Tag Archives: gastrointestinal

An Internship to Cure Obesity
Caleb Smith
Senior, Applied Health Science
Messiah College
2019 UGSRF Fellow

My Research Project

Trayagli & Anselmi (2016). Vagal Control of Gastric Functions

When exposed to a high-fat diet (HFD), both human and rat models show inflammation in the brainstem. The specific area of concern is called the dorsal vagal complex (DVC) which is responsible for maintaining homeostasis, or the balance, of energy and gut function. Specific neuronal cells in the brain, called astroglia, along with inflammation, help to control the DVC.

Previous studies have shown models that experienced a short period of energy regulation after a 24-hour period of excessive eating when exposed to a HFD. Therefore, the purpose of this study was to determine how that energy balance is restored during exposure to a HFD through the activation of the astroglial cells. As part of this study,  control and HFD chow were fed to a rat model for one, three, five and 14 days. The brainstems were removed and cut into thin slices and the astroglial cells were tagged with proteins that illuminated under specific lighting. This process, known as immunohistochemistry, allowed for the density of astrocytes and physical characteristics—like size and shape—to be analyzed. To process the role of the astrocytes in this energy metabolism regulation, small tubes called cannulae were surgically inserted into the DVC in order to directly administer fluoroacetate, a drug that inhibits the function of astrocytes. Once the rats recovered from surgery, a five-day control and HFD exposure were fed to the rats while food intake and body weight were measured twice daily.

While data was still being collected, preliminary data confirmed the role of astrocytes in metabolic regulation during HFD exposure. That meant astrocyte activation was necessary in controlling metabolic balance when exposed to HFD. Ultimately, painting the picture of how energy balance is controlled will be essential to producing a therapeutic drug that can help treat obesity.

Realities of Research

Working in a lab was similar to what I expected while still being very different. In many ways, research in a lab is exactly what you would expect: you make solutions, follow very strict procedures and analyze data for results. The techniques that I learned in high school and college labs were carried over with regards to safety, proper procedure, how to handle materials and how to pipette. I would come in and begin my day the same way by weighing and giving rats shots. In other ways, the lab was not what I expected it to be.

There were long periods of time, whether a few hours or days, where researchers were writing manuscripts to submit to journals, editing their own or other colleagues’ manuscripts, writing grant proposals or reading research happening in someone else’s lab to stay up to date on the current information. Not every minute was spent performing an experiment and analyzing data. The rest of my day involved one or two various procedures, so every day was different. Usually, I would have one or two main goals or techniques for the day. Some days it was immunohistochemistry or analyzing material under a confocal microscope. Other days I performed surgeries on rats or loaded brain tissue onto microscope slides. I was surprised by how similar this lab was to high school or college labs.

In some instances, we had to develop our own techniques. For example, we performed surgery on rats using the procedures and equipment we developed. Other procedures followed strict protocol that had been around for many years, like immunohistochemistry. The lab mentors had a strong understanding of their expectations for the outcome of an experiment, so we were able to successfully perform the experiments and get conclusive results that either supported or refuted the hypothesis. The results were what we had expected. We had a solid background understanding that allowed us to make a very scientifically guided hypothesis. However, that didn’t mean we didn’t have to start over in some cases. There were surgeries that did not go as planned, which resulted in having to start over and try again. Not everything in a lab runs perfectly or goes according to plan. Accidents happen, mistakes are made, and fresh starts were common. Luckily for me, no changes in the overall plan had to be made. Preliminary data suggested that we were going to receive conclusive results.

Life as a Scientist

Brain-Gut Laboratory Members at Milton S. Hershey Penn State University College of Medicine

Over the summer, I was able to dive into the life of a scientist and see what the day-to-day job was like. I was highly surprised by how much time was spent doing activities other than hands-on, standard research much like one would expect from high school or college labs. I couldn’t believe how much time each scientist spent doing work on a computer. In fact, my summer lab seemed to spend about half of the time performing procedures and the other half is spent on the computer doing activities like writing grant proposals, writing articles to be published in a journal, reviewing journal articles, ordering supplies and reading recent research. I just never realized how much time would be spent on these things, but the best part, was performing surgeries on rats. I was able to independently perform hands-on science in a way that, quite frankly, made me feel pretty cool. Who wouldn’t think it sounds impressive saying they’ve given a rat surgery before? I liked being involved in physical work instead of taking care of business on the computer. The surgeries were challenging enough that they required critical thinking, simple enough that I could feel confident in what I was doing and unique enough that every rat’s surgery was a little different. Plus, it was neat to see the success of the surgeries I performed. On the flip side, the worst part of the job was immunohistochemistry. The process was very tedious and with the large number of samples I had to do, it became quite exhaustive and—dare I say—boring. I would spend days at a time washing samples, mixing them in different solutions, transferring them between containers and very carefully plating them on microscope slides.

