生命早期双酚A暴露联合断乳后高脂饮食对小鼠糖脂代谢的影响及其机制

Effects of early-life bisphenol A exposure combined with post-weaning high-fat diet on glucose and lipid metabolism in mice and their mechanisms

目的:研究生命早期双酚A(BPA)暴露联合断乳后高脂饮食对小鼠糖脂代谢的影响,并探讨其发生机制。方法:在母鼠受孕第6天至子代小鼠断乳前按0.5 mg/(kg·d)对母鼠进行双酚A饮水染毒,分成未染毒组和BPA组,每组10只孕鼠。子代小鼠断乳后终止BPA染毒,并进行高脂饮食干预,分成普通饮食未染毒组、普通饮食BPA组、高脂饮食未染毒组和高脂饮食BPA组,每组10只,雌雄各半。在子代小鼠出生后第21天(PND21)和第91天(PND91)时处死小鼠并收集血液和胰腺组织样本,PND91小鼠在处死前进行葡萄糖耐量和胰岛素耐量试验,采用高分辨液质联用仪对小鼠胰腺进行非靶向脂质组学检测,实时荧光定量PCR法(qPCR)检测小鼠胰腺神经酰胺合成基因的mRNA表达水平,ELISA试剂盒检测小鼠血浆胰岛素和胰腺神经酰胺合成酶(Cers)活性,试剂盒检测小鼠胰腺丙二醛(MDA)含量和超氧化物歧化酶(SOD)活性。结果:PND21时,与未染毒组相比,BPA暴露组子代雄性和雌性小鼠均出现体质量增长加快。PND91时,与高脂饮食未染毒组相比,高脂饮食BPA组雄性小鼠体质量增加(P<0.05)。PND91小鼠的葡萄糖耐量和胰岛素耐量试验结果显示,与高脂饮食未染毒组相比,高脂饮食BPA组雄性和雌性小鼠血糖曲线下面积均显著增加(P<0.05)。计算小鼠胰岛素抵抗指数发现,与高脂饮食未染毒组相比,PND91高脂饮食BPA组雄性小鼠胰岛素抵抗指数升高(P<0.05)。胰腺的非靶向脂质组学检测结果表明,PND21小鼠中,与未染毒组相比,BPA组雄性和雌性小鼠神经酰胺(Cer)丰度显著升高(P<0.05);PND91小鼠中,与高脂饮食未染毒组相比,高脂饮食BPA组雄性小鼠神经酰胺(Cer)丰度显著升高(P<0.05)。胰腺qPCR检测结果发现,PND21小鼠中,与未染毒组相比,BPA组雄性小鼠Cers4和Cers6 mRNA的表达上调,雌性小鼠Cers4mRNA的表达上调(P<0.05);PND91小鼠中,与高脂饮食未染毒组相比,高脂饮食BPA组雄性小鼠Cers2、Cers4和Cers6 mRNA的表达上调(P<0.05)。胰腺神经酰胺合成酶活力检测结果显示,PND21小鼠中,与未染毒组相比,BPA组雄性小鼠Cers2、Cers4和Cers6活力显著增强(P<0.05);PND91小鼠中,与高脂饮食未染毒组相比,高脂饮食BPA组雄性小鼠Cers2和Cers4活力显著增强(P<0.05)。氧化和抗氧化指标检测结果显示,PND91小鼠中,与高脂饮食未染毒组相比,高脂饮食BPA组雄性小鼠胰腺MDA含量显著增加、SOD活力显著降低(P<0.05)。相关性分析表明,PND91雄性小鼠胰腺神经酰胺丰度与胰腺氧化和抗氧化比值存在显著的正相关关系(P<0.05)。结论:生命早期BPA暴露联合断乳后高脂饮食损害了子代小鼠葡萄糖耐量和胰岛素耐量,并以性别特异性方式增加子代雄性小鼠胰腺神经酰胺从头合成,后者与雄性小鼠胰腺氧化和抗氧化失衡密切相关。

OBJECTIVE:To investigate the effects of early-life Bisphenol A(BPA)exposure on glucose and lipid metabolism in post-weaning mice fed a high-fat diet.METHODS:Pregnant mice were exposed to BPA through drinking water at a concentration of 0.5 mg/(kg·d)from gestational day 6 until weaning of offspring.Twenty pregnant mice were divided equally into control and BPA groups.The weaned offspring were organized into,four groups:normal diet control,normal diet BPA,high-fat diet control,and high-fat diet BPA,with 10 mice per group,balanced by sex.Mice were sacrificed at postnatal day 21(PND21)and postnatal day 91(PND91)for the collection of blood and pancreas samples.Before sacrificing the PND91 mice,they were tested for glucose and insulin tolerance.Non-targeted lipidomics of the pancreas was analyzed using high-resolution liquid chromatography-mass spectrometry.qPCR was employed to measure mRNA expression levels of ceramide synthesis genes in the pancreas.Plasma insulin and pancreatic ceramide synthase activity were assessed using ELISA kits.Malondialdehyde(MDA)and superoxide dismutase(SOD)activities in the pancreas were measured using specific assay kits.RESULTS:At PND21,both male and female offspring in the BPA exposure group exhibited accelerated weight gain compared to the control group.At PND91,male mice in the high-fat diet BPA group showed increased body weight compared to the high-fat diet control group(P<0.05).Results of glucose tolerance and insulin tolerance tests at PND91 indicated that,compared to the high-fat diet control group,both male and female mice in the high-fat diet BPA group had significantly increased areas under the blood glucose curve(P<0.05).Calculating the insulin resistance index in mice,it was found that,male mice in the PND91 high-fat diet BPA group had an elevated insulin resistance index(P<0.05).Non-targeted lipidomics of the pancreas revealed that at PND21,compared to the control group,male and female mice in the BPA group had significantly increased ceramide(Cer)abundance(P<0.05).At PND91,compared to the high-fat diet control group,male mice in the high-fat diet BPA group had significantly increased ceramide abundance(P<0.05).Results from pancreatic qPCR showed that at PND21,compared to the control group,male mice in the BPA group had upregulated mRNA expression of the Cers4 and Cers6 genes,while female mice had upregulated Cers4 gene expression(P<0.05).At PND91,compared to the high-fat diet control group,male mice in the high-fat diet BPA group had upregulated mRNA expression of the Cers2,Cers4,and Cers6 genes(P<0.05).Analysis of ceramide synthase activity in the pancreas indicated that at PND21,compared to the control group,male mice in the BPA group had significantly enhanced Cers2,Cers4,and Cers6 activity(P<0.05).At PND91,compared to the high-fat diet control group,male mice in the high-fat diet BPA group had significantly enhanced Cers2 and Cers4 activity(P<0.05).Assessment of oxidative and antioxidative indicators showed that at PND91,compared to the high-fat diet control group,male mice in the high-fat diet BPA group had significantly pancreatic MDA content and significantly decreased SOD activity(P<0.05).Correlation analysis indicated aincreasedsignificantly positive correlation between ceramide abundance and the oxidative/antioxidative ratio in the pancreas of PND91 male mice(P<0.05).CONCLUSION:Early-life BPA exposure combined with post-weaning high-fat diet impaired glucose tolerance in offspring mice.It increased the de novo synthesis of pancreatic ceramide in male offspring mice in a sex-specific manner,which was closely related to the imbalance of oxidative and antioxidative status in the pancreas of male mice.