| Objective This study explored the effects of BPA exposure on insulin sensitivity and ceramide(cer)de novo synthesis in mice on the basis of HFD feeding,and used SPT inhibitors to clarify the specific role of Cer de novo synthesis in BPA-exposed HFD fed mice to induce IR.Method Three-week-old,specific pathogen free(SPF)-grade wild-type male C57 BL / 6 mice after one week of adaptive feeding were randomly divided into 5 groups: normal control diet(NCD)group,HFD group,HFD + 1000 n M BPA group,HFD + SPT inhibitor group,HFD + 1000 n M BPA + SPT inhibitor group,12 mice in each group,6 mice at each time point at 8 weeks and 16 weeks.BPA intake in drinking water of mice.0.3 mg/kg myriocin was injected into the SPT inhibitor group once every two days for the 8-week group was performed at 5-8 weeks,and the 16-week group was performed at 13-16 weeks of BPA exposure,15 times each.At 8 and 16 weeks,GTT and ITT experiments were performed.Mice in each group were sacrificed after anesthesia,take the tail blood of the mice,ELISA was to check the insulin contents in mouse.Epithelial adipose tissue was taken aseptically.Immunohistochemistry(IHC)was used to detect the expression of inflammatory factors IL-1β and TNF-α in adipose tissue of mice UPLC-MS was used to detect Cer content in adipose tissue of mice.Western blot detection of Cer de novo synthesis of mouse adipose tissue and content of insulin signaling pathway related proteins.Levels of Cer de novo synthesis related SPTLC1,SPTLC2 m RNA was checked by RT-PCR.Results 1 General state of mice The mice in each group grew well during the experiment.At the same time point,the mice in each group had no difference in average weekly water intake and food consumption(P> 0.05).2 Effects of SPT inhibitors on body weight of mice exposed to BPA exposed to HFD at different time points.2.1 Effects of SPT inhibitors on body weight of mice exposed to BPA exposed to HFD for 8 weeks.Groups of mice body weight increased progressively with increasing time.At the same time point,HFD + 1000 n M BPA group mice gained weight significantly higher than that NCD group from the 5th week(P <0.05).SPT inhibitor reduced the weight of HFD-fed mice at the 8th week(P <0.05);SPT inhibitor reversed the increase in body weight of BPA exposure HFD-fed mice from from the 7th week(P <0.05).2.2 Effects of SPT inhibitors on body weight of mice exposed to BPA exposed to HFD for 16 weeks.Groups of mice body weight increased progressively with increasing time.At the same time point,HFD group mice gained weight significantly higher than that NCD group from the 9th week(P <0.05).The body weight of mice in the HFD + 1000 n M BPA group graeter significantly than HFD group from the 14 th week.SPT inhibitor reduced the weight of HFD-fed mice at the 15 th week(P <0.05);SPT inhibitor reversed the increase in body weight of BPA exposure HFD-fed mice from from the 15 th week(P <0.05).3 Effects of SPT inhibitors on epididymal fat pad coefficient in BPA exposure HFD-fed mice The coefficient of epididymal fat pad in HFD group was higher than that in NCD group at 8 and 16 weeks(P <0.05).The epididymal fat pad coefficient of mice in the HFD + SPT inhibitor group reduced significantly,and the epididymal fat pad coefficient of mice in the HFD + 1000 n M BPA group raised significantly than that HFD group(P <0.05).The coefficient of epididymal fat pad in HFD + 1000 n M BPA + SPT inhibitor group was significantly lower than that in HFD + 1000 n M BPA group.At different time points,the epididymal fat pad of mice in HFD group and HFD + 1000 n M BPA group was significantly higher at 16 weeks than at 8 weeks(P <0.05).4 Effects of SPT inhibitors on FG,FI and HOMA-IR in BPA exposure HFD-fed mice The values of FG,FI and HOMA-IR were graeter in the HFD group than the NCD group at 8 and 16weeks(P <0.05).The FG,FI and HOMA-IR levels in the HFD + 1000 n M BPA group elevated significantly than HFD group at 8 and 16 weeks,while the FG,FI and HOMA-IR values in the HFD + SPT inhibitor group significantly reduced(P <0.05).The FG,FI,and HOMA-IR levels of mice in the HFD + 1000 n M BPA + SPT inhibitor group at week 8 