Abstract

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Obesity and diabetes are prevalent diseases across the U.S. and many deaths can be attributed to these diseases each year.  Although there are a number of drugs to treat diabetes  they do not address they do not address the underlying cause of obesity and so have been met with limited success. This proposal begins to address the issue. Currently the only truly effective cure for obesity and diabetes is gastric bypass surgery which is used only to treat the morbidly obese individual. How it reduces weight and cures diabetes in not well understood and this proposal begins to address and study the effects. It is thought that changes in hormonal control of nutrient absorption in conjunction with an imbalance of the microbial populations in the gut are the cause. These studies will begin to investigate the links between the host, the hormones that regulate nutrient absorption and the microbiota within the gastrointestinal tract.

More than one-third (35%) of US adults are obese (1) and are at a higher risk of heart disease, stroke, type 2 diabetes, and certain types of cancer compared to non-obese adults. To mitigate these risks and decrease the rate of obesity, it is important to more fully understand how the human body metabolizes and absorbs dietary nutrients, like glucose. Glucose-dependent insulinotropic polypeptide (GIP) is a hormone and regulatory factor of intestinal glucose absorption (2). Mice lacking a GIP receptor (GIPR-KO mice) fed a high fat diet were protected from obesity (3), and an incretin  effect following oral glucose challenge was not observed in these animals (4). Another important factor in nutrient availability and absorption in the gut is the diverse assemblage of microorganisms that live there (microbiome). These microorganisms play a significant role in breaking down
dietary compounds in food in a process called ‘energy harvest’. For example, the microbiome of obese individuals has an increased ability to harvest energy from food compared to the microbiome of lean individuals(5). It is clear that overabsorption of nutrients is involved in diet-induced obesity, but the mechanisms underlying overabsorbtion have not been adequately addressed and represent a critical knowledge gap for obesity research.

Our overarching hypothesis is that dysfunctional microbiome-GIP-nutrient interactions in the mammalian gut lead to overabsorption and diet-induced obesity. Gastric bypass surgery, a surgery that bypasses part of the jejunum, dramatically alters gut nutrient homeostasis. This surgery is associated with lower GIP plasma levels, reduced obesity, mitigation of diabetes, and restoration of a normal gut microbiome. Despite these health-promoting effects, half of gastric bypass patients fail to undergo remission, begin to gain weight, and develop diabetes again within five years (6). We believe a better understanding and improvement of the gastric bypass effect will improve outcomes, making this procedure more effective in a greater number of patients. We believe bariatric surgery has a three-fold effect: 1) it decreases the release of GIP production, resulting in reduced nutrient absorption and decreased weight, 2) it reduces insulin release that reduces insulin sensitivity and cures diabetes, and 3) it changes
nutrient dynamics (i.e. the amount and transit time of food in the gut), which resets an altered microbiome and promotes normal energy harvest. Together, these influences dramatically reduce pathologic weight gain and restore a non-obese state. This research is directly in line with the PI’s (Coon’s) long term goals to i) understand how absorption of nutrients is regulated by GIP and ii) expand therapeutic options for obese individuals. The proposed experiments were designed to test predictions and provide foundational research results for future R01-level NIH funding.

Specific Aims

  1. Quantify GIP levels under controlled nutrient conditions in the jejunal mucosa following Roux-en-Y gastric bypass surgery (RYGBS). We hypothesize that RYGBS restores normal nutrient absorption in mice by reducing GIP levels in the jejunal mucosa. Preliminary results support GIP’s involvement in glucose absorption in obese individuals and implicated GLUT2 as the glucose receptor through which GIP is acting. To test the above hypothesis and the necessity of GLUT2, we will use blocking drugs to abrogate their activity. Next, we will determine whether RYGBS restores normal (low) GIP levels and reverses the level of GLUT2 on the apical surface of jejunal tissue, as seen in non-obese mice. These experiments will leverage in vitro Ussing chamber experiments on murine jejunal tissue to precisely quantify GIP production while controlling for important nutrients, such as glucose. Tissue from pre- and post-RYGBS will be for these comparisons.
  2. Identify changes in gut microbiome associated with RYGBS and reduced GIP levels. We hypothesize that RYGBS promotes the development of a non-obese microbiome by changing both the amount and transit time of nutrients in the gut. Unfortunately, we cannot directly test this hypothesis during this project, as funds for accurate nutrient analyses are beyond the scope of this funding source. However, we can address a supporting hypothesis that the microbiome changes from obese to normal after RYGBS and that this change is associated in time and space with decreased jejunal GIP production (Aim 1). To test this first hypothesis, we will conduct a temporal microbiome analysis in RYGBS mice following surgery. To increase the generalizability of our results to human RYGBS, we will consider mice that host a human microbiome (i.e. humanized mice). Finally, germ free control mice will be included to understand whether the hypothesized GIP-GLUT2 axis under investigation in Aim 1 is at all affected by the presence-absence of a human microbiome.

Expected Results

Aim 1 will add to our preliminary results, but will help to uncover whether the observed increased levels of glucose absorption was due to GLUT2 on the apical surface of the jejunum. We expect that blockage of this receptor will abrogate the effect observed in flux analysis. If the GIP-GLUT2 axis proves to be the underlying factor, we expect RYGBS will have a potent GIP reducing role and that this will likewise reduce GLUT2 in the recovered mice. A primary point of interest in this experiment will be quantification of GIP and production of GLUT2 after RYGBS treatment, as we believe this holds high potential as a therapeutic target. Finally, we will examine the changes that develop in the human microbiome after RYGBS in mice. We expect that this surgical intervention will have a profound influence on the human microbiome, as has been seen in human patients. If so, these results will validate our approach for investigation host-microbiome interaction during bariatric surgery. We do not expect to sort causative pathways in Aim 2. Rather, we will validate our approach and lay a solid foundation for future investigations that sort cause and effect. The two proposed Aims
were designed to fill gaps in our current understand of jejunal nutrient absorption, to lay the groundwork for future studies, and to establish collaboration between the PI and collaborators at Montana State University. Such a stepping stone will be invaluable for this research program.

Potential Outcomes

It is our intention to maximize student involvement in this project. With some instruction and training, students can perform many of the proposed molecular biology techniques and Ussing chamber experiments. Dr. Walk and his staff will hold workshops at Fort Peck Community College on microbiome data generation and analysis. Graduate students at Montana State University will be paired with students at Fort Peck Community College to facilitate a low instructor-to-student ratio training experience. In addition, Fort Peck students will also travel to Bozeman to take part in mouse procedures and learn more about experiments with laboratory animals. Finally, students will be involved with presentation of research results and the manuscript writing process.

Primary Contact

Steven Coon SCoon@fpcc.edu