Preliminary Study On The Physiological Function Of Caveolin-3 Gene In Skeletal Muscle Cells And The Pathogenic Mechanism Of The Mutation Gene

Objective Caveolin-3(CAV3)protein is known to be expressed specifically in various myocytes,but its physiological function remains unclear.Skeletal muscle consumes most of the blood glucose as an energy source to maintain normal cell metabolism and function.CAV3,located at the cell membrane,may promote the sensitivity of the Akt signaling pathway,which is closely related to glucose metabolism and to cell growth and proliferation.In addition,the P104 L mutation in the coding sequence of the human CAV3 gene leads to the autosomal dominant disease limb-girdle muscular dystrophy type 1C(LGMD-1C).We previously reported that C2C12 cells transiently transfected with the P104 L CAV3 mutant exhibited decreased glucose uptake and glycogen synthesis after insulin stimulation.The present study aimed to examine whether the CAV3 and P104 L mutation affects C2C12 cell glucose metabolism,growth and proliferation without insulin stimulation.Methods C2C12 cells stably transfected with the CAV3 gene and CAV3-P104 L were established,and biochemical assays,Western blotting and confocal microscopy were used to observe glucose metabolism as well as cell growth and proliferation and to determine the effect of the CAV3 and P104 L mutation on the PI3K/Akt signaling pathway with no insulin stimulation.Results1.Establishment of a cell line that stabilizes CAV3 and its mutant gene P104 L using liposomes.2.After C2C12 cells were stable transfected with the mouse CAV3 gene,which increased CAV3 expression,unchanged the total amount of GLUT4 protein,but the abundance of the CAV3 and GLUT4 proteins on the cell membrane increased.Glucose uptake was increased,and this did not affect the glycogen synthesis,but the cell surface area and cell proliferation increased.While there were significant increases in p-Akt and p-p70s6 K,which is a downstream component of Akt signaling,the level of GSK3? protein,another component of Akt signaling did not change.3.C2C12 cells stably transfected with the P104 L CAV3 mutant exhibited decreased glucose uptake and glycogen synthesis,decreased CAV3 expression and reduced localization of CAV3 and GLUT4 on the cell membrane.The P104 L mutant significantly reduced the cell diameters,but accelerated cell proliferation.Akt phosphorylation was inhibited,and protein expression of GLUT4,p-GSK3? and p-p70s6 K,which are molecules downstream of Akt,was significantly decreased.Conclusions1.The muscle,CAV3 protein can activate Akt signaling,increase GLUT4 protein localization in the cell membrane,increase glucose uptake,and promote myocyte growth and proliferation.2.The CAV3-P104 L mutation inhibits glycometabolism and cell growth but accelerates C2C12 cell proliferation by reducing CAV3 protein expression and cell membrane localization,which may contribute to the pathogenesis of LGMD-1C.