It is well known that consuming fatty foods induces obesity, although relatively little attention is given to the effects of different types of fat. The neuroendocrine hormone leptin is known to reduce body weight and fat through inhibition of food intake and increased energy expenditure (Friedman, 2011). Leptin’s actions are mediated by leptin receptor (OB-r), which is expressed in the arcuate nucleus of the whole hypothalamus (Meyers et al., 2009; Swanson and Sawchenko, 1980). Leptin may rapidly inhibit food intake by altering the secretion of hypothalamic neuropeptides such as neuropeptide Y (NPY), a stimulator of food intake, and/or corticotropin-releasing hormone (CRH), an inhibitor of food intake. (Jang, et al., 2000). STAT3, a neuronal transcription factor involved in the hypothalamic leptin signaling pathway via OB-r is necessary for the effects of leptin on food intake and hepatic glucose metabolism (Buettner et al., 2006). In this study, male C57BL/6N mice were fed one of four iso-caloric diets for a duration of 17-27 weeks: a high fat diet (40%) made up of coconut oil (HFD), a high fat diet containing soybean oil (SO) which is high in polyunsaturated omega-6 fatty acid linoleic acid (LA-HFD), and a vivarium chow control (VC). We hypothesize that transcription levels of OB-r and STAT3 will be down regulated in HFD mice, and that transcription levels of NPY and CRH will increase in the HFD mice. Pre-designed oligonucleotide primers for GOIs will be used and optimized for efficiency and specificity. RNA will be isolated from whole hypothalamus brain samples from each diet using an RNA isolation kit. Gene analysis (qPCR), will be performed to measure transcription levels of each gene. We have been able to cut and section whole hypothalamus brain samples from each diet, and have run efficiencies on the OB-r and STAT3 primer. Our efficiency results so far have shown OB-r and STAT3 primers giving good melt curves and efficiency values that lie within our acceptable range of 90-110%. The next steps will be to run efficiencies on NPY and CRH, and afterwards run qPCR with the isolated RNA samples. This work will give further insight on the regulation of specific genes in different types of diet.
Going into this project, I had prepared to dedicate a lot of my time to being in lab. I was responsible for my own schedule, which allowed me at times to enjoy some shorter days in lab, while understanding that some days would require me to stay longer to get the required amount of work done. Often, things would not go as planned, as some of the experiments did not yield expected results. When that occurred, I took a step back for a closer look at all the possible sources of error in the experiment. Often, these errors were shown in the experiment’s results, so the source of error was more easily tracked. Sometimes, however, these errors resulted in having to change different aspects of the project. For this current project, we had to change some of the primers that we were using because some of them did not yield good band/efficiency results. Luckily, while this did change what primer was initially going to be explored, it did not change the region or the overarching hypothesis of this project.
Going into lab every day has changed my work ethic greatly. Being able to set my own schedule for experiments has been extremely nice for me, and has allowed me to see personally the progress I am making in my project and the experience I am gaining in lab, which is one of the many pros of being a scientist. This experience has made me more aware of the importance of setting my own schedule, and how flexible it needs to be to account for possible errors in the experiment and setting up various meetings with professors, lab mates, and technicians, which can be considered a con of being a scientist. Experiments will often not give good results, and it may sometimes be frustrating to have to go back and troubleshoot each individual experiment. Having lab mates there, however, was a huge help in overcoming some of the challenges I faced with my experiment. Working as part of a lab team has shown me that each member of the lab collaborates with their peers’ projects and helps each other when necessary. I have learned a lot about each of my lab mates, including their project layout and work ethic. Working together with my lab mates this past summer has allowed to connect with them and gain experience from their guidance and help.