Effecten van behandeling met insuline, tolbutamide of streptozotocine in perinatale vleeskippen

Different chicken studies revealed that there are considerable differences between mammals and chickens regarding the glucose-insulin metabolism. Briefly, chickens have blood glucose level being two-fold higher than that in mammals and the skeletal muscle and adipose tissue in broilers have a poor response to physiological insulin doses. Moreover, the effect of insulin on blood glucose concentrations seems to decrease with postnatal age in chickens. In addition, massive and near-lethal mammalian insulin doses are required to produce convulsions of hypoglycemia in chickens. However, few studies on the glucose-insulin metabolism in chicken embryos are reported. As the insulin sensitivity seems to be improved in chicken embryos compared to adult chickens, a fundamental research on the ontogeny of this metabolism in chicken embryos could gain new knowledge about metabolic disorders in humans such as diabetes type 2. Therefore, the first aim of this dissertation was to study the response of insulin (1 µg/g embryo weight (EW)), tolbutamide (known as an insulin-stimulated agent in chickens and mammals, 80 µg/g EW) and STZ (known as an agent to induce experimental diabetes in mammals, 50-700 µg/g EW) in broiler chicks during the perinatal period. These studies were performed by injecting these agents into eggs through a blood vessel of the chorioallantoic membrane (CAM) or via the vena jugularis of newly-hatched chicks. Results were compared to a saline- or NaOH-injected sham group and a non-injected control group. Firstly, plasma glucose levels of insulin-injected embryos on embryonic day (ED)18 recovered faster from their minimum level than those of insulin-injected ED16 embryos or newly-hatched chicks. In addition, at seven hours post-injection (PI) on ED16, ED18 and D0, the mRNA abundance of hepatic glucose transporter 2 (GLUT2) and fructose-1,6-biphosphatase 1 (FBP1) and phosphoenolpyruvate carboxykinase 2 (PCK2), two out of three regulatory enzymes of the gluconeogenesis, was decreased in the insulin group, being most pronounced when the insulin injection occurred on ED16. The hepatic mRNA abundance of GLUT1, 3, 8, 9 and 12, and PCK1, the third regulatory enzyme of the gluconeogenesis, remained unchanged at seven hours PI in insulin-injected ED16 and ED18 embryos and newly-hatched chicks. Secondly, after one tolbutamide injection on ED16 or on ED17, a prolonged (32h) decrease of plasma glucose level and a profound acute increase in plasma insulin level were observed. Thirdly, the STZ injection in ED16 embryos did not affect plasma glucose concentrations two, four and seven days later, even with near-lethal mammalian STZ doses. The pancreas weight decreased profoundly four days after the injection of STZ dosed at 700 µg/g EW. No changes were observed in plasma glucose levels after a tolbutamide challenge (160 µg/g EW) in embryos that were injected with STZ two days before. In addition, we also investigated STZ effects in broiler chicken embryos using an in vitro experiment. A cell culture of pancreatic cells isolated from ED18 broiler embryos was firstly established and then treated with STZ of different concentrations (0, 1, 2.5, 5, 10 and 20 mM) at different time intervals (30’, 2h, 4h, 8h, 10h and 24h). The glucose-induced insulin release in the pancreatic cells was changed after STZ treatment of 30 min, 2h, 4h and 10h. In addition, the caspase3/7 activity was not affected by STZ at each time interval. Furthermore, mammalian studies suggested that there is a correlation between a prolonged hypoglycemia during the late pregnancy and an increased risk for development of diabetes type 2 in postnatal life. However, these hypoglycemic fetuses are probably exposed to maternal endocrine factors, which could affect the fetal programming. In contrast, the chicken embryo develops independently from the hen and is accessible for manipulation, making them a suitable model for research on physiological imprinting effects. Therefore, the second aim of this PhD research was to investigate postnatal performance after the induction of four days long-lasting hypoglycemia in broiler embryos by three consecutive (daily) tolbutamide injections from ED16 to ED18. We observed that the fetal programming negatively affected the body weight (BW) and feed intake during the entire rearing period and the insulin tolerance on postnatal day (d) 42. In conclusion, it has been demonstrated that insulin had a regulatory role on the plasma glucose level and mRNA abundance of GLUT2, PCK2 and FBP1, but its role clearly differed with age of the broiler embryo/chick. The embryonic acute and chronic treatment of tolbutamide also changed plasma glucose and insulin concentrations in broiler embryos, and this chronic programming had affected the postnatal performance. Taken together, all these results are promising and invite to develop new fundamental research using our insulin and tolbutamide models. Finally, our results from the in vivo study on STZ were comparable with those from postnatal chicken studies, indicating that STZ seems to be unresponsive in broiler embryos. However, additional trials are still required to gain more insights about the regulation of STZ in chicken embryos.status: publishe