The Effect Of Maternal Hypothyroidism On Glucose Metabolism In Offspring Rats

Background:Since the theory of fetal origins of adult disease(FOAD)was posed by Barker,much effort has been directed at understanding the relationship between maternal hypothyroidism and adult degenerative and metabolic diseases.Maternal thyroid hormone play an important role for development and metabolism in offspring.Research has linked the intrauterine growth retardation(IUGR)as well as decreased muscle mass to maternal hypothyroidism,and studies have also reported deficiencies in the development of the nervous system,skin,lungs and skeletal muscle.Recently,the relationship between maternal hypothyroidism and glucose metabolic abnormalities in adult offspring has been increasingly emphasized.However,the underlying mechanisms remain poorly understood though.It is classically known that most bioactive effect of thyroid hormone are mediated by Thyroid hormone receptor(THR).Recently,Bioactive thyroid hormone T3 was verified to effect insulin secretion and ? cell proliferation through thyroid hormone receptor ?(TR?),but it has not been reported that if intrauterine hypothyroidism could affect the pancreas islet function in the offspring.Objective:To better understand the mechanisms by which maternal hypothyroidism affects metabolic functioning in offspring,we used a rat model of anti-thyroid drug PTU induced maternal hypothyroidism to study the effects of intrauterine hypothyroidism on islet development in offspring,glucose metabolism as well as the expression of TRs and other relevant genes in pancreas of offspring.And to investigate whether T3 could promote MIN6 cells proliferation,insulin secretion and the possible mechanism involved,which is provide scientific theoretical basis and experimental evidence for prevention,treatment,research and drugs development for this kind of diabetes mellitus.Methods:In animal study,female Sprague-Dawley rats weighing 180-220 g were divided into a maternal hypothyroidism(MH)group,which received water containing 0.02%6-propyl-2-thiouracil before and during pregnancy to induce intrauterine hypothyroidism,and a control group which consumed only tap water.Thyroid hormone thyroxine(T4)and thyroid stimulaton hormone(TSH)were monitored during gestational period to evaluate if the model was established successfully.Then the thyroid function was test in mother after delivery to evaluate the lasting stability of this model.Pregnant rate,weight gain during pregnancy,birth weight and thyroid function were measured to evaluate the outcome from maternal hypothyroidism.Offspring in different observation period were subjected to an oral glucose tolerance test(OGTT,2 g/kg glucose).The pancreas in two groups were abstracted,and the mRNA and protein expression of proliferation marker protein Ki67,insulin l,insulin2,thyroid hormone receptor ?1(TR?1),and TR?2 in the pancreas of offspring was evaluated at each time point by RT-PCR,immunohistochemistry and immunofluorescence methods to explore the effect of maternal hypothyroidism on glucose metabolism and possible mechanism in offspring rats.In cells experiment,MIN6 cells were cultured by conventional methods.Then incubated with T3 in different concentration(10-8,10-7,10-6,10-5,10-4,10-3,10-2 mM)for 24,48 and 72h,respectively.The cells viability was detected by cell counting kit-8(CCK-8)assay to determine the optimal effective time is 72 hours and the optimal concentration is 10-4 mM.TR? stable expressing MIN6 cell lines were constructed,and cultrured MIN6 cells were randomly divided into four groups:null vector transfection as the controlcontrol group(NC),NC+T3 group,TR?transfected group(TR ?),TR?+T3 group.After intervention for 72h,CCK-8 assay was tested to evaluate cell viability in each group,GSIS was performed to evaluate insulin secretion function in MIN6 cells,while Ki67 mRNA,Insulinl mRNA and Insulin2 mRNA level was test by RT-PCR to trace the potential mechanism.Result:In animal study,pregnant rate in MH group was 36.6%,which is less than that from normal group in 70%(p<0.05).Maternal weight gain reduced in MH group conpaired with normal group(118.5±9.5 vs 154.8±9.7g,p<0.05).The birth weight of offspring in MH group was significantly lower than that in control group(5.43±0.10 vs 7.30±0.12g,p<0.001).And the weight of offspring from MH group was lower than control group before since 0-8 weeks old.The weight between the 2 groups at 4-week and 8-week old was(74.01±1.75 vs 86.78±2.80g,P<0.01),(130.4±11.48 vs 202.4±9.28g,p<0.001)separately.When the offspring were born,difference of the two groups of serum T4 levels has no statistics significance,but TSH level in MH group was higher than that in control group(3.08±0.05 vs 2.37±0.28,p<0.05).However,there was no statistical difference in TSH and T4 level between the two groups when the offspring grew up to 4 weeks.Besides,random blood glucose level of the neonatal in MH group is 7.05±0.21 mmol/L,which is higher than that in control group(6.47±0.10mmol/L,p<0.05).In the OGTT test,the blood glucose level at each time point has no statistical difference between the two groups in offspring at 4 weeks old.But the fasting blood glucose in MH group was higher than that in control group at 8-week old(8.13±0.38 mmol/L vs 6.63±0.19 mmol/L,P<0.05).After glucose loading,glucose metabolism was also impaired in 8-week old offspring of the MH group compared with that of control group as demonstrated by peak value concentrations at 15 min in the OGTT(17.84±0.81 mmol/L vs 14.34± 1.21mmol/L,p<0.05).From birth to 8 weeks,the islet morphology and structure in offspring in MH group were similar with that in normal group observed in HE staining method.But offspring of the MH group showed lower gene expression levels of Ki67,insulin 1,insulin2,TR?1 and TR?2 in the pancreas compared with offspring of the control group(p<0.001)by RT-PCR.And maternal hypothyroidism results in impaired pancreatic insulin secretion and pancreatic cell proliferation in each observational period.Immunohistochemistry of pancreas showed that insulin secretion,Ki67 expression diminished in offspring in MH group at each observation point.And at each point,the Ki67,Insulinl,Insulin2,TR?1,TR?2 mRNA levels in pancreas maintained lower level in offspring of MH group than that in control group.In vitro study,T3 can apparently elevate the MIN6 cells viability using CCK-8 test,which is time and concentration dependent,and the optimal concentration is 10-4 mM.The viability of TR? overexpressed MIN6 cells in TR? group is higher than NC group.In RT-PCR test,Ki67mRNA?Insulin1mRNA?Insulin2 mRNA and TR? mRNA expression in NC+T3 group MIN6 cells is higher than that in NC group.Ki67mRNA level in TR?+T3 group cell is higher than that in NC group(p<0.05).In GSIS experiment,Insulin secretion level in TR?+T3 group is much higher than NC group(p<0.05)?Conclusion:Maternal hypothyroidism led to reduced pregnant rate and maternal weight gain,IUGR and development of offspring,and may have adverse effect on pancreas development in offspring.T3 could promote the proliferation of MIN6 cells,and intrauterine hypothyroidism could lead to TR? gene down-regulation in the pancreas,which impaired pancreatic insulin synthesis and pancreatic cell proliferation in neonatal offspring and subsequent glucose intolerance in young adult offspring.Thus,maternal thyroid hormone contributes to offspring pancreas proliferation and development,and the underlying mechanism of TR? pathway by which maternal hypothyroidism effect on offspring development warrant further study.