This summer I was fortunate enough to participate in the University of Pittsburgh’s Summer Undergraduate Research Program (SURP), which was generously funded by the American Society for Investigative Pathologists (ASIP). Over the summer I gained paramount experience working in the Department of Pathology under supervision of Dr. Wendy Mars. This incredible opportunity allowed me to gain insight into the world of liver pathologists and research professionals, which has since played a role in my future career aspirations. I attended lab meetings, seminars, and departmental lectures throughout the summer, which exposed me to a myriad of different disciplines and researchers. Not only was I able to meet talented and inspiring undergraduates during my program, but I also had the privilege to work alongside incredible professionals in a cutting-edge research lab, learning about liver microstructure and pathology.
The liver performs various functions essential for maintaining healthy lipid, carbohydrate and protein levels.Â A plethora of proteins are involved in regulating lipid metabolism, including the low-density lipoprotein receptor related protein 1 (LRP1) and Hepatocyte Nuclear Factor 4 alpha (HNF4Î±). My summer project focused on confirming that HNF4Î± is able to bind to LRP1 and analyzing the effect diet may have on the location and abundance of the complex. LRP1 is a multi-regulatory receptor that has a significant role in lipid homeostasis via its role as an apolipoprotein E and chylomicron remnant receptor. HNF4Î± is a nuclear receptor and transcription factor that regulates multiple genes connected with lipid metabolism. Previous data from the Mars lab showed that these two proteins are able to form a complex, and we hypothesized that the complex may regulate HNF4Î± target genes related to dietary metabolism.
I initially ran western blots on cytoplasmic- and nuclear-enriched extracts from livers of Plasminogen Activator Inhibitor type I (PAI-1) wild type (WT) and knockout (KO) mice fed either a normal chow (NC) or high fat diet (HFD) in order to confirm the presence of the complex and its location. We used PAI-1 mice because LRP1 controls PAI-1 turnover and there is evidence showing that the absence of PAI-1 in obese mice protects animals from developing type 2 diabetes. I later ran an immunoprecipitation (IP) on total cell lysates in which I immunoprecipitated LRP1 and probed with HNF4Î± to verify the two proteins were bound to each other. Immunohistochemistry (IHC) was also performed in order to visualize and interpret the results.
After I analyzed and quantified the western blots, it was apparent that in both the WT and KO mice, there was less HNF4Î± bound to LRP1 in the cytoplasmic-enriched HFD samples as opposed to the cytoplasmic-enriched NC samples. Furthermore, the IP confirmed the complex was in fact real and that overall, complex formation was more prevalent in HFD fed animals versus their NC counterparts. Interestingly, IHC also showed differences in the localization of HNF4Î±, with loss of nuclear staining for HNF4Î± in areas of fat deposition in the HFD fed KO animals. These findings confirm that LRP1 and HNF4a are forming a complex that is affected by diet and provide evidence that PAI-1 plays a regulatory role in the localization of HNF4a.
I am extremely grateful to have been a part of this incredible opportunity made possible through the University of Pittsburgh and ASIP. I was able to gain invaluable experience working in a lab performing revolutionary research that can potentially have clinical applications. As a result of SURP, I now know that I would like to incorporate research into my future career as a veterinarian and potentially receive a PhD in addition to a DVM. I was so lucky to be paired with Dr. Mars and she continues to mentor me through my undergraduate experience. Dr. Michalopoulos and the Department of Pathology were very accommodating and helpful, and I am appreciative that I was able to participate in a program that is so dedicated to making sure its students have the best experience possible. I can confidently say I am one step closer to being a veterinary scientist, thanks to ASIP and SURP.