Influence Of Triclosan On Glycometabolismin Pregnant And Non-pregnant Mice And The Underlying Mechanisms

Gestational diabetes mellitus(GDM)diagnostic criteria is that diabetes is firstly determined at the first prenatal visit and the whole duration of pregnancy,the women may have previously undiagnosed type 2 diabetes.More than 80% of diabetes duration of pregnancy is reported to be gestational diabetes mellitus.The prevalence of gestational diabetes mellitus may range from 4.3% to 17.5% of pregnant women in China.Glucose metabolism in GDM is most likely to return to normal after delivery,but it will increase the risk of type 2 diabetes in the future.The increase of persistent blood glucose levels can affect the development of the fetus and the metabolism of glucose after birth.The advanced maternal age,pre-pregnancy overweight or obesity and a family history of diabetes have been confirmed to be risk factors for GDM.Recent studies have focused on the effects of environmental factors on GDM.Triclosan(TCS),"dichlorophenol",is an antibacterial agent that is widely used in soaps,toothpastes,first-aid products,fabrics,and plastic goods.Epidemiological data suggest that TCS levels in patients with GDM were significantly higher than those in normal pregnancy.However,whether TCS exposure during pregnancy could cause abnormal glucose metabolism has not been reported yet with its mechanism.In non-pregnant women,TCS levels were negatively correlated with thyroid hormone levels.The treatment with TCS decreased total serum T3 and T4 in rats and mice that during pregnancy and non-pregnancy.Epidemiological data showed that GDM was associated with low thyroid hormone in the middle and late pregnancy,suggesting that hypothyroidism is one of the risk factors for GDM.Glucose transporter 4(GLUT4)is the predominant insulin-responsive glucose transporter isoform.It plays a key role in the process of transporting extracellular glucose into insulin-sensitive cells.Importantly,thyroid hormone is known to up-regulate the expression of GLUT4,thereby increasing insulin-stimulated glucose uptake.Previous studies in our laboratory have found that the exposure of TCS in pregnant mice reduces the activity and expression of the placental cell glucose transporter 2(GLUT2).Pregnant women undergoes significant physiologic changes in peripheral insulin resistance,decreases glucose tolerance and enhances insulin secretion,thus can be potentially sensitive and vulnerable to the influence from environmental estrogenic chemicals.Therefore,it is proposed that whether exposure to TCS during pregnancy can affect the glycometabolism through reducing thyroid hormones,to induce insulin resistance,a phenotype of gestational diabetes mellitus(GDM).Objective To elucidate influence of triclosan on glycometabolism when is exposed in pregnant and non-pregnant mice and the underlying mechanisms.Materials and methods 1.Experimental animal handling: ICR mice were given 1,4 and 8 mg/kg dose of TCS from gestation day(GD)5-17 as experimental group to build 1-TCS pregnant mice,4-TCS pregnant mice and 8-TCS pregnant mice.The non-pregnant mice were given 13 consecutive days of 8 mg/kg dose TCS,and an 8-tcs non-pregnant mouse model was constructed.The pregnant mice with the same amount of tea oil were treated as the control pregnant mice,and the non-pregnant rats were given the same amount of tea oil.Check the weight of the mice daily.Glucose metabolism was detected on the 17 th day of pregnancy.2.Enzyme linked immunosorbent assay(ELISA)was used to examine levels of serum glucose,t T4,f T4,t T3,estrogen(E2)and progesterone(P4).3.Measuring fasting insulin levels measure: Insulin levels were detected after fasting for 12 hours with glucose oxidase.We calculates ? cell function index(HOMA-?)and insulin resistance index(HOMA-IR).4.Glucose tolerance test(GTT): after fasting 12 hours,intraperitoneal injection of 2 g/kg glucose,respectively before glucose injection(0 minutes)and after glucose injection for 15,30,60,120 minutes from the tail vein blood measuring blood sugar levels,and calculate the area under the curve(AUC)5.Insulin tolerance test(ITT): after fasting 12 hours,intraperitoneal injection of 1 U/kg glucose insulin,respectively before glucose injection(0 minutes)and after glucose injection for 15,30,60,120 minutes from the tail vein blood measuring blood sugar levels,and calculate the area under the curve(AUC)6.The pancreatic tissue was fixed with 10% Bouin's solution,and paraffin embedding was carried out.Insulin-antibody was used for islet immunohistochemistry,and the light density(IOD)was calculated.7.Levels of Akt phosphorylation,and peroxisome proliferator activated receptor ?