| Background:Gestational diabetes mellitus(GDM)is a type of diabetes mellitus that occurs only during pregnancy when glucose metabolism is normal before pregnancy,it is a gestational complication that usually occurs in the middle and late pregnancy.Its frequency varies by race.It is reported that about 6%to 9%of pregnant women have diabetes,of which GDM accounts for about 90%.GDM has many adverse effects on pregnant women,fetus and newborn.For pregnant women,it can lead to miscarriage of pregnant women,increased incidence of hypertension during pregnancy,ketoacidosis,and polyhydramnios;For fetus,it can cause increased incidence of fetal macrosomia,fetal growth restriction,premature delivery,fetal distress,fetal malformation;For the newborn,it can increase the incidence of neonatal respiratory distress syndrome and neonatal hypoglycemia.At present,the incidence of GDM is increasing worldwide,but the clinical drugs for GDM are very limited.Resveratrol(RV)is a kind of natural compounds,which has been widely confirmed to be beneficial in treatment of type Ⅱ diabetes.However,there is still a lack of relevant fundamental research on whether resveratrol has beneficial effects on GDM mothers and offspring.Amp-activated protein kinase(AMPK)is a very important cell energy metabolism receptor.The activation of AMPK can inhibit gluconeogenesis and fat synthesis and promote fat catabolism.Therefore,AMPK can be an effective target for the treatment of diseases such as obesity and diabetes,and AMPK agonist is widely used in the treatment of type Ⅱ diabetes.However,there is a lack of relevant basic research on the effect of AMPK signaling pathway on GDM.The C57BL/KsJ-Lepdb/+(db/+)mouse is a model with a heterozygous mutation in the leptin receptor gene Lepr,which can closely simulate symptoms of GDM patients.At the non-pregnant state,their blood sugar and insulin resistance were normal.During pregnancy,the db/+females presented typical GDM symptoms including hyperglycemia,insulin resistance and obesity,which also have adverse effects on fetal development,such as increased body weight of offspring.In this study,we will investigate the effect and mechanism of resveratrol on maternal glycolipid metabolism and pregnancy outcomes of GDM mice by studying the db/+GDM mouse model.Methods:1.Controlled trials are taken between normal gestational placental tissue and GDM placental tissue,GLUTs expression was detected by RT-PCR and Western blot,and the expression of related protein of AMPK signaling pathway was analyzed.2.Establishment of gestational diabetes mouse model:C57BL/KsJ+/+(wild type)and C57BL/KsJdb/+(db/+)mice were divided into three groups according to different feeding methods:①wild type,free feeding group(WT group);②db/+paired feeding group(db/+group);③db/+paired feeding+RV group(RV group).3.Feeding and reproduction of mice:each group of mice were fed with the same diet as the WT group.During the study,mice in RV group were administered by oral gavage with resveratrol,solubilized in water.WT group and db/+group were also orally gavaged with water only daily,to serve as the control group.After the mice reached sexual maturity,the females in each group were mated with male mice of the same genotype,and the conception was recorded.The number of pregnant mice in each group was equal.4.Measurement of serum glucose,insulin and body weight:(1)Each group was accurately weighed by a top-loading balance;(2)Glucose tolerance and serum insulin levels test;(3)Insulin tolerance test.5.Determination of plasma triglyceride(TG)and leptin levels:blood was collected from the tail vein,plasma was obtained by centrifugation,and TG and leptin were determined by the ELISA kit.6.Determination of liver weight and TG in mice:(1)A fixed number of pregnant mice were randomly selected from each group to harvest the liver and weigh it;(2)Liver tissue lysis buffer was obtained and TG content was determined by the ELISA kit.7.Western blotting test of protein in mouse liver tissue:the expression of AMPK signaling pathway in the liver of the harvested mice was detected,while the remaining females completed pregnancy;the livers of the offspring were obtained by the same method;the activity of glucose-6-phosphatase(G6Pase)in the liver tissues of the offspring was detected.8.Reverse verification of AMPK signaling pathway effects:RV group were treated with AMPK inhibitor compound C(Compound C,CC)(db/+paired feeding+RV+CC group,RV+CC group).Blood glucose,serum insulin levels and body weight of mice in the RV+CC group were measured.The activity of G6Pase in the liver of all harvested pregnant mice was detected.After the completion of pregnancy,the number and birth weight of the offspring of each group were recorded.Results:1.The expression and activity of AMPK in GDM placenta decreased significantly compared with normal placentaRT-PCR and Western blot experiments of placental tissues showed that compared with the normal group,GLUT1 expression in placental tissues of the GDM group was significantly increased,GLUT4 expression was not significantly different,AMPK expression was significantly decreased,HDAC4 expression was significantly increased,and G6Pase expression was significantly increased.2.db/+mouse model is an effective animal model of GDMGlucose tolerance tests and serum insulin levels of the three groups of non-pregnant