The fact is that research does not always involve exciting and intriguing work. The other interesting aspect of working as a scientist was working as part of a lab team. Each person had independent projects that they were working on, but every project related back to the overall theme of the lab and contributed to the overall goal of the study. It was neat to be able to hear from other people about their findings and being able to learn as a collective. It was nice to be able to ask anyone in the lab for their input on a matter because person had an understanding of the science behind almost every project, even if it wasn’t their own. It allowed me to feel independent and like I was contributing my own work while having a support system in place in times of uncertainty or confusion. Ultimately, my summer research fellowship was a wonderful experience that allowed me to engage in hands-on research and experience the daily life of a scientist.

References:

Buckman,L.B. et al. Evidence for a novel functional role of astrocytes in the acute homeostatic response to high-fat diet intake in mice. Mol. Metab 4, 58-63 (2015).

Camilleri,M. Peripheral mechanisms in appetite regulation. Gastroenterology 148, 1219-1233 (2015).

Clyburn,C., Travagli,R.A., & Browning,K.N. Acute High Fat diet Upregulates Glutamatergic Signaling in the Dorsal Motor Nucleus of the Vagus. J. Amer. Physiol. Gastro. Liver Physiol. 314, 623-624 (2018).

Daly,D.M., Park,S.J., Valinsky,W.C., & Beyak,M.J. Impaired intestinal afferent nerve satiety signalling and vagal afferent excitability in diet induced obesity in the mouse. J. Physiol 589, 2857-2870 (2011).

de Lartigue,G., de La Serre,C.B., & Raybould,H.E. Vagal afferent neurons in high fat diet-induced obesity; intestinal microflora, gut inflammation and cholecystokinin. Physiol Behav. 105, 100-105 (2011).

Kentish,S. et al. Diet-induced adaptation of vagal afferent function. J Physiol 590, 209-221 (2012).

Janssen,P. et al. Review article: the role of gastric motility in the control of food intake. Aliment. Pharmacol. Ther. 33, 880-894 (2011).

Troy,A.E. & Browning,K.N. High fat diet decreases glucose-dependent modulation of 5-HT responses in gastrointestinal vagal afferent neurons. J Physiol 594, 99-114 (2016).

 

Caleb Smith is a senior majoring in applied health science with a pre-professional concentration at Messiah College in Mechanicsburg, Pennsylvania. He is a 2019 Undergraduate Summer Research Fellow (UGSRF) in the lab of Dr. Kirsteen Browning at the Penn State Hershey Medical Center’s College of Medicine in Hershey, Pennsylvania. Caleb’s fellowship is funded by the American Physiological Society. Upon graduating, Caleb hopes to continue into the medical field by becoming a physician assistant. 

Targeting NHE3 in the Hfe mouse model of hemochromatosis

Research Project

Over the summer I have been working in Dr. Bryan Mackenzie’s lab at the University of Cincinnati College of Medicine in the Pharmacology and Systems Physiology department. My focus has been on finding novel therapeutic targets in the treatment and/or prevention of iron overload disorders. Hemochromatosis is just one of several common hereditary disorders that result in iron overload and subsequent liver disease, heart damage, and a slew of other health disorders across several body systems. Since there is no regulated mechanism for iron excretion, iron homeostasis of the body is regulated by iron absorption. The main mechanism by which non-heme iron (the majority of iron humans consume from their diets) is absorbed from the small intestine is the divalent metal ion transporter-1 (DMT1) (3). DMT1 is already a validated therapeutic target in hereditary hemochromatosis. More recently, the Mackenzie lab identified a role for the Na+/H+ exchanger-3 (NHE3) in intestinal absorption of non-heme iron by DMT1.

