Abstract

Finalized by B250

     The PPAR-γ gene belongs to a family of nuclear receptor isoforms and is part of the “overeating matrix” in dopamine pathways (Kenny, 2011). This food storage and lipid-metabolizing gene resides on chromosome 3 in humans and promotes the behavioral effect of overeating. By creating homolog primers, PCR determined if the PPAR-γ gene was present in the spotted hyena, Crocuta crocuta. In this, polymerase chain reaction and gel electrophoresis with semi-log plot analysis for band verification was utilized to determine if DNA was correctly amplified (Lee, 2012). It was hypothesized that the PPAR-γ gene will be present in the spotted hyena, because genomic alignment of this gene between humans, cats, and other carnivorous species validates that the amplification of this gene with designed primers, using UCSC Insilico to identify the most conserved regions from FASTA sequencing, will elicit bands at 443 bp in the conserved region of the F. catus genome (Tyner, 2016).

     The hyena DNA elicited a band at 446 bp, indicating the presence of the PPAR-γ gene in this homologous species. A single band at 763 bp verified the presence of the PPAR-γ gene in humans, residing in the acceptable range of 793 bp, as predicted from published primers (Ahn, 2007). The purpose of the psychological experiment was to analyze the psychological implications of overeating in humans, relating the Profile of Mood States (Keith et al., 1991). Results demonstrated statistical significance amongst vigor, depression and tension emotional intensities from ANOVA analysis. Implications of these findings illustrate PPAR-γ as a model for overeating behavior in hyena clan feeding, along with potential genetic mechanisms for lipid metabolism and brain-reward systems.

Discussion

Finalized by B245

PPAR-γ gene and PCR Experimental Purpose

     Overeating is a complex behavior which is influenced by the PPAR-γ gene. Peroxisome proliferator-activated receptors (PPARs) are ligand-regulation transcription factors that control gene expression and lipid metabolism by binding to response receptors in promoter regions of a gene (Wang, 2014). Gene expression elucidates the potential for PPAR-γ to serve as a regulator of overeating behavior via brain reward systems and discrete fatty acid ligand sensors (Kenny, 2011). The question addressed is whether the PPAR-γ gene can be amplified and identified through PCR and gel electrophoresis experimentation in the species, Crocuta crocuta, spotted hyena, through designed primers based off genome alignment cross-referenced between various homologous species. We hypothesized that the PPAR-γ gene will be present in the spotted hyena because hyenas compete for large quantities of food due to scarcity of food and unpredictable feedings times (Holekamp and Smale, 1998).

Original Predictions for PCR Amplification of Crocuta crocuta

     By using gene alignment sequencing from various homologous species, including humans, dogs, cats and other carnivorous species, we predict that the forward and reverse primers can be designed for the spotted hyena because the UCSC Insilico identifies the most conserved regions in the PPAR-γ gene amongst various homologous species from FASTA sequencing (Tyner et al., 2016). Two different designed primers can amplify the conserved region of the PPAR-γ gene between 553,559,797bp and 53,560,239 bp in the F. catus genome and elicit a band of 443 bp during PCR (She et al., 2009). We predict that PCR amplification of PPAR-γ in H. sapiens DNA will elicit a band of 793 bp because forward and reverse primers bind to the conserved target region of this gene (Ahn et al., 2007).

Presence of PPAR-γ gene in Homo sapiens and Crocuta crocuta

     A 763 bp segment of the PPAR-γ gene was successfully amplified from isolated H. sapien DNA using the published H. sapiens primers from Ahn et al. (2007) (Figure 7). A semi-log plot (figure 7) and positive and negative controls were used to verify the band produced in the gel. The positive control was a 395 bp amplified segment of Lambda virus DNA used to verify proper migration distance of bands in relation to the 1kb Plus Ladder (Lee et al., 2011). The negative control contained all reaction components of PCR except the DNA and was used to verify if there was contamination in the amplified PCR samples. The band produced from the positive control appeared at 395 bp, supporting proper migration of the bands through the gel. The negative control yielded no results, thus refuting the possibility of sample contamination. Successful amplification of the human PPAR-γ gene supports its presence in H. sapiens and verifies the results published by Ahn et al. (2007).

     The genome of the C. crocuta has not been mapped, which created a problem when trying to design primers to amplify the PPAR-γ. Using FASTA or query (high scoring pairs) sequences between homologous species, we found a 443 bp conserved region between five species inserted into the UCSC Insilico genome browser (Tyner et al., 2016). The conserved region was found within the PPAR-γ gene of H. sapiens, thus successful amplification supports the presence of PPAR-γ gene in C. crocuta. The designed primers for the C. crocuta were successfully amplified the conserved region, producing bands at 446 bp (Figure 3). A semi-log plot and positive and negative controls were used to verify the band produced via PCR (Figure 3). Again, the 395 bp sequence of Lambda virus DNA was used as the positive control, and all the reaction components of PCR without the DNA was used as a negative control. The band produced from the positive control appeared at 395 bp, supporting proper migration of the bands through the gel. The negative control yielded no results, thus refuting the possibility of sample contamination. The bands seen in (Figure 3) were successfully reproduced four times, supporting the presence of the PPAR-γ in both H. sapiens and C. crocuta. This signifies that the human and hyena genome contains the PPAR-γ gene linking to overeating behaviors. Specifically, the dopamine reward circuitry is a neurophysiological mechanism for overeating with relation to the PPAR-γ variant (Le Foll et al., 2014). C. crocuta have been observed devouring a 400-pound antelope in less than 13 minutes (Holekamp and Smale, 1998). This overeating behavior could be linked to the activation of the PPAR-γ gene, as hyenas must compete with one another due to scarcity of food and unpredictable feedings times (Holekamp and Smale, 1998).

