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Selected Publication:
Nikodijevic, S.
Influence of Insulin on ER-stress in Human First Trimester Trophoblast
An in vitro study
Humanmedizin; [ Diplomarbeit ] Graz Medical University; 2021. pp. 37
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- Authors Med Uni Graz:
- Advisor:
- Desoye Gernot
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- Abstract:
- The placenta is the connection between the mother and the growing fetus. During pregnancy maternal metabolism changes, for example glucose metabolism, which translates into increased insulin resistance. These circumstances have an impact on the placenta and therefore on adverse pregnancy outcomes like gestational diabetes mellitus and pre-eclampsia. Furthermore, these diseases have not only acute consequences for the unborn but also increase the risks for cardiovascular diseases for mother and child. ER-stress plays an important role in pathophysiology of pregnancy and is induced by unfolded and misfolded proteins accumulated in the cell. To restore intracellular homeostasis, the cell responds with the so-called unfolded protein response (UPR). ER-stress sensors IRE, ATF6 and PERK regulate UPR. When homeostasis cannot be restored, apoptosis will be activated. Preliminary results have shown that pathophysiological insulin concentration decreases eIF2α phosphorylation suggesting that insulin might reduce ER-stress. The objective of this thesis is to investigate if insulin has an ER-stress reducing effect on first trimester trophoblast cells, which have been cultured with ER-stress inducing factors. The ACH-3P cell line was used as a model for first trimester trophoblast. It is a cell line of fused intravillous male trophoblast cells and the choriocarcinoma cell line AC1-1. Four passages of ACH-3P cell line were cultivated under physiological conditions of 6.5% O2 and 37°C in a medium containing pathological insulin of 10nM concentration and ER-stress inducers brefeldin A and tunicamycin. After 24 hours, the effect of insulin was determined by measuring specific ER-stress markers of different signaling pathway of UPR. GRP78, phospho- and total eIF2α as well as phospho- and total IRE1α protein levels were evaluated by Western blotting. Using RT-qPCR, mRNA levels of pro-apoptotic transcription factor CHOP as well as spliced and non-spliced XBP1 were measured. Statistical analysis of immunoblot and RT-qPCR could not show an effect of insulin on ACH-3P cells. ER-stress induction with tunicamycin (TM) and brefeldin A (BFA) could be shown. ER-stress marker GRP78 was increased (8.6-fold and 4.7-fold, respectively; p≤0.001) as well as total IRE1α (2.2-fold and 1.9-fold respectively; p≤0.01) and relative expression of CHOP (4.8-fold and 4.9-fold, respectively, p≤0.001), XBP1(1.7-fold and 1.6-fold, respectively; p≤0.0001 and p≤0.05) and its splicing (8.5-fold and 12-fold, respectively p≤0.001). Comparing the statistical results of ACH-3P cells incubated only with ER-stress inducers to cells incubated with ER-stress inducers TM and BFA with additional insulin, no significant difference was found. Under these circumstances, it can be considered that insulin has no effect on ER-stress in first trimester trophoblast cell line ACH-3P.