SamenvattingRational and Aim: Rapamycin (Rapa) is an immunosuppressive drug that suppresses cytokine-induced T-cell proliferation by inhibition of mammalian target of rapamycin (mTOR) signaling. Rapa is used for islet transplantation in type 1 diabetic patients because it is thought that Rapa has little net effect on beta cell function and peripheral sensitivity to insulin, which is an essential requirement for diabetic patients receiving human islet grafts. However, the early study showed that Rapa induced primary nonfunction of islet xenografts in a dose-dependent manner. Recent studies showed that prolonged exposure of rat and human pancreatic islets to high concentrations of Rapa in vitro induced apoptosis and inhibited glucose-induced insulin release. Since Rapa-effects were dose-dependent, it is important to examine its effects at a therapeutic concentration. The aim of this project was to study the effect of Rapa, at the concentrations in recipients of islet grafts, 1) on glycemic control in normal rats in vivo; 2) on purified rat beta cell survival and function in vitro. This study can help to understand beta cell graft function under the Rapa treatment. Safe and effective immunosuppressive therapy would greatly enhance the clinical utility of islet transplantation.
Materials and Methods: Adult Wistar rats were injected intraperitoneally with Rapa once a day for 3 weeks (0.5~1.0mg/kg.day) and control animals were injected with vehicle. Body weight, food and water intake, and urine volume were followed daily, and blood samples were taken weekly for analysis of Rapa trough level, fasting glycemia, insulin, total cholesterol, triglyceride and free fatty acid. Oral glucose tolerance tests (OGTT) were performed weekly. Be the end of 3wk-treatemt, pancreases were used for analysis of RNA, protein and insulin content, or used for islet isolation for studies of insulin synthesis and release. Some animals were followed for additional 9 weeks without drug administration to examine the reversibility of Rapa-effect. Pancreatic beta cells which had been isolated from control rats and cultured for 7 days with and without Rapa were used to study the direct effects of Rapa on beta cell survival and function.
Results: Therapeutic concentration of Rapa induced the impaired fasting glycemia (IFG) in 10 out of 26 rats and impaired glucose tolerance (IGT) in all animals after 3-week treatment. Rapa-induced IFG and IGT were associated with 2- to 5-fold increase in plasma insulin levels both after fasting and after glucose challenge, suggesting the presence of insulin resistance. However, Rapa-animals also exhibited a lower early insulin response to glucose challenge during OGTT, indicating that both impaired beta cell function and insulin sensitivity were responsible for the development of IFG and IGT. The impaired beta cell function resulted, at least in part, from the direct inhibition of Rapa on insulin synthesis which was demonstrated by culture of beta cells with Rapa, and from the 50% reduction in pancreatic insulin content as a consequence of overstimulation of insulin release by hyperglycemia and insulin resistance. Rapa-treatment also increased plasma cholesterol (37%) and triglyceride levels (53%) and reduced free fatty acid (34%). The Rapa-induced metabolic abnormalities partially restored at 9w-post-treatment. This slow recovery was related to the slow clearance of the drug which blood trough level returned to base line only after 7wk stopping drug administration.
Conclusions: Rapamycin-treatment in normal rats, at the concentrations in recipients of islet transplantation in human, causes beta cell dysfunction and insulin resistance, leading to impaired glucose tolerance and impaired fasting glycemia. This disturbed glucose homeostasis is associated with dyslipidemia and slow growth. The relevance of this finding in human should be further studied.
|Zhidong Ling (Promotor)