Presenter(s): Shawn Melendy − Biochemistry
Faculty Mentor(s): Carrie McCurdy, Byron Hetrick
Poster 75
Research Area: Natural/Physical Science
Funding: UROP mini-grant, American Physiological Society Undergraduate Summer Research Fellowship Program
Type 2 Diabetes, an increasingly prevalent disease worldwide, is partially caused by a progressive loss of insulin response in adipose tissue and skeletal muscle. Multicolor flow cytometry is a powerful tool that can be used to measure multiple signaling events simultaneously in specific cell types within mixed populations. The objective of this study is to design a sensitive and high-throughput assay to measure key points of regulation in the insulin signaling pathway for myocytes using flow cytometry. We have developed a multicolor flow cytometry panel to measure the insulin stimulated phosphorylation of Akt(S473) and the transport of the insulin responsive glucose transporter, GLUT4, to the plasma membrane. C2C12 myoblasts were stained with primary conjugated antibodies for pAkt(S473) and an extracellular region of GLUT4, indicative of translocated GLUT4 present
in the plasma membrane. Both C2C12 myoblasts, an immortalized cell line, and primary myoblasts, isolated from non-human primate muscle, responded to insulin with increased pAkt(S473) and plasma membrane GLUT4 with an EC50 of <10nM, similar to physiological response. Future work will expand the panel to measure phosphorylation of insulin receptor substrate and phosphoinosital 3-kinase (PI3K) activity by quantitating phosphoinosital (3,4,5) phosphate (PIP3) production. The sensitivity of the assay will be demonstrated by inhibiting key insulin-activated kinases including PI3K by Wortmannin, and Akt activation by MK- 2206 and measuring insulin signaling at points up and downstream of inhibition. We anticipate that this will provide a powerful method to rapidly dissect the insulin signaling cascade for a specific cell type within mixed populations of cells.