Insulin signalling is an essential process in humans by which the level of plasma glucose is maintained within the physiologically healthy range. Insulin activates the phosphoinositide 3 kinase (PI3K) signalling pathway that generates the phospholipid messenger PtdIns(3,4,5)P3, which in turn enhances the activity of two important proteins, AKT and Rac1. This then leads to increase the presence of the glucose transporter 4 (GLUT4) at the plasma membrane that enhances the intake of glucose, particularly in skeletal muscle cells and adipocytes. Insulin signalling also triggers interconversion of several other phosphoinositides (PIs) which play pivotal roles in different steps of glucose regulation. PtdIns5P is an important PI that is robustly increased after insulin treatment in the skeletal muscle cell line, L6 myotubes. Many of PtdIns5P`s functions are not fully understood. To gain more knowledge of the role of PtdIns5P in insulin signalling in muscle cells, the PtdIns5P kinase phosphatidylinositol-5-phosphate 4-kinase a (PIP4K2a) was over-expressed in L6 myotubes as a way of removing PtdIns5P, and the consequences in insulin signalling were studied. Although PtdIns5P is converted by PIP4K2a to PtdIns(4,5)P2 which is a precursor of the potent PI PtdIns(3,4,5)P3, previous studies revealed that the increase in PtdIns(3,4,5)P3 induced by insulin in control cells is diminished in cells overexpressing PIP4K2a, for unknown reasons. Additionally, although the phosphorylation of the serine/threonine protein kinase AKT was not affected in these L6 cells, glucose uptake was attenuated. The current study investigates the possible causes of attenuating glucose uptake in PIP4K overexpressing myotubes by examining the small GTPase Rac1 which plays an important role in the cytoskeleton re-arrangement that is necessary for GLUT4 translocation. Furthermore, the possible roles of PI phosphatases that may cause the disturbance on the levels of PIs in response to insulin were evaluated. Additionally, the potential role of PtdIns5P in Rac1 activation in L6 myotubes was further investigated by delivering synthetic PtdIns5P using a carrier-based delivery approach. The results showed that the attenuation of glucose uptake documented in previous studies occurred as a result of a defect in the process of translocating GLUT4 from intracellular storage to the plasma membrane. Rac1 activity was significantly reduced in cells expressing PIP4K2a. Quantifying the level of PIs suggested that PIP4K2a expression increases the removal of PtdIns(3,4,5)P3 by the PI 5-phosphatase, SKIP. Silencing the expression of SKIP by siRNA restored the level of PtdIns(3,4,5)P3 but Rac1 activity and the attenuation GLUT4 translocation were not rescued possibly as a result of removing PtdIns5P by PIP4K2a. On the other hand, exogenous delivery of PtdIns5P in L6 myotubes activates both Rac1 and GLUT4 translocation in the absence of insulin. However, activating GLUT4 translocation by the exogenous PtdIns5P requires PI3K activity since redistribution of GLUT4 to the plasma membrane is inhibited by the PI3K inhibitor, wortmannin. Removing PtdIns5P reduces Rac1 activity and stimulates SKIP that inhibits PtdIns(3,4,5)P3 increase which attenuates GLUT4 translocation and hence glucose uptake. These results emphasise the critical role played by PtdIns5P which seems to serve as a regulator of insulin signalling, both directly and/or by regulating other enzymes involved in the metabolism of PIs.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:764692 |
| Date | January 2018 |
| Creators | Al-Abri, Abdulrahim |
| Contributors | Wang, Xin ; Hinchliffe, Katherine |
| Publisher | University of Manchester |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | https://www.research.manchester.ac.uk/portal/en/theses/investigating-the-effect-of-pip4k2a-overexpression-in-insulin-signalling-in-l6-myotubes(1dd2d1dd-c765-4830-9b66-cf32a64d7de9).html |
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