mice showed similar blood glucose and insulin levels.Glucose tolerance test conducted after pregnancy in mice showed that compared with WT group mice,glucose level in db/+group mice was significantly increased,indicating the presence of significant glucose intolerance,while RV treatment can significantly alleviate the glucose intolerance of mice in the RV group.Insulin levels of mice in db/+group was significant lower than that of WT group,which further demonstrated glucose intolerance.RV administration can restored the insulin response of RV group to the level as the WT group.Glucose tolerance test and serum insulin level detection in db/+group mice further proved that db/+mouse model is an effective animal model of GDM.3.RV can improve glycolipid metabolism and insulin resistance in pregnant db/+mice(1)Compared with mice in WT group,the glucose level of mice in the db/+group increased significantly after the first glucose injection,indicating the presence of significant glucose intolerance,while RV treatment can significantly alleviate the glucose intolerance of mice in the RV group.Insulin levels of mice in db/+group was significant lower than that of WT group,and RV administration can restored the insulin response of RV group to the level as the WT group.An acute insulin tolerance test in all three groups of mice proved that the mice in db/+group exhibited significantly higher blood glucose levels than the WT group following the initial insulin injection,indicating the presence of insulin resistance.While the RV treatment markedly reduced glucose levels in the RV group mice to similar levels as the WT group.Related indicators of fat metabolism of the three groups of mice were examined,including:body fat percentage,liver weight and liver triglycerides,plasma triglycerides,plasma free fatty acids and plasma leptin levels,and found that these indicators of the db/+group mice are significantly higher than that of the WT group,this indicated that the fat metabolism was abnormal,and the relevant indicators of RV group were similar to those of WT group,indicating that RV had an effect on improving fat metabolism of GDM mice.(2)The steady state levels of blood glucose and insulin levels were also measured throughout the pregnancy.It was found that mice in db/+group showed significantly elevated blood glucose levels,indicating the typical hyperglycemia symptom of GDM.However,blood glucose levels of mice in RV group stayed at the same as the WT group through the entire duration of pregnancy,indicating the alleviating effect on hyperglycemia by RV.Steady state insulin levels in db/+group mice were also significantly lower than WT group,which could be fully restored in RV group by RV administration.Maternal body weight gains in all three groups of mice were almost the same throughout the pregnancy.4.RV increased liver AMPK activation in pregnant db/+miceBy western blotting of the liver of mice,it was found that,AMPK activation was attenuated in mice of db/+group,contributing to higher HDAC4 activation,which in turn elevated G6Pase expression in the liver.RV administration can fully reversed the trend of activation profile of the related proteins,eventually reducing G6Pase levels of mice in RV group to almost the same as in WT group.5.RV reduced liver G6Pase activity in offspring of db/+miceIn this study,offspring of mice from each group were selected and their livers were collected for G6Pase activity determination.It was found that G6Pase activity of offspring of mice in db/+group was significantly increased,while G6Pase activity of offspring of mice in RV group was reduced due to the use of RV,which was similar to that in WT group.6.RV improved reproductive outcome of pregnant db/+miceBy calculating the number of offspring of the three groups of mice,it was found that:the number of offspring of mice in the RV group was very close to the WT group,and the number of offspring of mice in the db/+group was significantly reduced;The birth weight of the offspring in all three groups was further recorded and it was found that mean body weight of offspring born by mice in the db/+group was significantly higher than those by WT group,whereas body weight of offspring from mice in the RV group was almost the same as WT group.7.AMPK inhibitors can counteract the effect of RV on GDMIn this study,AMPK inhibitor CC was used to treat the RV group,and it was found that the blood glucose level,insulin level,liver G6Pase activity,offspring number and offspring weight of the RV+CC group mice were close to those of the db/+group mice.Therefore,AMPK inhibitors can counteract the improvement effect of RV on GDM,further proving that RV alleviates the phenotype of GDM by enhancing AMPK activity.Conclusions:1.Decreased AMPK activity in the placenta of pregnant women with GDM leads to increased HDAC4 expression,which in turn leads to increased G6Pase expression,which is one of the important manifestations of GDM.2.db/+mouse model is an effective animal model of GDM.RV greatly improved glycolipid metabolism,insulin tolerance and reproductive outcome of the pregnant db/+females.3.RV relieved GDM symptoms through enhancing AMPK activation,which in turn reduced production and activity of G6Pase in both pregnant db/+females and their offspring.AMPK inhibitors can counteract the improvement effect of RV on GDM. |
