Genetic Links to Obesity: a Neurological Approach
A Neurological Approach to Understanding Obesity
Thomas Jefferson High School for Science and Technology
This article was originally included in the 2018 print publication of Teknos Science Journal.
On my first day at Yale University’s Discovery to Cure program, I watched as my mentor worked with mouse brains, slicing them until each layer was as thin as a hair, immersing them into a vial of solution, and ultimately, used a paintbrush to gently whisk them away onto a slide for microscopy. Having watched her repeat the process for 50 different brain sections, I felt ready to try it out myself. Yet, after attempting to guide my first brain section with the paintbrush, I stared in disbelief at the misshaped slice, a single tear straight down the center nearly splitting the sample in two. I had managed to tear a precious 50-micrometer-thick brain section that could potentially offer the key to unlocking the relationship between the brain and obesity. That section contained vital information that would help study a protein found in the brain called Melanocortin Receptor Accessory Protein 2 (MRAP2), and its effect on obesity.
Over the past decade, obesity has garnered increased attention, not only in the United States, but around the world. Despite the ongoing initiatives to improve nutrition and physical activity, the prevalence of obesity still skyrockets worldwide, with the Center for Disease Control reporting that 40% of American adults and 20% of American children are obese (Gussone, 2017). In order to combat this disease, which can lower life expectancy by up to 14 years, scientists have begun to delve into its causes and tackle the issue at its roots (Kitahara et al., 2014). As such, every year, the scientific community acquires new information on the fundamental role of diet and exercise in combating obesity, but more recently, another potential underlying factor has been uncovered: genetics.
Scientists have discovered that deleting the gene for MRAP2 in mice leads to severe obesity, and that mutations in MRAP2 have been documented in obese humans (Novoselova et al., 2016). MRAP2 mutations are reported to affect the Melanocortin 4 Receptor Protein (Schonnop et al., 2016). According to J. Sebag, MRAP2 interacts with the Melanocortin 4 Receptor (MC4R) in the paraventricular nucleus of the hypothalamus, which is responsible for controlling both food intake and energy expenditure (personal communication, Jan. 15, 2018). Mutations in MC4R have been noted to be the most common cause of monogenic early onset obesity, meaning the deletion of this gene alone is sufficient to trigger early onset obesity (Rouault, Srinivasan, Yin, Lee, & Sebag, 2017). A protein called proopiomelanocortin (POMC), which is located in a separate region of the hypothalamus, known as the arcuate nucleus, activates MC4R, located in the paraventricular nucleus of the hypothalamus (Rouault et al., 2017). POMC interacts with MC4R neurons in the hypothalamus, signaling to the body to decrease food intake and energy expenditure (Rouault et al., 2017). Thus, POMC neurons are associated with reduced food intake and are counteracted by agouti-related protein (AGRP) neurons, which are associated with increased food intake (Rouault et al., 2017).
Although we know that MRAP2 is present alongside MC4R, mice without MC4R and mice without MRAP2 do not develop the same levels of obesity (Chaly, Srisai, Gardner, & Sebag, 2016). This indicates that MRAP2, which is located outside of the paraventricular nucleus, away from MC4R, could also play a role in energy homeostasis (Chaly et al., 2016). That being said, the exact mechanism through which MRAP2 regulates energy homeostasis and the mechanism through which mutations in MRAP2 lead to obesity still remain uncertain (Novoselova et al., 2016).
My research mentor hypothesized that MRAP2 could regulate metabolism through interactions with POMC (neurons that reduce food intake) and AGRP (neurons that increase food intake). MRAP2’s interactions with MC4R could be explained if increased MRAP2 levels resulted in increased POMC production and thus decreased food intake, or if increased MRAP2 resulted in decreased AGRP production, preventing overeating. To test her hypothesis, we bred two different types of mice: mice with standard MRAP2 levels, and MRAP2 knockout mice, or mice without MRAP2. While it has already been established that MRAP2 knockout mice will become obese, we wanted to see if MRAP2 affects obesity by altering POMC or AGRP production. In order to measure these, we sacrificed our mice to analyze their brains. We took vertical sections of the brain, so that the paraventricular nucleus of the hypothalamus and the arcuate nucleus would be present in 10 different brain slices, allowing each brain to provide us with 10 different data points. The brain sections were then stained for our proteins of interest: POMC, AGRP, and c-Fos, a transcription factor. This means that when c-Fos is present in a cell, the cell is being activated. Staining is the first step in immunohistochemistry, a laboratory technique that allows us to quantify the amounts of the specific proteins in the brain.
