Insulin signaling in rabbit preimplantation embryos

Abstract

Addition of insulin during In-vitro-culture improves embryonic development by increasing cell proliferation and reducing apoptosis. The insulin receptor (IR) signaling involves two major pathways, the mitogenic-activated protein kinase (MAPK) and the phosphatidylinositol 3-kinase (PI3-K). In muscle and adipocyte cells insulin stimulates glucose uptake mainly by activation of the PI3-K pathway and translocation of glucose transporter (GLUT) 4. We have currently shown the expression of two insulin-responsive GLUT (4, 8) in rabbit embryos. The mechanism of insulin action on embryonic cells, however, is still not clear. In the present study the expression of IR-isoforms (IR-A, IR-B) and the activation of the PI3-kinase and the MAPK signaling pathways were studied in rabbit preimplantation embryos. The rabbit mRNA sequences for the coding region of IR-A and -B were cloned and sequenced. Using specific primer combinations for RT-PCR the expression of both isoforms was analyzed. The IR expression was detectable from the blastocyst stage onwards, with an IR-isoform specific pattern for embryoblast (Em) and trophoblast (Tr) cells. Whereas IR-A was found in both Em and Tr cells, the IR-B-isoform was detectable only in the trophoblast. In cultured blastocysts the effect of insulin stimulation (170 nM) was studied by analysis of Akt and Erk phosphorylation, GLUT4 translocation and measurement of 3-OMG glucose transport. Insulin stimulated the phosphorylation of the mitogenic activated protein kinase Erk1/2, but not the phosphorylation of the PI3-K dependent protein kinase Akt. Glucose uptake and GLUT4 localization were not influenced by insulin stimulation. Activation of the MAPK signaling pathway in the absence of GLUT4 translocation and of a glucose transport response suggest that in the rabbit preimplantation embryo insulin is acting as a growth factor rather than a component of glucose homeostatic control. The possible function of insulin-responsive GLUT isoforms and the cell specific expression of IR-A and -B in rabbit preimplantation embryos need further investigation.