Psychosocial Implications of Overeating Experiment

     Overeating falls under the category of eating disorders, according to the Diagnostic and Statistical Manual for Mental Disorder (Kupfer et al., 2013). This obsessive behavior resembles a drug addiction due to physiological and psychological dependence on foods high in fats and sugars (Le Foll et al., 2014). Overeating results from countless neurological pathways, namely the dopamine pathway, because, like alcohol, MDMA etc. food is rewarded and stimulates release of neurotransmitters, like serotonin and dopamine (Singh, 2014).

     A 28 day experiment studying the psychological effects of overeating was conducted, using the Profile of Mood States (POMS) questionnaire which is used to measure anger, confusion, depression, fatigue, tension, and vigor (Keith et al., 1991). We hypothesize that vigor POMS scores will increase, while tension, depression, and anger scores decrease because over consumption of food stimulates the release of dopamine and serotonin from the central nervous system (Singh, 2014). Vigor was categorized by adjectives like cheerful and energetic (Keith et al., 1991). The results of the POMS self-assessment questionnaire were analyzed using an ANOVA statistical test to determine the effects of overeating. Genetic correlates also link obesity and overeating, like PPAR-γ dysregulation, through dopamine reward systems (Le Foll et al., 2014).

     A statistically significant decrease tension (p=0.048) was recorded on Day 28 of the experiment (Figure 8A). Additionally, a significant decrease in depression (p=0.03) was seen between Days 14 and 28, however, the change was not significant between Days 1 and 28 (Figure 8D). Experimental subjects experienced extreme fluctuations in anger between Day 1 and 14, with average POMS scores ranging from 1 to 7.5 highlighting the mood alteration that overeating induced, however, the fluctuations were not statistically significant (Figure 8B). No other statistical significance was observed during the experiment. The significant decrease in tension on Day 28 suggests that overeating may be linked to the brain reward system. The intake of food releases dopamine and serotonin, rewarding the central nervous system, thus reducing tension. Further research must be conducted to determine the psychological effects of overeating. One experimental subject had to refrain from overeating after Day 14, due to health concerns, which decreased the sample size (n=3) for Day 28.

Future Directions

     Our study of the psychological effects of overeating was limited by sample size (n=4). We had two in our control group and two in our experimental group, which could have skewed our data. A larger sample size would have given us flexibility with diets, like high fat, high carb, low fat, etc. and been more accurately representative of the sample groups. Clinical studies have shown that lipid intake releases leptin from adipose tissue, which then activates PPAR-γ (Nolte et al. 1998). This activation of PPAR-γ turns on target gene transcription for adipogenesis and lipid and glucose metabolism (Nolte et al. 1998). A study of a high fat diet would reveal the psychological effects of the activation of PPAR-γ, which a larger sample size would allow.

     Figure 7 displays the successful amplification of the human PPAR-γ gene using PCR and gel electrophoresis. In addition to the bands seen in the gel, there also appears to be nonspecific binding, however, we tried on my occasions to rise the annealing temperature, yet this did not improve the nonspecific binding of the the primers. We believe that the DNA may have been highly fragmented which would have occurred during DNA isolation. If we were given more time, a different DNA isolation protocol would be tested to see if longer fragments of DNA could be isolated. If the nonspecific binding displayed in the gel is a result of fragmented DNA, it would not be present if longer fragments were isolated.

    Lastly, in all our trials, our C. crocuta bands were always faint. Increasing the DNA concentration (up to 7µl) to obtain brighter bands and maximize reactions per tube, which had an insignificant effect on the band's vibrance. The Holekamp lab provided purified blood cultures with a low concentration of DNA which would need to be increased to get brighter bands. If we had more time, different samples of hyena DNA would have been attained and run through PCR.

