was significantly increased(P<0.Ol),and the brown fat/body mass coefficient was decreased(P<0.05);HFD group,0.05 mg/kg.bw DEHP+HFD,500mg/kg.bw DEHP+HFD group showed significant increase in epididymal fat and abdominal wall fat(P>0.01),but there was no significant difference between the groups(P>0.05);The brown fat of the experimental animals was increased(P<0.01).Compared with the control group,0.05mg/kg.bw DEHP liver and spleen weight increased(P<0.05,P<0.01);500mg/kg.bw DEHP group 500mg/kg.bw DEHP group increased liver and kidney weight(P<0.05,P<0.01),testis was significantly reduced(P<0.05,P<0.0 1).HE staining of adipose tissue paraffin sections revealed that DEHP can increase the volume of white fat cells.Liver oil red O staining showed that 0.05 mg/kg.bw DEHP significantly increased fat deposition in the liver.Biochemical tests showed that serum CHOL levels were significantly higher in the HFD group,0.05 mg/kg.bw DEHP+HFD group and 500 mg/kg.bw DEHP+HFD group than in the control group(P<0.01).Compared with the control group,the HDL-C of the 0.05 mg/kg.bw DEHP group and the 500 mg/kg.bw DEHP group and all the high fat diet group were decreased(P<0.01),and the higher the DEHP dose,the HDL-The lower the level of C;the change of animal serum LDL-C is exactly the opposite.In the 0.05mg/kg.bw DEHP group and the 500mg/kg.bw DEHP group compared with the control,the LDL-C level increases with the increase of DEHP dose.They gradually increased(P<0.05,P<0.01),and the levels of LDL-C in the high-fat diet animals were much higher than those in the control group(P<0.01).2.The effect of DEHP on glucose tolerance and insulin sensitivity in mice:GTT showed that the AUC in the experimental animals in the HFD group,0.05 mg/kg.bw DEHP+HFD group and 500 mg/kg.bw DEHP+HFD group was significantly higher than that of the control group(P<0.05,P<0.01),0.05mg/kg.bw and 500mg/kg.bw DEHP exposure can significantly increase the blood glucose level of experimental animals at 15min,30min and 60min under high-fat diet(P<0.05,P<0.01).Under normal feed feeding conditions,0.05mg/kg.bw and 500mg/kg.bw DEHP had no significant effect on glucose tolerance.Statistical analysis showed that there was a synergistic effect between the DEHP of 0.05 mg/kg.bw and 500 mg/kg.bw and the high-fat diet on blood glucose levels at 30 min and 60 min(P<0.05).The ITT results showed that the HFD could lead to insulin resistance in experimental animals,in which 0.05 mg/kg.bw and 500 mg/kg.bw DEHP were simultaneously exposed,and the insulin resistance of the experimental animals was more pronounced.Under normal feed feeding conditions,0.05 mg/kg.bw and 500 mg/kg.bw DEHP had no significant effect on insulin sensitivity.Statistical analysis showed a synergistic effect on the level of glucose in the blood at 30 min between DEHP at 0.05 mg/kg.bw and 500 mg/kg.bw and a high-fat diet(P<0.05).3.The effect of DEHP on adipogenic differentiation of 3T3-L1 cells:The differentiation rate of 3T3-L1 cells in DEHP was higher than that in the control,and the increase in the 12.5?M/L DEHP group was the most obvious,and the elevated level was close to 38%(P<0.01).By observing the time series of adipogenic differentiation of cells,it was found that the fat droplets in the 12.5 ?M/L DEHP group appeared at the earliest,about the fifth day of the replacement of the induction solution 1,and the other groups basically observed more fat droplets on the sixth day..The 3T3-L1 differentiated cells stained with oil red O were stained with isopropanol,and the triglyceride,free fatty acid and glycerol were measured on the eluted components.The results showed that the three indicators of DEHP exposed cells were higher than the control;The 12.5?M/L DEHP group had the most significant increase,and the difference was statistically significant compared with the control group(P<0.01)4.The effect of DEHP on the protein related to cell fat decomposition pathway in experimental animals and in vitro culture:Western Blot results of abdominal adipose tissue showed that DEHP down-regulated the expression of PKA,and the 500 mg/kg.bw DEHP group was down-regulated by 35.7%.The difference was statistically significant(P<0.01).The phosphorylation level of HSL was decreased,p-HSL(ser660)and p-HSL ser563 were significantly down-regulated compared with the control,and the p-HSL(ser563)of 0.05 mg/kg.bw DEHP group was down-regulated by nearly 100%(P<0.01).The p-HSL(ser660)in the 500 mg/kg.bw DEHP group was down-regulated by 27.2%(P<0.05).Compared with the control group,the level of UCP1 in the white fat of DEHP-treated animals was significantly down-regulated,and the 0.05 mg/kg.bw DEHP group and the 500 mg/kg.bw DEHP group were down-regulated by 46.1%(P<0.01)and 62.8%,respectively.0.01).The expression of various proteins in brown fat and epididymal fat of mice was not significantly changed.Immunofluorescence staining of 3T3-L1 cells showed that the level of UCP1 in the cells treated with 25?M/L and 50 uM/L DEHP was lower than that of the control.Conclusion1.Long-term exposure to DEHP leads to an increase in body weight and white adipose tissue in mice,leading to insulin resistance and elevated blood lipids.2.DEHP can promote the differentiation of pre-adipocytes into fat cells and increase the body fat.3.DEHP decrease the lipolysis,leading to fat deposition in the body.