Development of Flow Cytometry Based Methods to Analyze Cellular Insulin Response Adetokunbo Martins, Byron Hetrick & Carrie McCurdy | Department of Human Physiology | University of Oregon Abstract Insulin Stimulated 2-NBDG Uptake Glut- 4 Membrane Translocation Obesity is a prevalent health issue in the United States and results in many detrimental diseases such as hypertension and diabetes. Insulin resistance is a primary characteristic of obesity and is thought to be the underlying cause of obesity-associated metabolic diseases. Insulin is responsible for homeostasis of blood glucose levels and involves a cellular signaling pathway that activates enzymes phosphatidylinositol 3-kinase (PI 3-K) as a secondary kinase, and AKT as the downstream effector. The objective of our study was to develop flow cytometry based methods to rapidly analyze cellular insulin response in single cell assays. A flow cytometer is an instrument that analyzes fluorescently labeled cellular components one at a time. We have tested several methods for glucose uptake in C2C12 myoblasts, an insulin responsive and physiologically relevant cell line that was derived from mouse skeletal muscle. We have developed methods to measure (1) uptake of a fluorescent glucose analog, 2-NBDG,(2) intracellular analysis of insulin stimulated Akt phosphorylation, and (3) Glut-4 translocation to the cell surface, the final step in the insulin signaling pathway.  Once we have developed and verified a reliable method of assessing insulin action at different stages of the signaling pathway, our future goal is to investigate the role of the most abundant regulatory subunit of PI 3Kinase (p85ɑ, and isoforms p55ɑ, p50ɑ,) in controlling PI3K localization and enzyme activity in response to insulin. B. A. A. 2-Deoxyglucose NBD C. D. B. C. D. E. A. Representation of GLUT 4 translocation to cellular membrane upon insulin stimulation. B. Histogram of cell number vs. intensity of GLUT4 antibody stain in C2C12 cells treated and untreated with the anti GLUT4 stain. C. Bar graph comparison of the mean fluorescence intensities of C2C12 cells that have been permeabilized, unstained or stained with anti – GLUT4 antibodies. Permeablized cells displayed the highest intensity for GLUT-4 translocation. D. Bar graph of C2C12 cells stained with anti–GLUT4 antibodies mean fluorescence intensities vs. insulin stimulation at 1nM, 10nM and 100nM. Surprisingly, cells treated with 10nM insulin displayed the highest intensity. Insulin Signal Transduction Pathway A. Structural figure of the fluorescent glucose analog, 2-NBDG. B. Forward scatter vs. side scatter plot of C2C12 myoblasts. C. Cell count vs. 2-NBDG fluorescence intensity histogram of 100nM insulin stimulated vs. unstimulated cells incubated with 10μM 2- NBDG. The low intensity peak represents cells that did not take up 2-NBDG while the high intensity peak represents cells that did. Insulin stimulated cells displayed an increase in the number of high intensity events, indicating more cells imported 2-NBDG. D. Histogram of cell count vs. fluorescence intensity of cells incubated with 10μM, 100μM, and 1000μM 2-NBDG. E. 2-NBDG concentration dependence on mean fluorescence intensity of the high fluorescence peak. Increased fluorescence intensity with 2-NBDG concentration indicates that 2-NBDG uptake is the cause of the higher fluorescence intensity. Summary Developed flow cytometry based methods to measure cellular functions downstream of insulin signaling. Glucose uptake AKT phosphorylation GLUT4 translocation Advantages of Flow Cytometry based methods Rapid analysis Quantitative measurements Will allow broad response understanding of insulin response in single samples Can be used on mixed populations of cells Akt Phosphorylation A. B. C. PI3K -pAkt +pAkt Akt Y P α Phospho Thr306 Akt Y Insulin signal transduction cascade. The insulin signaling pathway is activated upon an increase in blood glucose levels, normal blood glucose levels range from about 4 to 7mM in a healthy individual. Beta cells located in the pancreas secrete insulin and a long change of phosphorylation occurs through the use of tyrosine kinase receptors. Phosphorylation of essential enzymes including phosphatidylinositol 3-kinase (PI 3-K) and AKT results in the translocation of glucose transporter GLUT4 to the cell membrane, permitting the import of extracellular glucose. AF555 α Rabbit D. E. C2C12 Myoblasts & Insulin Treatment     Fruman, David A. "Result Filters." National Center for Biotechnology Information. U.S. National Library of Medicine, n.d. Web. 02 June 2016. Inukai, K., M. Funaki, T. Ogihara, H. Katagiri, A. Kanda, M. Anai, Y. Fukushima, T. Hosaka, M. Suzuki, B.-C. Shin, K. Takata, Y. Yazaki, M. Kikuchi, Y. Oka, and T. Asano. "P85  Gene Generates Three Isoforms of Regulatory Subunit for Phosphatidylinositol 3-Kinase (PI 3-Kinase), P50 , P55 , and P85 , with Different PI 3-Kinase Activity Elevating Responses to Insulin." Journal of Biological Chemistry 272.12 (1997): 7873-882. Web. McCurdy, Carrie E., Simon Schenk, Michael J. Holiday, Andrew Philip, Julie A. Houck, David Patsouris. "Attenuated Pik3r1 Expression Prevents Insulin Resistance and Adipose Tissue Macrophage Accumulation in Diet-Induced Obese Mice | Diabetes." Attenuated Pik3r1 Expression Prevents Insulin Resistance and Adipose Tissue Macrophage Accumulation in Diet-Induced Obese Mice | Diabetes. Diabetes Journal, n.d. Web. 02 June 2016 A. Akt is phosphorylated downstream if insulin interactions with insulin receptor. Akt phosphorylation can be detected in a flow cytometer by a pAkt specific antibody and a AF555 conjugated secondary antibody. B. Cell count vs. intensity in AKT antibody stain channel in unstained C2C12 myoblasts. C. Cell count vs. intensity AKT antibody stain in stained C2C12 myoblasts. An increased number of events in the high fluorescence region indicates detection of pAkt. D. Bar graph of mean fluorescence intensity of C2C12 cells stained vs. unstained with pAKT primary and secondary antibodies. E. Bar graph of comparison of the mean fluorescent intensities of 1nM vs. 100nM insulin concentration in C2C12 cells stained for AKT phosphorylation. The cells stained treated with 100nM concentration of insulin displayed a higher mean fluorescence intensity. Error bars are the SEM from counting 3-5,000 individual events. Gundry RL, et al,. “The mouse C2C12 myoblast cell surface N-linked glycoproteome: identification, glycosite occupancy, and membrane orientation”. Insulin responsive and physiologically relevant cell line Do not have to differentiate Muscle glucose uptake is the strongest in response to insulin Makes for rapid experiments and analysis