Studies On Insulin-like Role And Mechanisms Of Recombinat Bombyxin Ⅱ In Vitro On Mammalian Cells

Bombyx (Bbx) is the insect insulin, which has been first identified in invertebrates, withthe molecular weight5000Da. The amino acid composition of Bbx Ⅱprotein is highlyhomologous with human insulin. In insects' organism, Bbx is capable of regulating themetamorphosis, involving in the regulation of glucose metabolism, promoting cellproliferation of blood-forming organs and ovarian development and other physiologicalfunctions. Our workgroup previously synthesized Bbx Ⅱgene and constructed recombinantBbx Ⅱeukaryotic expression vector, achieved transient expressiong the Bbx Ⅱprotein in293FT cells. Through recombinant Bbx Ⅱprotein acts the hepatoma cell line HepG2cells,we initially confirmed the insulin-like effects of Bbx Ⅱprotein in mammalian cells.On the basis of the above studies, this paper targets the hepatoma cell line HepG2andpreadipocyte cell line3T3-L1, which are the target cells of insulin, and prepares stableexpression of Bbx Ⅱprotein via gene transfection in target cells. The Bbx Ⅱprotein isexpected to play a sustained biological role in target cells. On this basis,(1) to further verifythe action of Bbx Ⅱprotein in sugar metabolism in mammalian cells,(2) to explore theglucose metabolism signaling pathway related to Bbx Ⅱprotein in mammalian cells,(3) toexplore the effect of Bbx Ⅱprotein on the molecule expression and function involved inglucose metabolism pathways in mammalian cells. All these studies above intend to furtherclarify the Bbx Ⅱprotein affects glucose metabolism in mammalian cells and themechanisms. The findings in this paper are promising to lay the experimental and theoreticalbasis for Bbx Ⅱto develop the new anti-diabetic drug.The results of this paper are as follows:(1)Bbx Ⅱgenes were stably expressed in HepG2cells and3T3-L1cells.Bbx Ⅱexpression plasmids were transfected into HepG2cells and3T3-L1cells,respectively, using liposome-mediated gene transfer technology, and then cells withgenetically stable expression were selected out. The expression of Bbx Ⅱin the cells wasdetected by RT-PCR technology, immunocytochemistry and Western blotting, respectively.The results showed that two cell clones were selected by G418and they could passage.RT-PCR showed Bbx Ⅱgene fragments were detected in the two selected clones; not in cellstransfected with blank liposome or blank plasmid. Immunocytochemistry showed that the visible brown particles appeared in the cytoplasms in the two selected clones, not in the cellstransfected with blank liposome or blank plasmid. Western blots showed that the culturesupernatant and the cytolysate of the two selected clones were found to present clear proteinbands in the size of about5000Da in gel. These results demonstrated that Bbx II genes werestably expressed in both HepG2cells and3T3-L1cells. These product cells provide theexperimental basis for further studying the insulin-like effects of Bbx II gene on the two celllines.(2) Insulin-like effect of recombinant Bbx II on HepG2cells and3T3-L1cellsThe MTT method was used to plot cell proliferation curves, and flow cytometrydetected cell cycle to determine the impact of recombinant BbxII on the ability of cellproliferation. Glucose uptake test and glycogen synthesis tests measured the glucosemetabolism which was affected by recombinant Bbx II. The results showed that Bbx IIpromoted the proliferation of selected HepG2and3T3-L1cell clones, and increased thefraction of S phase in cell cycles of the two cell clones. Bbx II protein promoted the glucoseuptake of selected HepG2and3T3-L1cell clones, and the glycogen synthesis in selectedHepG2cell clones. These results demonstrated that recombinant Bbx II has the insulin-likeeffect to promote cell proliferation and regulate glucose metabolism in mammalian cells.The finding provides a theoretical basis for further study of the molecular mechanism.(3) The insulin-like role of the recombinant Bbx II through the PI3K and MAPKpathwaysCell proliferation, glucose consumption and glycogen synthesis was measured after thetwo selected cell clones exposed to the PI3K and MAPK inhibitors, respectively. The resultsshowed that the PI3K inhibitor decreased the glucose uptake and glycogen synthesis in thetwo selected HepG2and3T3-L1cell clones, indicating that the action of Bbx II on theglucose metabolism in mammalian cells can be blocked by the PI3K inhibitor. The MAPKinhibitor decreased markedly the cell proliferation of the selected3T3-L1cells, indicatingthat the action of Bbx II on the proliferation in mammalian cells can be blocked by theMAPK inhibitor. These findings demonstrated that the recombinant Bbx II may play aninsulin-like role through the PI3K and MAPK pathways.(4) The recombinant Bbx II action on the phosphorylation level of moleculesinvolved in the PI3K and MAPK pathways, and the molecular mechanism ofinsulin-like effectsWestern blotting was performed to detect the phosphorylation levels of AKT, GSK-3β and FoxO-1protein molecules involved in the PI3K pathways of in HepG-2cells of eachgroup. The results showed that the selected HepG-2cells increased the phosphorylationlevels of AKT and GSK-3β, significantly higher than HepG-2cells untransfected with BbxII gene, and the phosphorylation level of FoxO-1protein lowered. Western blotting wasperformed to detect the phosphorylation level of AKT and FoxO-1protein involved in thePI3K pathway in3T3-L1cells of each group. The results showed that the selected3T3-L1cells increased the phosphorylation levels of AKT and FoxO-1proteins, significantly highthan3T3-L1cells untransfected with Bbx Ⅱgene. Insulin can increase the phosphorylationlevels of AKT, GSK-3β and FoxO-1protein molecules involved in the PI3K pathway inHepG-2cells and3T3-L1cells. Western blotting was performed to detect thephosphorylation levels of MEK and ERK protein molecules involved in the MAPK pathwayin3T3-L1cells of each group. The results showed the selected3T3-L1cells increased thephosphorylation levels of MEK and ERK protein molecules, significantly high than3T3-L1cells untransfected with Bbx Ⅱgene. These results demonstrated that the recombinant Bbx IIplays the insulin-like role to affect to some extent the phosphorylation levels of proteinmolecules involved in the PI3K and MAPK pathways. While, there was a different impacton the phosphorylation level of certain protein molecule in comparison to insulin, suggestingthat the Bbx Ⅱprotein might have some different biological functions than insulin.The innovation of this paper is to clarify the recombinant Bbx Ⅱplays the insulin-likerole in aspects of regulating glucose metabolism and cell proliferation in mammalian cells bystable expression of Bbx Ⅱgene in HepG2and3T3-L1cells; on this basis, confirm Bbx IIplays its insulin-like effect for mammalian cells through the PI3K-AKT-GSK-3β andMAPK-MEK-ERK pathways. The findings of this paper are promising to provideexperimental and theoretical foundation for the development of insulin analogues,anti-diabetic drugs.