and 16 were significantly lower than those in the HFD + 1000 n M BPA group(P <0.05).5 Effects of SPT inhibitors on GTT and ITT in BPA exposure HFD-fed mice Mice were injected with glucose intraperitoneally,the blood glucose concentration of mice in all groups enhanced significantly at 8 and 16 week,and reached a peak at 15 min,then decreased gradually.At 120 minutes after glucose injection,the blood glucose content of mice in NCD group,HFD + SPT inhibitor group,and HFD + 1000 n M BPA + SPT inhibitor group all returned to baseline levels.The blood glucose concentration in the HFD group and HFD + 1000 n M BPA group was still higher than the baseline level.At 8 and 16 weeks,the area under the GTT curve was graeter in the HFD group than the NCD group(P <0.05).The AUC of GTT in mice of the HFD + 1000 n M BPA group elevated significantly,while that in the AUC of GTT in the mice in the HFD + SPT inhibitor group decreased significantly at 8 and 16 weeks(P <0.05).At 8 and 16 weeks,the AUC of GTT in HFD + 1000 n M BPA + SPT inhibitor group was significantly lower than that in HFD + 1000 n M BPA group(P <0.05).Mice were injected with insulin,the blood glucose concentration of mice in all groups reduced significantly at 8 and 16 week,and then decreased to the lowest value at 30 minutes,then improved gradually.The blood glucose content of mice in NCD group,HFD + SPT inhibitor group,and HFD + 1000 n M BPA + SPT inhibitor group all returned to baseline levels at 120 minutes.The blood glucose concentration in the HFD group and HFD + 1000 n M BPA group was still higher than the baseline level.At 8 and 16 weeks,the area under the GTT curve was still graeter in the HFD group than the NCD group(P <0.05).The AUC of GTT in mice in the HFD + 1000 n M BPA group elevated significantly,while that the HFD + SPT inhibitor group lower than HFD group at 8 and 16 weeks(P <0.05).At 8 and 16 weeks,the AUC of GTT in HFD + 1000 n M BPA + SPT inhibitor group was significantly lower than that in HFD + 1000 n M BPA group(P <0.05).6 Effects of SPT inhibitors on inflammatory cell infiltration in epididymal adipose tissue of BPA exposure HFD-fed mice Compared to the NCD group,the adipocytes of the mice in the HFD group improved significantly,and the infiltration of intercellular inflammatory mediators enhanced significantly at 8 and 16 weeks.Compared to the HFD group,the infiltration of inflammatory mediators in the adipocytes in the HFD + 1000 n M BPA group was significantly worsed,while that in the HFD + SPT inhibitor group was significantly reduced at 8 and 16 weeks(P <0.05).At 8 and 16 weeks,the size of adipocytes and infiltration of inflammatory mediators in adipose tissue in HFD + 1000 n M BPA + SPT inhibitor group were significantly lower than those in HFD + 1000 n M BPA group(P <0.05).The same group at different time points,the infiltration of interstitial inflammatory mediators in the adipose tissue of mice in the HFD group and HFD + 1000 n M BPA group at 16 weeks was more serious than that in 8 weeks,but there was no difference in the NCD group,HFD + SPT inhibitor group,and HFD +1000 n M BPA + SPT inhibitor group.7 Effects of and SPT inhibitors on expression of inflammatory factors in epididymal adipose tissue of BPA exposure HFD-fed mice Mouse epididymal adipose tissue inflammatory factors IL-1β and TNF-α are mainly expressed in the intercellular space of adipocytes.The IL-1β and TNF-α contents of mice adipose tissue with HFD group were more graeter than NCD group at 8 and 16 weeks(P <0.05).The content of inflammatory factors IL-1β and TNF-α in mice adipose tissue of the HFD + 1000 n M BPA group was enhanced,while that in the HFD + SPT inhibitor group significantly reduced at 8 and 16 weeks(P <0.05).At different time points,the expression of inflammatory factors IL-1β and TNF-α in adipose tissue of mice in the HFD group and HFD + 1000 n M BPA group at 16 weeks was more serious than that in 8 weeks,but there was no difference in the NCD group,HFD + SPT inhibitor group,and HFD +1000 n M BPA + SPT inhibitor group.8 Effects of SPT inhibitors on the expression of key enzymes in the de novo synthesis of Cer