Tag Archives: obesity

The Pursuit of the Insulin Signaling Pathway

This summer, I’ve been working full time in the Obesity and Metabolism Lab, within the Human Physiology Department at the University of Oregon. The lab as a whole is pursuing several research objectives related to how human health is impacted by obesity and high fat diet (HFD), which affect more people today than ever before. The objective of my research is to understand how insulin-resistance, the hallmark symptom of type-II diabetes, develops as a result of obesity and HFD.

Insulin is a hormone produced in the pancreas that is delivered throughout the body, signaling cells when they need to take up glucose from the bloodstream for energy. Cells respond to this communication with a so called “signaling cascade.” Insulin binds to a receptor protein at the surface of the cell, sending the signal via more protein interactions inside the cell, toward the nucleus. This eventually leads to new proteins being made from the cell’s DNA, which will help with glucose uptake. These pathways can be very complex, and have “cross-talk” with other pathways for different cellular functions. If the exact mechanisms of the entire insulin signaling pathway where known, then it would be much easier to see exactly where diabetes causes a disruption, and potentially develop better treatments or a cure.

My project looks at just one protein in the insulin signaling pathway, called phosphatidyl inositol-3-kinase and one of its regulatory subunits called p55a. I’m looking to see if fat cells (called adipocytes) behave differently when p55a is upregulated. When there are relatively high amounts of this regulatory subunit, does the cell become more sensitized to insulin, or less? Does it take up glucose better, or worse? These observations will hopefully allow me to infer how p55a modulates PI3K’s role in the insulin signaling pathway.

What did you learn working in the lab?

As my first real research experience, I’ve learned a lot about the process of laboratory work. The biggest thing I’ve noticed so far is that my experiment isn’t moving as quickly as I had anticipated. Much of my time has been spent troubleshooting. I’ll try a procedure, it won’t quite work as it should, and I’ll have to re-trace my steps to determine how I can optimize the process or correct problems. It seems like each week something new arises that needs to be re-done two or three times. For example, I’ve been growing mouse cells in flasks which I will use for my experiment once they have “matured” into adipocytes. This process requires about two weeks of careful cell maintenance, and I’ve had to start over once because they didn’t develop properly. The other researchers in my lab have told me that they, too, still spend a lot of time troubleshooting, and that being able to recognize necessary adjustments is a crucial skill for a scientist to possess.

Life as a Scientist

My day-to-day life as a research scientist is quite different than it was in all my previous jobs. The work is very much self-supervised, and I have a freedom to plan out my daily tasks in an order that I choose. On the other hand, certain time-sensitive procedures dictate my schedule very strictly, such as changing out the growth media for my cells. This needs to be done every three days, or the cells can die. When juggling multiple projects such as this, I’ve found that making a specific schedule is key. I often write out game plans which allocate time to each of my required tasks for the coming days. This keeps me working efficiently, without losing sight of the broader goals for my ten-week fellowship.

Working as part of a lab team is my favorite part. Every day, I’m learning valuable new skills from my co-workers and asking them interesting questions. They’ve all been very accommodating and willing to teach me. We’ve discussed all kinds of topics, ranging from protein function to the application process for graduate school. The lab is an intellectual environment in which I’ve definitely enjoyed spending my summer.

Shawn Melendy is a junior majoring in Biochemistry at the University of Oregon in Eugene, OR. He is a 2017 APS STRIDE Fellow working in Dr. Carrie McCurdy’s Obesity and Metabolism research lab at the U of O. Shawn’s research is funded by the APS and a grant from the National Heart, Lung and Blood Institute (R25 HL115473-01), as well as Dr. McCurdy’s grant from the NIH (R01 DK095926). After finishing his bachelor’s degree at Oregon, Shawn plans to attend graduate school and earn a PhD. in biochemistry, pursuing a career as a research scientist.

Impact of Soybean Oil High Fat Diet on Hypothalamic Feeding Circuits in Mice

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.

At the Interface of Physiology and the Pleckstrin Homology Domain

While the prevalence of obesity increases each year and continues to underlie many serious health conditions in children and adults alike, the genetic aspects and pathophysiology associated with the disease are not fully understood. Consequently, the obscurity of the mechanisms underlying obesity makes treating affected patients difficult. Now more than ever, as we face an intercontinental obesity pandemic, our knowledge about the disease must grow faster than the climbing obesity rates.

