Knockdown of Exocyst Sec10 in Mouse Skeletal Muscle Cells and Analysis of Insulin‐Stimulated GLUT4 Exocytosis

Abstract

Type II diabetes is a metabolic disorder characterized by insulin resistance and high glucose in the blood. The GLUT4 receptor is the major glucose transporter that allows insulin‐stimulated glucose uptake into metabolic tissues such as adipose and skeletal muscle. In cultured adipocyte 3T3‐L1 cells, the eight‐protein exocyst complex has been shown to exocytose GLUT4 vesicles to the plasma membrane when stimulated by insulin. Our lab has extensively studied the activity of the exocyst trafficking complex in renal epithelial cells, and we have successfully generated a conditional knockout mouse for the exocyst subunit Sec10. Previous studies have been limited to cultured adipocytes, but skeletal muscles are responsible for taking in a higher percentage of glucose, and it has been shown that exercise helps maintain glucose levels to prevent the onset of type II diabetes.To evaluate whether the exocyst also plays a role in GLUT4 regulation in skeletal muscle cells, we used shRNA to generate stable Sec10 knockdowns in C2C12 mouse skeletal muscle cells. After confirming the knockdown, we measured glucose uptake using two different methods. First, we measured the amount of glucose in the media of Sec10 knockdown and control cells over the course of 2 weeks. Second, we compared insulin‐stimulated glucose uptake using a fluorescent glucose analog, 2‐NBDG. Based on preliminary findings, knockdown of Sec10 in mouse skeletal muscle cells results in decreased glucose uptake. Ongoing work with skeletal muscle‐specific Sec10 knockout with our new mouse model will help with better characterization of the role of the exocyst in insulin signaling and GLUT4 exocytosis that may reveal new therapeutic targets for human type II diabetes and pre‐diabetic insulin resistance.