CRISPR Modifications of Gluconeogenic Genes in Primary Hepatocytes

microscope and computer workstation

Students

Faculty

Associate Professor of Physiology

Project Summary

Through PURM, I was able to work under Dr. Joseph Baur at the Institute for Diabetes, Obesity and Metabolism in the Perelman School of Medicine.

Our project focused on using primary hepatocytes to study liver metabolism. These cells are useful for studying liver specific functions that are not modeled in hepatoma cell lines, such as glucose production. However, normally primary hepatocytes lose their morphological structure and function within 72 hours of culture, which is insufficient time for genetic modifications. Therefore, our first objective was to determine whether primary cultures could survive long-term and still be able to produce glucose. Using optimized media supplemented with growth factors, we were able to extend the lifespan of these hepatocytes to up to 8 days while still maintaining glucose production.

Next, we wanted to use these hepatocytes to study the effect of metformin, an antidiabetic drug, on glucose metabolism to understand how metformin works. It was previously theorized that metformin activated the AMP-activated protein kinase (AMPK). AMPK is an enzyme expressed in the liver that plays an important role in insulin signaling, whole body energy balance and the metabolism of glucose and fats. It was thought that the activation of AMPK was necessary for metformin to inhibit liver glucose production. However, currently in mouse models in which AMPK have been knocked out in hepatocytes, the effects of metformin were not eliminated. Instead, we believe that metformin may be working through LKB1, a kinase upstream of AMPK. Therefore, we decided to use CRISPR-based modifications to knockdown LKB1 in order to determine whether or not metformin works through LKB1.

As a biology major, working in the Baur lab this summer gave me valuable experience and knowledge that I would have not been able to learn in a lecture hall or a lab course. It showed me the value of what I was learning in the classroom by allowing me to apply what I learned in class to my project and research. In addition, it introduced me to the field of diabetes, obesity and metabolism which I didn’t know much about before and taught me what research really entailed. Working in such a research-intensive environment allowed me to take everything in and learn not just from my own research projects but from what others were working on as well. From going to lab meetings every week to just talking with the fellow researchers who were always willing to help me, this really added to the learning experience. I was able to learn many techniques such as cell culturing and maintenance, mouse caring and handling, RNA extraction, cDNA synthesis, bicinchoninic acid/ protein assays and glucose hexokinase assays. Having wanted to be a physician since I was a little kid, this experience exposed me to whole new side of medicine that I had never knew about and makes me want to continue research here at Penn.