Analysis And Research Caveolin-3 K15N Mutation Gene Protein In Type2 Diabetes Patients

[Objective] The genetic mechanisms of Type 2 diabetes mellitus (T2DM) is closely related to gene polymorphism. Caveolin-3(Cav-3) i-s muscle specific membrane protein, which regulates the insulin signaling pathway. Our previous studies have found that there were several mutati ons in Cav-3 gene exon of T2DM patients. In order to explore whether t-he Cav-3 gene mutation influence the glycometabolism of muscle cells, which takes part in the pathogenesis of T2DM. We will transfect the Cav-3 K15N mutation into C2C12 cells, and then observe the function of m-utant proteins.[Method] Predicted the secondary protein structure of K15N (KU971296), D16H and N33N in the Cav-3 first exon by bioinformatics methods, and then selected the K15N missense mutation to further study. We constructed negative control group GFP vector, expression vector of wild type and the K15N mutation of Cav-3 gene, and transfected them into C2Cl2 cells, respectively. After screening of G418, stimulating by insulin 30min, we used Western Blot and IF technique to determine the protein expressionsof Cav-3, AKT, pAKT and GLUT-4. In the 12h and 24h, we detected the glucose absorbtion and glycogen synthesis of C2C12 cells.[Result] 1. Bioinformatics:analysised K15N, D16H and N33N three mutations in the first exon in T2DM patients and showed that secondary structure of Cav-3 protein was changed from random curl into a helix structure in the K15N and D16H mutations.2. We got stable expression cell lines using G418 screening wit-h 400 μg/ml, which was confirmed by Western blot. There wasn’t signifi-cant difference in the transfection efficiency between each groups.3. Compared with the wild type group, the K15N mutation reduced the total expression of cell Cav-3 protein, which also decreased in cell membrane, but enriched in the cell nucleus. Downstream signal molecule AKT phosphorylation decreased, but total AKT protein level had no obvious change. Cellular GLUT-4 protein was not only decreased in the overall level of expression, but also gather into clusters on the cell membrane.4. Cav-3 K15N mutation protein led to reduced glucose uptake of C2C12 cells. In 12h, when K15N mutation group compared with negative control groups and the wild type groups,p=0.042 and 0.000, respectively. In 24h, when K15N mutation groups compared with wild type group p=0.010, that had significant statistical difference, but p=0.366 when compared with negative control groups, that had no statistical significance. In 36h, Glycogen synthesis decreased in K15N mutation groups (p< 0.050).[Conclusion] K15N Cav-3 mutation leads to decrease of Cav-3 expression in C2C12 cell membrane, which affects the phosphorylation of AKT and translocation of GLUT-4 protein in insulin signaling pathway, resulting in impaired glucose uptake and glycogen synthesis. We hypothesized that reduced glucose utilazation of skeletal muscle cell might be one of the mechanisms that lead to the development of hyperglycemia and T2DM.