Browsing by Subject "Schwangerschaftsdiabetes"

Now showing 1 - 1 of 1

Untersuchungen zur Bedeutung des Sulfonylharnstoffrezeptors 1 für die Modulation von Apoptose durch 17 beta-Estradiol in rekombinanten HEK (Human Embryonic Kidney) 293-Zellen und in pankreatischen beta-Zellen
(2009) Ackermann, Stefanie; Bode, Christiane
The sulfonylurea receptor (SUR) 1 forms the regulatory subunit of pancreatic ATP-sensitive potassium channels (KATP channels) which are essential for triggering insulin secretion in the beta-cell. Insulin secretion is modulated by additional KATP channel-independent pathways and by adaptive variation of beta-cell mass due to apoptosis, proliferation and/or neogenesis of beta-cells. Apoptosis of beta-cells is assumed to be involved in the pathophysiology of diabetes type 1 and 2. Previously it has been shown, that the insulinotropic sulfonylurea glibenclamide and the natural compound resveratrol can induce enhanced apoptosis and that this effect is specifically linked to the expression of SUR1. In the present work, it has been investigated whether there are substances that are more potent in inducing apoptosis than glibenclamide and resveratrol. Thereby the main focus was put on 17beta-estradiol which shows structural and functional analogies to the ?phytoestrogen? resveratrol. Like resveratrol, this naturally occurring estrogen is able to induce apoptosis in different experimental systems. Furthermore, it is known that 17beta-estradiol is able to decrease KATP channel activity in beta-cells acting as a KATP channel blocker. It is still discussed whether 17beta-estradiol directly interacts with KATP channels or whether it binds to so far unidentified ?non-classical? plasmalemmal estrogen receptors which are linked to KATP channels via an intracellular signaling cascade. In heterologous competition experiments, Ackermann et al. (2008) were able to show that 17beta-estradiol is a specific ligand of SUR like glibenclamide and resveratrol. Obviously SUR1 can act as a ?non-classical? estrogen receptor. In the present work it was investigated whether 17beta-estradiol induces apoptosis that is specifically linked to the expression of SUR1. Furthermore, the role of the SUR-isoforms SUR1 and SUR2 and of several SUR-mutants in the induction of apoptosis by 17beta-estradiol was investigated. Therefore, experiments were performed with recombinant HEK293-cells expressing the different isoforms of SUR. Cells that were transfected with empty pcDNA expression vector (pcDNA-cells) were used as control cells. By quantification of different apoptotic parameters such as cell detachment, changes in nuclear morphology as well as increased activity of caspase-3, it was shown that 17beta-estradiol induces specific apoptosis in cells expressing SUR1. In cells expressing the pancreatic isoform SUR1, treatment with 17beta-estradiol resulted in massive apoptosis while cells expressing the cardiac isoform SUR2A or the vascular isoform SUR2B as well as sham-transfected control-cells were less affected. Furthermore, 17beta-estradiol is more potent in inducing apoptosis in cells expressing SUR1 than glibenclamide or resveratrol. The pancreatic KATP channel consists of the regulatory subunit SUR1 and the pore-forming unit Kir6.2. In the present work, it has been proven that this SUR1-dependent effect of 17beta-estradiol was not significantly modified by coexpression with Kir6.2. These data show that apoptosis induced by 17beta-estradiol does not require the existence of functional pancreatic KATP-channels (formed by SUR1 and Kir6.2 subunits). These results provide evidence for an additional function of SUR1 apart from regulating electrical activity of the pancreatic KATP channels. SUR1 might be specifically involved in an adaptive change of the beta-cell mass and could contribute to the regulation of insulin secretion via influencing beta-cell mass. Additional experiments with cells from the clonal beta-cell lines HIT-T15 and RIN-m5F, endogenously expressing SUR1, showed that treatment with 17beta-estradiol can induce apoptosis in these cells. In pancreatic islet cells from mice aged 20-32 weeks, a clear induction of apoptosis after treatment with 17beta-estradiol was observed. Beta-cells of Langerhans also express SUR1 endogenously. Treatment of islet cells from wildtype mice with 17beta-estradiol resulted in intensive changes in nuclear morphology while islet cells from SUR1 knockout (SUR1KO) mice of the same age as well as untreated or solvent-treated islet cells from wildtype and SUR1KO mice did not show any marked signs of apoptosis. In contrast to islet cells from elderly mice aged 20-32 weeks (male or female), clear anti-apoptotic effects were detected in islet cells from young mice aged 5-7 weeks (male or female) after treatment with 17beta-estradiol. In untreated or solvent-treated islet cells from young mice (male or female) apoptosis was measured to a large extent, which was reduced by treatment with 17beta-estradiol. These results provide evidence that age is obviously an important factor which can influence the effect of 17beta-estradiol. The apoptotic effect of 17beta-estradiol in elderly mice as well as the anti-apoptotic effect in younger mice is specific to the expression of SUR1 as it was missing in experiments with islet cells from SUR1KO mice. During pregnancy, plasma concentrations of 17beta-estradiol in humans markedly increase. In the third trimester of pregnancy, 17beta-estradiol concentrations between approximately 50 and 100 nM can be readily achieved. At this point of time, the concentration of maternal serum 17beta-estradiol can be elevated up to more than 100 times compared to serum concentrations during the normal menstrual cycle (follicular phase: approx. 0.1-1.0 nM; luteal phase: approx. 0.5-2.0 nM). Changes in beta-cell mass mediated by 17beta-estradiol might contribute to the etiology of gestational diabetes mellitus (GDM). GDM is defined as glucose intolerance that appears or is first recognized during the last trimester of pregnancy. Estron, another endogenously occurring estrogen, also shows the ability to induce apoptosis in HEK293-cells expressing SUR1 as well as in cells from the SUR1 expressing clonal beta-cell lines HIT-T15 and RIN-m5F. However, the extent of apoptosis after treatment with estron is much lower than after treatment with 17beta-estradiol, although estron differs from 17beta-estradiol only in lacking one hydroxyl group. This hydroxyl group seems to be important for this pronounced SUR1-specific induction of apoptosis by 17beta-estradiol. In the present work, also the role of different SUR-mutants was examined. SUR1(M1289T) is a mutant, in which the amino acid methionine at position 1289 in transmembrane helix 17 (TM17) of SUR1 was exchanged by the corresponding amino acid of SUR2B (threonine). The experiments indicate that the amino acid methionine at position 1289 in TM17 obviously plays an important role in apoptosis which is induced by 17beta-estradiol and is specific for the expression of SUR1. This apoptotic effect after treatment with 17beta-estradiol is abolished by this single mutation. To investigate whether this apoptotic effect after treatment with 17beta-estradiol was linked to a correct function of the nucleotide binding folds, experiments with the mutants SUR1(R1379C) and SUR1(R1379L) were performed. Both mutations are located in the second nucleotide binding fold of SUR1 and result in an enhanced ATP-hydrolysis at the NBFs. These naturally occurring mutations were found in patients with neonatal diabetes with some of them showing a family history in adult-onset type 2 diabetes or GDM. The expression of these mutants in HEK293-cells leads to a much stronger induction of apoptosis than the expression of SUR1. The observation that apoptosis induced by 17beta-estradiol can be influenced by certain mutations of SUR could be of particular importance for the pathophysiology of diseases like cancer or diabetes. The enhanced activity of caspase-9 in cells expressing SUR1 after treatment with 17beta-estradiol suggests that the mitochondrial pathway might play a major role in this apoptotic process.