Sources: NCHS, National Health Examination Survey and National Health and Nutrition Examination Surveys.

The Src homology 2 B adaptor protein 1 (SH2B1) has recently been identified as a gene associated with obesity, and SH2B1 mutations have been identified in a large cohort of patients with severe early-onset obesity. Three of the human mutations identified in the obese patients are located in the pleckstrin homology (PH) domain of SH2B1. The Carter-Su Lab is currently studying one of these point mutations (P322S) using a CRISP-Cas9 generated mouse model. Along with the P322S mouse model, the Carter-Su Lab has also generated a mouse model with a six base-pair deletion that causes a 2 amino acid deletion (ΔP317, R318) in the PH domain of SH2B1. This mouse model is important because it explores the function of the PH domain, which seems to be important for the function of SH2B1, yet has not been fully investigated. For my summer research project, I have been working on determining the metabolic phenotype of the ΔP317, R318 mice. I have been measuring the food intake and body weight of a cohort of 48 animals, as well as performing glucose tolerance tests and insulin tolerance tests to investigate whether the six base-pair deletion affects glucose metabolism. I am also performing blood draws to examine levels of insulin and leptin, the latter of which is referred to as the “satiety hormone.” My project using the ΔP317, R318 mouse model will help investigate the function of SH2B1 in regulating energy homeostasis as well as provide insight into the function of the PH domain of SH2B1, which is conserved across species. This is all extremely important since understanding how SH2B1 works will provide insight that may enable identification of new therapeutic targets for obesity.

Wild-type mouse (left) pictured next to a SH2B1β ΔP317,R318/ΔP317,R318 mouse (right).

Doing research in a lab is an incredibly rewarding experience. I have made plenty of mistakes along the way (my lab can verify this), but each day I am getting better. What surprised me the most about working in a research lab this summer was how much other people were truly willing to teach me — not because I asked them to or because they had to, but because they knew I wanted to learn. In research there is no “one size fits all” policy, and that is okay. It is all about finding what works for you. The reality is that the process of doing research is not perfect; things will go wrong, and that is a fact. The honest truth is that many experiments will have to be done…and redone, but in my case, all of the experiments I have performed in my 7 weeks so far have been successful in the end. My preliminary results are mostly what my research host and I expected, some of the phenotypes being even more extreme that originally hypothesized (cue obese mouse below!)

SH2B1β ΔP317,R318/ΔP317,R318 female on a scale weighing more than three times the amount of a wild-type female of comparable age.

There are days when I feel extremely frustrated, days when I think I’ll be stuck in lab all night, and days when I feel like the luckiest person in the world just to be where I am, doing what I’m doing. The day-to-day life of a scientist is different every day, and that is what draws many of us to science in the first place. Science allows us to be innovative and creative and to spend each day with other people who share similar interests, working on different pieces of our own puzzle. Working as part of a lab team has provided me with an amazing support system, and I could not be more thankful for that. As a member of a team, you are responsible for pulling your own weight, which is both a blessing and a curse. When something goes right, that is on you, but if something goes wrong, you must take responsibility for that too. The biggest struggle that comes with this is managing your time effectively, especially when faced with so many time sensitive issues. Your animals will not stop aging; your cells will not live forever if they are not split. The life of a scientist is a roller coaster, but it is one that I would choose to ride each day.

References

Overweight & Obesity Statistics | NIDDK. [online] National Institute of Diabetes and Digestive and Kidney Diseases. https://www.niddk.nih.gov/health-information/health-statistics/overweight-obesity [13 Jul. 2017].

Lauren DeSantis is a junior double majoring in Neuroscience and Spanish at the University of Michigan in Ann Arbor, MI. She is a 2017 Undergraduate Summer Research Fellow (UGSRF) doing research in Dr. Carter-Su’s lab in the Molecular and Integrative Physiology Department at the University of Michigan Medical School in Ann Arbor, MI. Lauren’s fellowship is funded by the APS. After graduation, she plans on pursuing her MD-PhD to become a neurosurgeon-neuroscientist.