Recipient Organization
VIRGINIA POLYTECHNIC INSTITUTE
(N/A)
BLACKSBURG,VA 24061
Performing Department
Animal Poultry Sciences
Non Technical Summary
Problem Statement: As the human population continues to grow, the consumption of animal meat products will increase. Current trends suggest that the consumption of animal products is trending to become much greater than the total products available1,2, leading to a shortage of food products. One method that farmers have focused on to meet this increased demand for animal products is by increasing animal weight. However, due to the interconnected nature of brain regions controlling appetite, hormone production, motivation and emotion, increasing animal weight can inadvertently lead to other production-related issues, including reproduction. For example, while the hypothalamus controls appetite, it is also highly involved in the reproduction process through control over hormone levels. However, the amygdala, which regulates emotional and motivational states, has been shown to regulate hormone release in the hypothalamus via direct synaptic connections. This suggests that any changes in the functioning of the amygdala could in term effect cellular processes within the hypothalamus, such as appetite, hormone regulation and reproduction. Thus, a better understanding of how weight gain changes biochemistry in relevant brain regions is critical in order to prevent these potentially negative changes in animal motivation, hormone production and reproductive behavior.Relevance to advancing VA/Region and the U.S.: Virginia is home to a thriving poultry industry, with an economic impact higher than $8 billion and more than 17,000 jobs3. Thus, understanding how selective breeding of chickens may be inadvertently changing their neurobiology could have significant impacts in terms of improving and/or maximizing chicken product production, resulting in potentially significant economic impacts. Additionally, the Appalachia region has one of the highest rates of obesity in the United States4. Understanding how obesity alters brain neurobiology can ultimately lead to the development of novel therapeutics that could treat the underlying pathophysiology of obesity, which could potentially result in reducing the obesity rate in Appalachia and the rest of the U.S. population. Approach: In this proposal, we will examine how animal weight gain alters activity of a major cellular pathway within the three main regions of the limbic system: the hypothalamus, amygdala, and hippocampus, which control appetite, motivation/emotion and memory, respectively. The cellular mechanism of interest is the ubiquitin-proteasome system, a highly conserved pathway present in all eukaryotes that controls protein degradation (turnover) in cells. In Phase 1, we will use a diet-induced model of obesity (weight gain) in combination with sophisticated molecular assays to assess changes in ubiquitin-proteasome functioning in the hypothalamus, hippocampus and amygdala of chickens and rats, the latter of which will be used as a positive control for our assays. We will compare these results with those obtained in Phase 2, in which we will examine ubiquitin-proteasome functioning in the same brain regions using a genetically-inherited model of obesity/weight gain in chickens. Collectively, these studies will determine how gaining weight within a single generation (Phase 1) or across generations (Phase 2) alters functioning of critical cellular mechanisms within brain regions controlling appetite, motivation, memory and reproductive behavior.Anticipated Outcomes and Impacts: Such information could have significant implications for agriculture where selective breeding based on weight is a common practice and reproductive behaviors in these animals is essential to maintain the optimal genetic line for meat production. Additionally, the information obtained in this study could lead to the development of targeted therapeutics which could reverse any adverse changes that occur in ubiquitin-proteasome functioning as a result of selective breeding for bodyweight. Furthermore, as this study focuses on cellular mechanisms of weight gain, the results obtained here will have important implications to human health and disease as well.
Animal Health Component
0%
Research Effort Categories
Basic
100%
Applied
0%
Developmental
0%
Goals / Objectives
Goals and ObjectivesThe overall goal of this project is to understand how the evolutionarily conserved ubiquitin-proteasome system is altered in animals following weight changes.Objective 1: We will examine how diet-induced weight gain is associated with changes in ubiquitin-proteasome expression, activity and function in various brain regions involved in weight control, feeding behavior and motivation.Objective 2: Using chicken lines selectively bred for high or low bodyweight, we will examine how a genetically-inherited predisposition for higher or lower body weight is related to changes in ubiquitin-proteasome expression, activity and function in various brain regions involved in weight control, feeding behavior and motivation
Project Methods
Objective 1, Rationale: Here, we will examine how diet-induced weight gain alters subcellular ubiquitin-proteasome expression, function and activity in the limbic system within a single generation. We will examine this in chicken lines allowed free access to food vs those on a controlled diet. As a positive control for our biochemical analyses, a rodent model will also be used here since 1) it is a well-established model of animal weight gain, and 2) our subcellular fractionation and ubiquitin-proteasome activity assays have already been optimized for this species. Additionally, the use of a rat model will allow us to determine the generalizability of our findings across species that are vastly different, which is important for understanding how ubiquitin-proteasome function is involved in weight gain in animals.Experimental Design: Forty Cobb-500 broiler chickens (half male, half female) will be divided into two groups, Controlled Diet (n = 10 per sex) and Ad Libitum Diet (n = 10 per sex). Forty rats (half male, half female) will be divided into two groups, Control Diet (n = 10 per sex) and High Fat Diet (n = 10 per sex). Food in the high fat diet is 60% fat while the control diet will be standard rat chow. These group sizes are based on prior work from our group using similar methodology and sample sizes. Animals will remain on the diet for 6 weeks, which is