Use of Insulin to Increase Epiblast Cell Number: Towards a New Approach for Improving ESC Isolation from Human Embryos
Schematic of insulin signalling and its regulation of Nanog expression and pluripotency. Green arrows indicate reactions with a stimulatory effect on their target, and red closed bars indicate reactions with a retarding effect on their target. P marks reactions where phosphorylation occurs, and Ub marks reactions where ubiquitination occurs. Insulin binds the insulin receptor (IR), a tyrosine kinase which is then able to phosphorylate the IRSs. PI3K is able to bind to the phosphorylated IRSs by its SH2 domains, resulting in activation. PI3K phosphorylates the phospholipid PIP2, producing PIP3, which can be bound by the pleckstrin homology domains of PDK-1 and Akt. Results in  show that activation of PI3K is necessary for insulin to increase the number of Nanog positive epiblast cells during embryo culture. When PDK-1 and Akt are colocalised to the cell membrane, PDK-1 is able to phosphorylate and activate Akt. Active Akt can phosphorylate GSK3, inactivating it. When active, GSK3 is able to phosphorylate β-catenin, Hedgehog, and c-Myc; all factors which safeguard pluripotency through interactions with other second messengers. Additionally, active GSK3 phosphorylates and protects the intracellular domain of Notch, promoting differentiation. Further, inactivation of GSK3 is necessary for insulin to increase the number of Nanog positive epiblast cells during embryo culture . Akt is also able to phosphorylate and activate MDM2 which ubiquitinates the proapoptotic factor p53, causing its inactivation and removal from the nucleus, where it would bind to the Nanog promoter and suppresses its expression. Inactivation of p53 is necessary for insulin to increase the number of Nanog positive epiblast cells during embryo culture . GSK3 and p53 are able to form a dimer, resulting in the phosphorylation of p53 and the increased activity of both factors. GSK3 is also able to phosphorylate and activate MDM2. However, despite these outcomes, the interaction of GSK3 and p53 do not have a significant effect on Nanog positive epiblast cell number during embryo culture .