Through the use of a fluorescent and a confocal microscope, we were able to compare the number of POMC- and AGRP-producing neurons in the arcuate nucleus in wild-type MRAP2 and knockout MRAP2 mice. We also quantified the projections from the arcuate nucleus to the paraventricular nucleus of the hypothalamus, allowing us to quantify the interactions between the POMC and AGRP neurons in the arcuate nucleus and MC4R in the paraventricular nucleus of the hypothalamus. While staining the brain sections for POMC and AGRP allows researchers to count how many POMC and AGRP neurons are in the arcuate nucleus, it does not necessarily show how many of them are active. C-fos is a transcription factor found in active neurons, so by overlapping the C-fos pictures with the POMC and AGRP pictures of a brain section, we can count the number of cells that express both POMC and C-fos or both AGRP and C-fos, thus providing us with the total number of active POMC/AGRP neurons.
Ultimately, while none of my results were statistically significant, my experiment still helped the scientific community take a significant step towards not only understanding MRAP2, a protein undeniably linked to obesity, but also eradicating genetic predispositions to obesity. Like the torn brain section from my first day, the result was by no means ideal; however, neither result was a failure. Instead, they were both stepping stones, whether it was a stepping stone to help me learn the technical procedure, or a stepping stone to bring the scientific community one step closer to preventing obesity.
Chaly, A. L., Srisai, D., Gardner, E. E., & Sebag, J. A. (2016). The Melanocortin Receptor Accessory Protein 2 promotes food intake through inhibition of the Prokineticin Receptor-1. eLIFE, 5. https://doi.org/10.7554/eLife.12397.001
Gussone, F. (2017, October 13). America’s obesity epidemic reaches record high, new report says. Retrieved January 14, 2018, from NBCnews website: https://www.nbcnews.com/health/health-news/america-s-obesity-epidemic-reaches-record-high-new-report-says-n810231
Kitahara, C. M., Flint, A. J., Berrington de Gonzalez, A., Bernstein, L., Brotzman, M., MacInnis, R. J., . . . Adami, H. O. (2014). Association between class III obesity (BMI of 40–59 kg/m2) and mortality: A pooled analysis of 20 prospective studies. PLOSMedicine. https://doi.org/10.1371/journal.pmed.1001673
Novoselova, T., Larder, R., Rimmington, D., Lelliott, C., Wynn, E. H., Gorrigan, R. J., . . . Chan, L. F. (2016). Loss of Mrap2 is associated with Sim1 deficiency and increased circulating cholesterol. The Journal of Endocrinology, 230(1), 13-26. https://doi.org/10.1530/JOE-16-0057
Rouault, A. A.J., Srinivasan, D. K., Yin, T. C., Lee, A. A., & Sebag, J. A. (2017). Melanocortin Receptor Accessory Proteins (MRAPS): Functions in the melanocortin system and beyond. Biochimica et Biophysica Acta (BBA) - Molecular Basis of Disease, 1863(10), 2462-2467. https://doi.org/10.1016/j.bbadis.2017.05.008
Schonnop, L., Kleinau, G., Herrfurth, N., Volckmar, A.-L., Cetindag, C., Muller, A., . . . Hinney, A. (2016). Decreased melanocortin-4 receptor function conferred by an infrequent variant at the human melanocortin receptor accessory protein 2 gene. Obesity, 24(9), 1976-1982. https://doi.org/10.1002/oby.21576