The Molecular Mechanism Of Growth Arrest-specific6(Gas6) Regulating Plasma Glucose Uptake In Mice

Background&Aims:Arrest-specific gene product6(Gas6) is a vitamin K-dependent growth factorand is the ligand of receptor tyrosine kinase family of TAM (Tyro-3, Axl and Mer). Alarge number of domestic and foreign researches have shown that Gas6has similarfunctions with plasma anticoagulant protein S, in addition Gas6/TAM systeminvolved in Pathological and physiological process such as cell survival, adhesion,proliferation and migration, release of inflammatory cytokines and stabilizinghemagglutination. It plays the role in clotting, infection, autoimmune disease, cancerand vascular, reproductive and neurological disorders. The affinity between Gas6andAxl was significantly higher than the other members, so we often refer to them asGas6/Axl pathway. Gas6combines with Axl and forms a dimer which leading thephosphorylation of the intracellular tyrosine kinase domains and activates the PI3K,AKT proteins. In the recent years, several clinical investigations have found that theplasma concentration of Gas6was significantly correlated with the risk of type2diabetes mellitus (T2DM). However, molecular mechanisms of Gas6/TAM system inthe development of Type II diabetes are unknown. The receptors of Gas6and Insulinboth are the members of tyrosine kinase family, and they can activate the cytoplasmicproteins such as PI3K and AKT. We hypothesized that Gas6likely participate in thesignaling of insulin regulating organism glucose metabolism, especially promote thetranslocation of glucose transporter4in skeletal muscle cell, thus participate in theregulation of blood glucose. Hence, in this study we establish the experimental modelusing mice and cell to explore the possible role and mechanisms of Gas6/Axlpathways in T2DM. Methods:1. Compare the differences in glouse and lipid metabolism-related physiologicaland biochemical indicators between the Axl knockout (Axl-/-) mice and wild-type(Axl+/+) mice. To analyse Gas6signal blocked (Axl gene knockout) whether affectfundamental blood glucose values, the coefficient of fat, four indicators of bloodlipids, the mRNA expression levels and the proteins phosphorylation levels of insulinsignaling proteins PI3K, AKT and the glucose transporter4(GLUT4) in skeletalmuscles.2. Establish the experimental T2DM model with the above two genotypes micefeeding high fat diet (HFD)5w combining with streptozotocin (STZ). Compare thesuccessful T2DM modeling rate with the two genotypes of mice. Then analyse theconcentration of Gas6protein in serum between each genotype of mice and whichrelated T2DM model mice. To detect whether Gas6affects glucose tolerance in eachgroup when we gave Gas6or insulin to intervene the mice of two genotypes and theircorresponding T2DM model.3. Analyzed the levels of glucose tolerance before and after treating spontaneousT2DM model (db/db mice) mice with Gas6or insulin. Detect whether Gas6affectsglucose tolerance and the phosphorylation levels of AKT, AS160and other keyproteins in the skeletal muscle and adipose tissue of db/db mice treated with differentdrugs.4. Treat cells with Gas6, insulin or Gas6+R428(Axl inhibitor), then comparethe changes in the rate of glucose absorption, and the phosphorylation levels of AKTand AS160in each group.Results:1. Compared with wild-type mice, the major receptor Axl of Gas6knockout mice showed the plasma total cholesterol levels and high-density lipoprotein levelsdecreased significantly, and the Aliphatic index increased significantly. Meanwhile,the based glucose values of mice in Axl-/-group is significantly higher than inwild-type group. Third, the mRNA levels of PI3K, AKT and GLUT4are increased insome extent, while the phosphorylation level of the corresponding protein are reducedin Axl-/-mouse skeletal muscle.2. The success rate of the artificial establish T2DM models in Axl-/-mice wassignificantly higher than wild-type mice, the concentrations of plasma Gas6in T2DMgroups were significantly lower than the corresponding group of non-T2DM animals.The blood glucose levels were significantly reduced in wild-type mice andcorresponding T2DM mice after treated with Gas6, while it has no significantlychange in Axl knockout mice and corresponding T2DM mice.3. When detected the glucose tolerance after the db/db mice treated with Gas6orinsulin, we found that the blood glucose was all significantly reduced. Thephosphorylation levels of AKT and AS160which are the key proteins promoting theuptake of glucose in skeletal muscle were obviously increased after the db/db micetreated with Gas6.4. The rate of glucose absorption and the phosphorylation levels of AKT andAS160were significantly increased when the cells treated with Gas6, but the cellstreated with Gas6+R428(the inhibitor of Axl), the effect of Gas6disappeared.Conclusion:Research results preliminary demonstrated that by means of gene knockoutblocking Gas6/Axl signaling pathway may increase the incidence of T2DM in mice,lead to fat deposition and increase blood glucose levels in a certain extent. Gas6regulating blood glucose may be by increasing phosphorylation levels of PI3K/AKTprotein to activate AS160, increasing GLUT4translocation to the cell membrane, and increasing promote glucose uptake ultimately. Thus, activation of the Gas6signalpathway T2DM help improve the sensitivity of insulin signal in skeletal muscle ofT2DM mouse.