enough time to allow for significantly higher weight gain in the experimental groups. At the end of the diet, all animals will be euthanized, brain extracted and the hypothalamus, hippocampus and amygdala will be dissected out as described previously. All brain tissue will be split by hemispheres, which will be counterbalanced across extraction conditions. For each brain region, from one hemisphere a crude synaptic fraction will be collected; nuclear and cytoplasmic fractions will be collected from the other hemisphere. All fractions for the three collected brain regions will then undergo extensive biochemical analysis, including measuring:Proteasome activity using an in vitro proteasome activity assayProteasome number using western blottingOverall protein ubiquitination levels using western blottingDegradation-specific protein ubiquitination levels using GST-pull down assays and western blotting with antibody against K48 ubiquitinDegradation-independent protein ubiquitination levels using western blotting and linkage-specific antibodies against K63 and M1 ubiquitinSubcellular Fractionation: All collected tissue will undergo subcellular fractionation as described previously.Proteasome Activity Assay: Proteasome activity will be measured as described previously.GST-Pull Down Assays: To measure degradation-specific protein ubiquitination, protein samples will be mixed with a recombinant S5a protein fused to GST (GST-S5a); S5a is the subunit of the proteasome which recognizes and binds proteins that have the proper chain linkage and length for degradation. All captured proteins will be purified out of the sample with glutathione agarose, as described previously.?Western Blotting: Western blotting for linkage-specific protein ubiquitination will be performed as described previously. Proteasome number will be assessed using antibodies against 20S alpha and beta subunits and various 19S subunits.Statistical Analysis: Chicken and rat data and different subcellular fractions will be analyzed separately as no direct comparisons can be made across species or fractions. All data will be analyzed with independent samples t-tests using Graphpad Prism v8.0. An experiment wide threshold of p<0.05 will be used to establish statistical significance across all comparisons.Equipment: The equipment to perform such experiments exists within my lab in Litton-Reaves Hall.Expected Results: Considering evidence that ubiquitin-proteasome changes in response in weight changes in other parts of the body, we expect that animals fed a high fat diet will show decreased proteasome function in all subcellular compartments, which will occur simultaneously with a reciprocal increase in degradation-dependent and degradation-independent protein polyubiquitination levels. Proteasome expression is expected to remain constant. These results are expected in both chickens and rats and across subcellular compartments.Limitations and Alternative Plans: Our tissue collection procedure is thoroughly vetted and applicable across a broad range of species and tissue types. Thus, we do not expect any issues in tissue processing. However, we still may not observe changes in ubiquitin-proteasome expression and function across groups. While unexpected, such a result still provides important information since it would suggest that diet-induced weight gain does not alter this major cellular signaling mechanism in the brain of a given animal. This would have implications for understanding the long-term physiological consequences of selective feeding strategies in animals.Objective 2, Rationale: Virginia Tech has chicken lines that have been selectively bred for high and low bodyweight for over 60 generations. Not only are these chickens agriculturally relevant, but any brain molecular process that has changed as a result of the selective weight breeding has likely been inherited across generations. Thus, these chickens serve as an ideal model for understanding how selective breeding based on weight, a common practice in agriculture, has led to changes in ubiquitin-proteasome functioning in brain regions critical for appetite, motivation and reproduction. In combination with Objective 1, results from this experiment will identify how diet-induced vs genetically inherited bodyweight changes are associated with altered UPS function.Experimental Design: Sixty chickens (half male, half female) will be used in this experiment, randomly selected from one of three selective breeding groups: Control/intercrossed line, High Weight and Low Weight (n = 10 per group, per sex); these group sizes are based preliminary work from our group using similar methodology and breeding conditions. Immediately after hatch, chickens will be killed, brain extracted and the hypothalamus, hippocampus and amygdala dissected out; this will be done to prevent any unintended effects of diet on the genetically-inherited changes in the UPS. Hemispheres will be split, subcellular fractions collected and biochemical analyses performed as described in Objective 1.Subcellular Fractionation: See Objective 1.Proteasome Activity Assay: See Objective 1.GST-Pull Down Assays: See Objective 1.Western Blotting: See Objective 1.Statistical Analyses: All data will be analyzed with Analysis of Variance (ANOVA) with bonferonni post hoc tests using Graphpad Prism v8.0. An experiment wide threshold of p<0.05 will be used to establish statistical significance across all comparisons.Equipment: See Objective 1Expected Results: Considering evidence that ubiquitin-proteasome changes in response in weight changes in other parts of the body, we expect that chickens selectively bred for high or low body weight will have decreased proteasome activity in all three brain regions relative to controls. This will occur simultaneously with increased degradation-dependent and degradation-independent protein polyubiquitination levels. Proteasome expression is expected to remain constant. These results are expected across subcellular compartments.Limitations and Alternative Plans: Even with success of tissue collection, we may not observe changes in ubiquitin-proteasome expression and function across groups. While unexpected, such a result still provides important information since it would suggest that selective breeding based on weight does not alter this major cellular signaling mechanism in the brain of offspring. This would have implications for understanding the long-term physiological consequences of weight-based selective breeding strategies.