This summer I had the opportunity to work under the leadership of Dr. Paul Titchenell’s lab with Megan Stefkovich, a graduate student in the lab, in the Institute for Diabetes, Obesity, and Metabolism affiliated with the Department of Physiology at the Perelman School of Medicine. The Titchenell lab studies metabolic regulation with a special emphasis on the hormone insulin. 

The overarching goal of this project was to explore how the protein glucokinase (GCK) is acutely activated by insulin in hepatocytes. GCK is a hexokinase found in liver (and pancreatic) cells that phosphorylates glucose to glucose-6-phosphate in glycolysis. GCK is sequestered in the nucleus during fasting by glucokinase regulatory protein (GKRP) but translocates to the cytoplasm after a meal, where it binds with phosphofructokinase 2 (PFK2) and becomes catalytically active. Preliminary data has shown that PFK2 is phosphorylated in an AKT dependent manner. This process of GCK translocation and subsequent activation is increased by insulin, although the mechanism through which this occurs is unknown. We hypothesize that insulin signaling via AKT stimulates GCK cytoplasmic translocation by promoting increased binding with PFK2.

My project contained three major experiments. First, preliminary western blot analysis showed phosphorylation of PFK2 at the Ser486 phosphorylation site in response to insulin. The aim was to observe whether this phosphorylation would be observable in skeletal muscle. Results showed that phosphorylation of PFK2 was observed in liver but was not seen in skeletal muscle, suggesting specificity to hepatocytes. Next, preliminary immunofluorescence data of hepatocytes showed that cytoplasmic translocation of GCK is promoted by insulin through an AKT-dependent mechanism. The goal of this experiment was to replicate previous results using a lower concentration of glucose that was reflective of a naturally occurring environment with high glucose levels. The results were found to be replicable with a lower glucose concentration suggesting that the mechanism is relevant under physiological conditions. Finally, a nested PCR technique was used to attempt to clone and tag GCK, GKRP, and PFK2 from rat CDNA, of which GCK and GKRP were successfully cloned. The long term goals of this cloning experiment would be to overexpressed the tagged proteins in an immortalized cell line which would be used to observe how PFK2 affects translocation. This would be done through mutation of the Ser486 phosphorylation site. 

I appreciate the opportunity provided by CURF to work on this project as well as the members of the Titchenell lab who provided me with an invaluable introduction to research at Penn.