 

Intestinal iron uptake (redrawn from reference 1)

The Mackenzie lab demonstrated that NHE3 generates the driving force for iron absorption via DMT1 by transporting protons into the small intestine (2). My project this summer has built upon this prior knowledge using an Hfe knockout mouse model. Rendering this single gene inoperative in mice results in a very good model of iron overload. We are then able to test the hypothesis that iron overload in the Hfe KO mouse can be corrected by ablation of NHE3. The idea is that without NHE3, the driving force for DMT1-mediated iron uptake is reduced, thereby reducing iron absorption. If the data supports this hypothesis, we can conclude that ablating NHE3 prevents iron loading in the Hfe mouse model of hereditary hemochromatosis. Even further, we can reveal intestinal NHE3 as a potential therapeutic target in the treatment of iron overload.

Realities of Research

Throughout the summer I have learned countless basic research techniques and have mastered various skills that are good to possess in the Mackenzie Lab. I have learned how to ween and care for our colonies of mice. I now know how to genotype them using tail clippings and polymerase chain reaction (PCR). This step is especially important because it determines which mice are our experimentals, or the mice we collect blood and tissue samples from on experiment days. I was placed in charge of the mouse experiments, learning how to effectively set-up and delegate responsibilities. Being my first full-time experience in a research lab, I appreciated this opportunity to take charge and have ownership over such a large task. I also had the task of running serum iron assays and non-heme iron assays on freezers full of samples that have been collected. This task proved to be challenging and slightly stressful at times, as I found errors in our established assay protocols and analytical procedures. For example, some mice had very little blood collected on experiment days, meaning I had very little serum to run the assays. When the protocols had detrimental errors, I felt I had wasted irreplaceable serum at times. Luckily, I have corrected our protocols and have finally organized/assayed most of our samples. I have also not only learned how to operate a software program called SigmaPlota to analyze my data, but have learned how to edit our labs established transformations within this software. This summer full of learning techniques, troubleshooting, and analyzing my data has been an invaluable experience. I have transformed into a proficient, independent, and productive lab member in just a few months’ time.

Life of a Scientist

I have enjoyed my day-to-day life in the lab this summer. I was pleased to learn that a 9-to-5 workday is certainly possible, with proper planning. I also understood that some late nights/early mornings are unavoidable, especially when things didn’t go as planned and required a little extra time. I discovered that the environment in the lab was much more relaxed than I anticipated. I was able to control my day to day activities with little intervention, unless I needed assistance or extra guidance. This assistance from other lab members is something I really appreciated. Although everyone has their own projects and own experiments to run, a helping hand was always there when I needed it. I liked this collaborative, team-based environment and believe it greatly increased all I was able to learn in the lab this summer. The only con in the life of a scientist that I can think of is the down time that sometimes occurs, as I’m the type of person that likes to stay busy. Luckily, I was able to fill my time by reviewing relevant literature in the field or discussing my progress with other lab members. Overall, I really enjoyed getting to experience the life of a scientist for a summer and look forward to finishing up my project/exploring new directions in the lab.

 

References

  1. Mackenzie B, Garrick MD. Iron Imports. II. Iron uptake at the apical membrane in the intestine. American Journal of Physiology-Gastrointestinal and Liver Physiology289, 2005.
  1. Shawki A, Anthony SR, Nose Y, Engevik MA, Niespodzany EJ, Barrientos T, Öhrvik H, Worrell RT, Thiele DJ, Mackenzie B. Intestinal DMT1 is critical for iron absorption in the mouse but is not required for the absorption of copper or manganese. American Journal of Physiology-Gastrointestinal and Liver Physiology309, 2015.
  1. Shawki A, Engevik MA, Kim RS, Knight PB, Baik RA, Anthony SR, Worrell RT, Shull GE, Mackenzie B. Intestinal brush-border Na /H exchanger-3 drives H -coupled iron absorption in the mouse. American Journal of Physiology-Gastrointestinal and Liver Physiology311, 2016.
Sydney Stone is a junior majoring in Medical Sciences at the University of Cincinnati in Cincinnati, OH. She is a 2018 Short-Term Research Education Program to Increase Diversity in Health-Related Research Fellow (STRIDE) working in Dr. Bryan Mackenzie’s lab at the University of Cincinnati College of Medicine. Her summer of research was supported by APS and a grant from the National Heart, Lung, and Blood Institute (Grant #1 R25 HL115473-01). Upon graduation, Sydney plans to pursue an MD/PhD or MD/MS in order to become a physician scientist. She hopes to combine proficiency in physiology with invaluable access to patients in order to follow her passion for preventing and curing human disease.