diet | APS Undergraduate Researcher

The Western diet is high in fats and sugar and can lead to an increase in metabolic diseases, which cause a chronic state of peripheral inflammation (1). My project this summer aims to observe the effect of diet on brain inflammation. We used a mouse model of tagged peripheral monocytes (3). Monocytes turn into macrophages, which target inflammation in the body and brain (2). These mice were fed either a diet high in fat and fructose or a normal diet for 5 weeks. Then the blood from their brain was washed out, and the brain was sliced. The slices were stained for the genetic tag for the peripheral macrophages. Peripheral macrophages found in the brain suggest that chronic inflammation weakens the blood-brain barrier, allowing peripheral macrophages to cross where they increase brain inflammation. This may cause damage and may have links to diseases such as Alzheimer’s Disease and Parkinson’s Disease, which show increased inflammation in the brain (4). This project would further support the idea that a healthy diet could be a key factor in prevention of brain diseases.

This project, as well as science in general, had many obstacles that I had to overcome. Originally, I planned to analyze a different genetic mouse that modeled Alzheimer’s disease. Those mice were also going to be fed a high fat high fructose or control diet, and were going to be compared to see if there was an increase in peripheral macrophages in the brain in diet treated mice. However, those brains didn’t have the blood cleared from the brain, which limited our ability to see the stain. To overcome that problem, we used the new mouse type that had peripheral monocytes tagged, which had the blood removed from it. With this new mouse model, I would have a smaller number of animals, but I could better test my hypothesis.

I enjoy the day-to-day life in research. I was expecting it to be somewhat repetitive, but that was far from the case. I had many problems that I had to solve and was constantly learning, which made the time fly by. My day was broken up by working on different parts of my experiment, writing and reading literature, and meeting and talking with my lab members. The best part for me was that I constantly learned new things. There were many hiccups in my summer experience, which were disheartening at times. However, solving these problems and further learning more made it rewarding as well. The Tansey lab has many members who have been very helpful in solving these problems. I enjoyed being a part of a larger team, as there were so many projects going on that I could learn from.

References

De Sousa Rodrigues, M. E., Bekhbat, M., Houser, M., Chang, J., Walker, D., Jones, D. P., Oller do Nascimento,C., Barnum, C. J. & Tansey, M. Chronic psychological stress and high-fat high-fructose diet disrupt metabolic and inflammatory gene networks in the brain, liver, and gut and promote behavioral deficits in mice. Brain, Behavior, and Immunity 59: 158-172, 2017.
Khoury, J. E., Toft, M., Hickman, S.E., Means, T. K., Terada, K., Geula, G., & Luster, A. D. Ccr2 deficiency impairs microglial accumulation and accelerates progression of Alzheimer-like disease. Nature Medicine 13: 432-438, 2007.
Saederup, N., Cardona, A. E., Croft, K., Mizutani, M., Cotleur, A. C., Tsou, C.-L., Ransohoff, R. M., & Charo, I. F. Selective chemokine receptor usage by central nervous system myeloid cells in CCR2-red fluorescent protein knock-in mice. PLoS ONE 5: e13693, 2010.
Selkoe, D. J. The therapeutics of Alzheimer’s disease: where we stand and where we are heading. Annals of Neurology 74: 328-336, 2013.

Lindsey Sniffen is a senior majoring in Neuroscience and Behavioral Biology at Emory University in Atlanta, GA. She is a 2017 Integrative Organismal Systems Physiology (IOSP) Fellow in Dr. Malu Tansey in the Department of Physiology at Emory University in Atlanta, GA. Her fellowship is funded by the APS and a grant from the National Science Foundation Integrative Organismal Systems (IOS) (Grant #IOS-1238831). After graduation, she plans to pursue a Ph.D. in pharmacology, and then work in the pharmaceutical industry.

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.

Overcoming Challenges

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.

Edward Truong is a junior majoring in Cell, Molecular, Developmental Biology at the University of California in Riverside, CA. He is a Short-Term Research Education Program to Increase Diversity in Health-Related Research (STRIDE) Fellow working in Dr. Margarita Curras-Collazo’s lab at the University of California in Riverside, CA. Edward’s fellowship is funded by the APS and a grant from the National Heart, Lung and Blood Institute (Grant #1 R25 HL115473-01). After graduation, Edward plans to attend medical school and pursue a career as a physician.