(PPAR?)protein were examined by western blotting analysis.8.Levels of PPAR? and GLUT4 m RNA were measured by RT-PCR.9.Drug treatment: L-T4 was injected with the dose of 20ug/kg.The PI3 K inhibitor LY294002 was administered with a dose of 10mg/kg.For the purpose of using 5mg/kg dosage form,we can use the dosage form.Both pregnant and non-pregnant rats were treated with the same amount of solvent.Results 1.In comparison with 0-TCS G-mice and 0-TCS Ng-mice,8-TCS G-mice showed a significant increase in fasting blood glucose level,while 1-TCS G-mice and 4-TCS G-mice and the 8-TCS Ng-mice did not show the changes.2.The results of glucose tolerance test(GTT)showed that the glucose removal time in 8-TCS G-mice was significantly longer than that in 0-TCS G-mice,while it was changes in 1-TCS G-mice and 4-TCS G-mice and the 8-TCS Ng-mice.The results indicate that exposure to TCS(8mg/kg)during pregnancy results in reduced glucose tolerance.3.In comparison with 0-TCS G-mice and 0-TCS Ng-mice,8-TCS G-mice showed a significant increase in fasting serum insulin levels,while 1-TCS G-mice and 4-TCS G-mice and the 8-TCS Ng-mice did not.By islet immunohistochemical staining with insulin antibody,we observed an elevation of the insulin immunoreactive IOD in 8-TCS G-mice.HOMA-? in 8-TCS G-mice is reduced.4.The results of the insulin tolerance test(ITT)showed that the glucose level after insulin injection in 8-TCS G-mice was significantly higher than that in 0-TCS G-mice,but 1-TCS G-mice and 4-TCS G-mice and 8-TCS Ng-mice did not significant difference from controls.In addition,the decline of HOMA-IR was found in8-TCS G-mice.The results indicate that exposure to TCS(8mg/kg)during pregnancy results in caused insulin resistance.5.In comparison with 0-TCS G-mice and 0-TCS Ng-mice,the levels of serum t T3,t T4,and f T4 in 4-TCS G-mice and 8-TCS G-mice or 8-TCS Ng-mice were reduced,which were associated with the changes in the levels of E2 and P4.6.The treatment with thyroid hormone(L-T4)for 13 days in 8-TCS G-mice can reduce the levels of glucose and insulin,and recovered the HOMA-? and HOMA-IR and restored the glucose clearance and insulin tolerance.7.The levels of PPAR? m RNA and PPAR? protein in muscle and adipose tissue were significantly reduced in 8-TCS G-mice,but not in 1-TCS G-mice and 4-TCS G-mice or 8-TCS Ng-mice.The treatment with L-T4 in 8-TCS G-mice could recover the expression of PPAR?.The results indicate that exposure to TCS(8mg/kg)during pregnancy through reducing thyroid hormones depresses the expression of PPAR?.8.The level of Akt phosphorylation in muscle and adipose tissue was reduced in 8-TCS G-mice,but not in 1-TCS G-mice and 4-TCS G-mice or 8-TCS Ng-mice.The administration of the PPAR? agonist rosiglitazone or L-T4 in 8-TCS G-mice corrected the decline in the level of Akt phosphorylation.9.The level of GLUT4 m RNA in muscle and adipose tissue was reduced 8-TCS G-mice,but not in 1-TCS G-mice and 4-TCS G-mice or 8-TCS Ng-mice.The administration of L-T4 in 8-TCS G-mice corrected the decrease in the GLUT4 expression,but rosiglitazone had no effect.10.In comparison with 0-TCS Ng-mice,the levels of PPAR? and GLUT4 expression or Akt phosphorylation in muscle and adipose tissue were unchanged in 8-TCS Ng-mice.Conclusion(1)The exposure to TCS(8mg/kg)during pregnancy through reducing thyroid hormones suppresses the levels of PPAR? and GLUT4 expression leading to insulin resistance,a phenotype of gestational diabetes mellitus(GDM).(2)Although the exposure to TCS(8mg/kg)reduces thyroid hormones in non-pregnant mice,it does not influence the glucose clearance and insulin tolerance.