The Effects And Mechanism Of Caveolin-3 Gene P47L And P104L Mutations On Glucose Metabolism In Muscle Cells

Objective Caveolin-3 (CAV3)protein is a muscle specific protein, which plays an important role in regulating insulin signaling pathway, glucose metabolism and maintaining the physiological structure and function of muscle. The pathogenesis of type 2 diabetes mellitus (T2DM) clearly has a genetic component. We previously found that there were some mutations in the exons of CAV3 in T2DM patients. CAV3 gene mutation is also associated with a variety of muscle diseases, among which novel autosomal dominant form of limb girdle muscular dystrophy-1C (LGMD-IC) in humans is due to P104L mutation within the coding sequence of the human CAV3 gene and is characterized by muscle weakness. Nevertheless whether the pathogenesis of T2DM and LGMD-IC is due to the loss of glucose metabolism in skeletal muscle cells as well as the decrease of glucose utilization and energy deficiency caused by CAV3 mutations remains unknown. The aim of this study was to investigate the effects of P47L mutation of T2DM in CAV3 gene and P104L mutation of LGMD-IC in CAV3 gene on insulin signaling pathway and glucose metabolism in skeletal muscle cells at cellular level; and to investigate: the pathogenesis of T2DM and LGMD-1C.Methods Bioinformatic was used to analyze P104L mutation of LGMD-1C and three missense mutations within the coding sequence of CAV3 gene in T2DM patients:K14N, D15H and P47L. Subsequently, the expression vectors of P47L mutation and P104L mutation were constructed on the basis of the wild-type CAV3, and the vectors were transfected into C2C12 myoblast cells by liposome transfection kit Lipofectamine3000. Then four groups of cell lines expressing different genotypes were established:the negative control group (NC) cell line was transfected with empty vector, the wild-type group (WT) cell line was transfected with wild-type CAV3 gene and the mutant groups cell lines were transfected with P47L or P104L. Glucose consumption and glycogen synthesis in differentiated C2C2 myotubes were detected by glucose oxidase-peroxidase method (GOD-POD) and glycogen assay kit, respectively. Western blot was performed to detect the protein expression of insulin-stimulated glucose uptake related signaling molecules, while the cellular localization and expression of CAV3 and GLUT4 were observed by confocal microscopy.Results (1) According to analysis of the bioinformatics, K14N, D15H and P47L mutations affected the secondary structure of protein construction, and P47L mutation was located in the conserved amino acids of caveolin (CAV) protein family, while P104L was located in the transmembrane domain.(2) The glucose consumption and glycogen synthesis of P47L and P104L mutations were significantly decreased compared with those in WT group after insulin stimulation (P<0.05); compared with the NC group, the overexpression of wild-type CAV3 promoted glucose consumption. (P<0.05) and glycogen synthesis.(3) Western blot analysis showed that compared with the WT group, decreased expressions of CAV3, CAV1 and phospho-Akt (pAkt) were observed in P47L and P104L group. However, there was no obvious difference in total GLUT4 and Akt expression between the four groups. The result of confocal immunomiscrocopy indicated that compared with WT group, the CAV3 and GLUT4 expression in plasma membrane was reduced in P47L group; while the GLUT4 expression in plasma membrane in WT group was significantly enhanced compared with NC group. P104L mutantion reduced CAV3 and GLUT4 in plasma membrane that were mainly accumulated near the nuclear.Conclusion CAV3-P47L and P104L mutations resulted in the reduction of glucose uptake and glycogen synthesis in C2C12 myoblast cells, probably due to the mutations that reduced the expression of CAV3 on the membrane, and the subsequent inhibition of Akt in insulin signaling pathway, which resulted in the blocking of GLUT4 translocation to the plasma membrane,and therefore reduction in the uptake of glucose and glycogen synthesis. In addition, the two mutations reduced CAVl, the other subtype of CAV family, which plays a crucial role in insulin dependent glucose uptake in skeletal muscle cells. Impaired glucose utilization and glucose supply in skeletal muscle cells may be involved in the pathogenesis of T2DM and LGMD-1C.