in the epididymal adipose tissue of BPA exposure HFD-fed mice The protein expression of SPTLC2 in mouse adipose tissue was not statistically different in each group at 8 and 16 weeks(P> 0.05).The protein expression of SPTLC1 in HFD group was greater than that of NCD group at 8 and 16 weeks(P <0.05).The protein content of SPTLC1 in mice adipose tissue of the HFD + 1000 n M BPA group was enhanced,while that in the HFD + SPT inhibitor group was reduced at 8 and 16 weeks(P <0.05).At the same time,the protein expression of SPTLC1 in mice adipose tissue with HFD + 1000 n M BPA + SPT inhibitor group was significantly lower than that of HFD + 1000 n M BPA group at 8 and 16 weeks(P <0.05).9 Effects of SPT inhibitors on epididymal adipose tissue SPTLC1 and SPTLC2 m RNA expression in BPA exposure HFD-fed mice The m RNA expression of SPTLC2 in mouse adipose tissue was not statistically different in each group at 8 and 16 weeks(P> 0.05).The m RNA expression of SPTLC1 in mice adipose tissue was greater in HFD group than NCD group at 8 and 16 weeks(P <0.05).The m RNA expression of SPTLC1 in mice adipose tissue in the HFD + 1000 n M BPA group was elevated,while that in the HFD + SPT inhibitor group was reduced at 8 and 16 weeks(P <0.05).At the same time,the m RNA expression of SPTLC1 in mice adipose tissue with HFD + 1000 n M BPA + SPT inhibitor group was significantly lower than that of HFD + 1000 n M BPA group at 8 and 16 weeks(P <0.05).10 Effects of SPT inhibitors on Cer content in epididymal adipose tissue of BPA exposure HFD-fed mice The attack time of C16 Cer(d18: 1/16: 0),C17 Cer(d18: 1/17: 0),C18 Cer(d18: 1/18: 0),C20 Cer(d18: 1/20: 0),C24: 1 Cer(d18: 1/24: 1(15Z))and C24 Cer(d18: 1/24: 0)is 5.00 s,5.22 s,5.43 s,5.82 s,6.21 s,6.71 s respectively.The content of C16 Cer(d18: 1/16: 0)and total Cer in mice adipose tissue was more in HFD group than NCD group at 8 and 16 weeks,while that of C20 Cer(d18: 1/20: 0)was greater at 16 weeks(P <0.05).The content of C16 Cer(d18: 1/16: 0),C24: 1 Cer(d18: 1/24: 1(15Z)),C24 Cer(d18: 1/24: 0)and total Cer content in adipose tissue of mice in the HFD + 1000 n M BPA group increased significantly at 8 and 16 weeks,while that of C20 Cer(d18: 1/20: 0)was diferent at 16 weeks,moreover,the contents of various Cer and total Cer in adipose tissue of mice in the HFD + SPT inhibitor group decreased significantly at 8 and 16 weeks(P <0.05).At the same time,the contents of various Cer and total Cer in adipose tissue of mice in HFD + 1000 n M BPA + SPT inhibitor group were lower than those in HFD + 1000 n M BPA group at 8 and 16 weeks(P <0.05).11 Effects of SPT inhibitors on expression of insulin signaling pathway proteins in epididymal adipose tissue of BPA exposure HFD-fed mice The protein expression levels of IRS1,PI3 K,and AKT in mice adipose tissue in each group were not different at 8 and 16 weeks(P>0.05).The protein expression of p-IRS1,p-PI3 K,and p-AKT in adipose tissue of mice in HFD group was significantly smaller than that in NCD group at 8 and 16 weeks(P<0.05).The p-IRS1,p-PI3 K,and p-AKT protein expression in mice adipose tissue of the HFD + 1000 n M BPA group was significantly reduced,while that in the HFD + SPT inhibitor group was significantly elevated at 8 and 16 weeks(P<0.05).At the same time,the protein expression of p-IRS1,p-PI3 K,and p-AKT in mice adipose tissue with HFD + 1000 n M BPA + SPT inhibitor group was significantly higher than that of HFD + 1000 n M BPA group at 8 and 16 weeks(P<0.05).Conclusions 1 Long-term low-dose BPA exposure promotes weight gain,increases adipose tissue infiltration of inflammatory cells,and up-regulates the expression of proinflammatory cytokines IL-1β and TNF-α in HFD-fed mice.2 Long-term low-dose BPA exposure reduces insulin sensitivity in HFD-fed mice,up-regulates SPTLC1 expression,stimulates Cer de novo synthesis,and leads to Cer accumulation in adipose tissue in HFD-fed mice.3 SPT inhibitors down-regulate SPTLC1 protein expression in adipose tissue of mice,inhibit Cer from de novo synthesis,reverse BPA exposure to IR induced by HFD-fed mice,improve glucose intolerance,and increase insulin sensitivity. |
