title: Insulin increases epiblast cell number of in vitro cultured mouse embryos via the PI3K/GSK3/p53 pathway.
abstract: High-quality embryos give rise to embryonic stem cells (ESCs) at greater efficiencies than poor-quality embryos. However, most embryos available for human ESC derivation are of a reduced quality as a result of culture in relatively simple media up to 10 years earlier, before cryopreservation, or before compaction. In the present study, we used a mouse model to determine whether a culture with insulin from the 8-cell stage could increase the number of ESC progenitor epiblast cells in blastocysts, as well as endeavor to determine the molecular mechanism of the insulin's effect. Culture in media containing 1.7 ρM insulin increased epiblast cell number (determined by Oct4 and Nanog co-expression), and proportion in day 6 blastocysts. The inhibition of phosphoinositide 3 kinase (PI3K) (via LY294002), an early second messenger of the insulin receptor, blocked this effect. The inhibition of glycogen synthase kinase 3 (GSK3) or p53, 2 s messengers inactivated by insulin signaling (via CT99021 or pifithrin-α, respectively), increased epiblast cell numbers. When active, GSK3 and p53 block the transcription of Nanog, which is important for maintaining pluripotency. A simultaneous inhibition of GSK3 and p53 had no synergistic effects on epiblast cell number. The induced activation of GSK3 and p53, via the inhibition of proteins responsible for their inactivation (PKA via H-89 and SIRT-1 via nicotinamide, respectively), blocked the insulin's effect on the epiblast.From our findings, we conclude that insulin increases epiblast cell number via the activation of PI3K, which ultimately inactivates GSK3 and p53. Furthermore, we suggest that the inclusion of insulin in culture media could be used as a strategy for increasing the efficiency with which the ESC lines can be derived from cultured embryos.
authors.affiliation: Centre for Stem Cell Research, School of Paediatrics and Reproductive Health, University of Adelaide, Adelaide, South Australia. jared.campbell@adelaide.edu.au
authors.forename: Jared M
authors.initials: JM
authors.lastname: Campbell
authors.forename: Mark B
authors.initials: MB
authors.lastname: Nottle
authors.forename: Ivan
authors.initials: I
authors.lastname: Vassiliev
authors.forename: Megan
authors.initials: M
authors.lastname: Mitchell
authors.forename: Michelle
authors.initials: M
authors.lastname: Lane
mesh_terms: D000818:Animals; D052160:Benzothiazoles / Q000494:pharmacology; D002452:Cell Count; D002867:Chromones / Q000494:pharmacology; D003470:Culture Media / Q000378:metabolism; D046149:Embryo Culture Techniques; D004622:Embryo, Mammalian / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D047108:Embryonic Development; D053595:Embryonic Stem Cells / Q000166:cytology / Q000378:metabolism; D004789:Enzyme Activation; D005260:Female; D005855:Germ Layers / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D038362:Glycogen Synthase Kinase 3 / Q000037:antagonists & inhibitors / Q000378:metabolism*; D018398:Homeodomain Proteins / Q000235:genetics / Q000378:metabolism; D007150:Immunohistochemistry; D007328:Insulin / Q000494:pharmacology*; D007546:Isoquinolines / Q000494:pharmacology; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D009025:Morpholines / Q000494:pharmacology; D000071317:Nanog Homeobox Protein; D050814:Octamer Transcription Factor-3 / Q000378:metabolism; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism*; D000081082:Phosphoinositide-3 Kinase Inhibitors; D011725:Pyridines / Q000494:pharmacology; D011743:Pyrimidines / Q000494:pharmacology; D011972:Receptor, Insulin / Q000378:metabolism; D015398:Signal Transduction; D013449:Sulfonamides / Q000494:pharmacology; D014050:Toluene / Q000031:analogs & derivatives / Q000494:pharmacology; D014158:Transcription, Genetic; D016159:Tumor Suppressor Protein p53 / Q000037:antagonists & inhibitors / Q000378:metabolism*
chemical_list: D052160:Benzothiazoles; D002867:Chromones; D003470:Culture Media; D038362:Glycogen Synthase Kinase 3; D018398:Homeodomain Proteins; D007328:Insulin; D007546:Isoquinolines; D009025:Morpholines; D000071317:Nanog Homeobox Protein; D050814:Octamer Transcription Factor-3; D019869:Phosphatidylinositol 3-Kinases; D000081082:Phosphoinositide-3 Kinase Inhibitors; D011725:Pyridines; D011743:Pyrimidines; D011972:Receptor, Insulin; D013449:Sulfonamides; D014050:Toluene; D016159:Tumor Suppressor Protein p53; C473711:Chir 99021; C475540:Nanog protein, mouse; C494174:Pou5f1 protein, mouse; C085911:2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one; C121565:pifithrin; C063509:N-(2-(4-bromocinnamylamino)ethyl)-5-isoquinolinesulfonamide