COMPARISON OF THE EFFECT OF MATERNAL HYPOTHYROIDISM ON CARBOHYDRATE METABOLISM IN YOUNG AND AGED MALE OFFSPRING IN RATS. Karbalaee1 Narges Karbalaee*,Saleh Zahediasl, Asghar Ghasemi, Farzaneh Faraji Shahrivar Endocrine Research Center, Research Institute for Endocrine Sciences, Shaheed Beheshti University of Medical Sciences, Tehran, Iran

Introduction Critical periods of development (Composite data are from rodents and humans)

Hyperlipidemia Reproductive disorders Insulin resistance Hypertension adulthood Ischaemic heart disease Renal failure Intrauterine growth retardation Glucose intolerance Type 2 diabetes Obstructive pulmonary disease

CNS Metabolic Activities in Most Tissue Cardiovascula r Hemodynamic Reproductive system Liver function Immune System Growth and Development There are evidences that maternal hormonal status markedly influence intra-uterine growth and developmen

Thyroid hormones act as metabolic and maturational signals, any changes in the levels of these hormones in uterus alters fetal development and has long term consequences for cardiovascular, reproductive,and metabolic function. There is a relationship between serum thyroid hormone levels and diabetes risk and also various abnormalities in carbohydrate metabolism occur in patients with thyroid disease. So abnormalities in carbohydrate metabolism may take place in fetal hypothyroidism. Karbalaee6

Aim This study aims to assess the glucose tolerance and insulin secretion capacity of islets in young and aged male offspring of mothers who were hypothyroid during pregnancy. Karbalaee7

Animals and study design 8 Pregnant femaleWistar rats Control Pregnant rats consumed tap water during gestation Fetal Hypothyroid Pregnant rats received water containing 0.02 % of PTU during gestation Male offspring (3 and 12 month) Delivery Mother and Offspring (Blood) Thyroid hormones (T3, T4) Assessed for Male and female Wistar rats were crossed for 24 h

Material and Methods Intravenous Glucose Tolerance Test (IVGTT)  In young and old male offsprings, after over night fasting (12 h), IVGTT was carried out by intravenous infusion of 50% glucose (0.5 g/kg body weight)  The plasma glucose and insulin levels of rats were assayed at 0, 5,10,15, 20, 30, and 60 minutes after an intravenous glucose load. 9

Material and Methods Glucose-stimulated insulin secretion (GSIS) ASSAY  Islet isolation  Incubation with different glucose concentrations (5.6, 8.3, 16.7 mM) ( 60 min, 37°C,95 % O 2 / 5 % CO 2 )  Collection of supernatant for insulin determination Karbalaee10

Measurements  Weight  Plasma T3 & T4(ELISA kits )  Plasma Glucose concentration(glucose oxidase method )  Plasma Insulin concentration(ELISA method ) Karbalaee11

Results Karbalaee12

Table I. Plasma T 3 and T 4 concentration in the fetal hypothyroid of control groups at birth and adulthood (3 and 12 months ), and their mothers at the time of delivery. OffspringMothers At the time of birthAdulthood 3 months 12 months At the time of delivery Hormones Control (n=10) Fetal hypothyroid (n=9) Control (n=15) Fetal hypothyroid (n=15) Control (n=7) Fetal hypothyroid (n=8) Control (n=12) Hypothyroid (n=12) Triiodothyronin e (T 3, ng /dl) 69.1 ± ± 5.5 * * 98.8 ± ± ± ± ± ± 4.9 *** Thyroxine (T 4, µg /dl) 0.98 ± ±.06 * 3.5 ± ± ± ± ± ± 0.03 *** Karbalaee13 Values are means ± SEM. * p< 0.05, *** p< and *** p< Significant difference compared to controls

Table II. Effect of hypothyroid during pregnancy on reproductive performance. Parameter (units)Control groupFH group Gestation length (day)21.7 ± ± 0.3  Percent maternal weight gain (%) a 49.2± ± 2.0 Litter size10.2±0.89.6±0.4 Offspring mortality rate (%) *** Birth weight (gr) b 6.0±0.04.6±0.1 *** Karbalaee 14 a Percent weight gain relative to the weight on day 1 of pregnancy. Day 1 weight in all groups ranged from g. b All pups in a litter were weighed and the average pup weight of a litter computed and reported as a single point. This was done for all litters in a group and the mean of these points is reported as the group mean in the table. Values are means ± SEM. * p< 0.05 and *** p<0.001 Significantly from control

Body weight in control and Fetal hypothyroid (FH) male offspring rats. Values are mean ± SE ; n= Karbalaee

Plasma glucose and insulin cocentrations and their AUC during intravenous glucose tolerance test in the young ( a, c n = 11) and old ( b, d n = 12) rats of fetal hypothyroid and control groups. Values are mean ± SE. 16

Comparison of the islets insulin secretion in the fetal hypothyroid and control groups in old ( a) and young (b) rats. Values are means ± SE Karbalaee17

Conclusions The results of this study have shown that maternal hypothyroid during pregnancy leads to:  Intrauterine growth restriction in rat fetuses  Glucose intolerance and lower insulin secretion capacity in their adult off spring particularly in aged animals. From the results of this study we can conclude that carbohydrate metabolism in the offspring of fetal hypothyroid subjects is a matter of concern particularly in the old ages. Karbalaee18

Karbalaee19

Ghasemi20

Ghasemi21

 Definition of term `programming‘ A stimulus or insult during a critical or sensitive period of development can have long-term or lifetime effects on an organism’, as well as with the ‘predictive adaptive response’. ( Holness,2000; Hales and Barker (2001 Lucas A, 2000; Gluckman PD,,2006)  Disturbance in the metabolic intrauterine environment alters the development of the endocrine pancreas and the insulin sensitive tissues.  lPerturbed nutritional and hormona environment are responsible for the altered β-cell mass. Ghasemi22

Diagram illustrating the relationships between nutritional state, hormone concentrations, metabolism and tissue accretion and differentiation in the fetus. (A L Fowden and A J Forhead(2004)