Figures

Finalized by B135

Figure 1: A) Results for PCR amplification of the PPAR-γ gene in hyena and human DNA and gel electrophoresis. B) Semi-log plot for hyena (C.c.) and human (H.s.) gel electrophoresis band verification. A) Designed hyena primers: (F: 5’-TTA TCT ATG ACA TGA ATC-3’) and (R: 5’-TCC ACT GAG AAT AAT GAC-3’) (Tyner, 2016). Human primers: (F: 5'-TGA TAT CGA CCA GCT GAA CC-3') and (R: 5'-GTC CTC TCA GCT GTT CGC CA-'3) (Ahn, 2007). PCR was used to amplify a target region of the PPAR-? gene. Hyena and human PCR cocktails included: 37.75µl of ddH2O with 1µl DNA, 36.75µl of ddH2O with 2µl DNA, 35.75µl of ddH2O with 3µl DNA, 7µl of 10X PCR buffer without MgCl2, 1µl of 10mM dNTPs, 1µl of each primer at 10µM, 5µl MgCl2, 0.25µl of GoTaq, 5µl 5X colorless GoTaq Flexi Buffer. The lambda positive control included: 37.75µl of ddH2O, 1µl of template DNA, 0.25µl of Taq, 1µl of each primer with remaining ingredients synonymous to human/hyena PCR cocktails. The cocktails were run in the thermocycler with initial denaturation set to 95°C for 3 minutes then 45 seconds for the other 30 cycles, annealing at 58-60°C gradient for human, 58°C for lambda and 40°C for the hyena DNA for 1 minute and elongation at 72°C for 1 minute with a final elongation for 5 minutes. A 1% agarose gel was made using 0.5 g agarose, 45 mL DI water, 5 mL 10X TBE Buffer and 5µl SYBR. 3µl of DNA/ladder, 1µl of 6X blue loading dye and 2µl of ddH2O with totalled 6µl of sample were pipetted into each well. Gel electrophoresis was run at 100V for 30 minutes and analyzed under UV light. Lane 1 had the 1Kb Plus ladder. Lanes 2-4 contained 1µl, 2µl and 3µl of female hyena DNA, respectively. Lanes 5-7 contained 1µl, 2µl and 3µl of male hyena DNA, respectively. Lanes 8-9 had 3µl of Qiagen isolated human DNA. Lane 10 had the positive control. B) Migration distance vs. molecular weight of 1Kb Plus ladder used to verify PCR products. The molecular size of the bp in the ladder were known. Migration distances were measured using pixel distance in a computerized preview of the gel. A line of best fit derived from the exponential function was used to analyze the migration distance of the amplified DNA. The migration distance of the spotted hyena, human and lambda positive control bands were plugged into the x-variable, producing a y-value representing the base pair of the amplified DNA in the PPAR-γ gene. Lane 10 had successful positive control amplification at 395 bp, to verify hyena/human bands. Lanes 8-9 with the Qiagen human DNA had the brightest bands, at 751 bp, which is close to the predicted 793 bp (Ahn, 2007). Lanes 2-7 of hyena DNA had dim bands at 446 bp, which can be remedied by increasing DNA concentration. The predicted band length for the hyena was 443 bp, and bands obtained from this experiment demonstrate that the PPAR-γ gene is potentially present in this species (Tyner, 2016).

Finalized by B255

Figure 2: Results of the Profile of Mood States (POMS) questionnaire for control and experimental subjects A) Tension B) Anger C) Confusion D) Depression E) Vigor F) Fatigue. On Days 1, 14 and 28 control (n=2) and experimental groups (n=2) took the Profile of Mood States (POMS) survey, which measured anger, confusion, depression, fatigue, tension, and vigor (Keith et al., 1991). The survey contained 65 adjectives and statements that are rated on a 5-point scale ranging from “not at all (1)” to “extremely (5)” (Keith et al., 1991). Week 1’s caloric intake for control subjects was averaged and set as basal caloric intake. The experimental subjects ate beyond their basic caloric intake by multiplying their averaged calories for week 1 by 1.4. This value was determined because 140% energy is required to maintain weight (Apolzan, 2013). Experimental subject 2 had to refrain from overeating after Day 14, due to health concerns. Therefore, the resulting POMS averages from Days 1 to 14 between both experimental subjects were most significant to note. The mood graphs obtained have a x-axis representing days of sampling and a y-axis of emotion intensity based on the POMS survey results (Keith et al., 1991). Error bars were created in Excel based on standard deviation values from average mood intensities (variation from mean) as well as ANOVA statistical analysis. A) Experimental and control groups had a steady tension throughout Days 1, 14 and 28 with non-overlapping error bars signifying statistical significance (p=0.047). B) Experimental group’s anger increased during the experiment, but due to a small sample size (n=2), error bars overlapped demonstrating no statistical significance (p=0.25). The negative error bar for the experimental group on Day 1 resulted from a small sample size and the negative mood scale ranging from -32 to 200 on the POMS survey (Keith et al., 1991). C) Overlapping error bars demonstrate no statistical significance (p=0.12), where confusion peaked at Day 1 then leveled off. D) Depression intensity remained level for both groups, and non-overlapping error bars led to statistical significance (p=0.026). E) Vigor was categorized from adjectives, such as “happy, cheerful, energetic, etc.” in POMS survey (Keith et al., 1991). Vigor expressed no statistical significance (p=0.057), despite large gaps between error bars. This may be caused due to a small sample size (n=4). F) Experimental group fatigue peaked at Day 14 and partial overlap between error bars led to no statistical significance (p=0.47).

Finalized by B255

Fig. 3. Our documentary, Into the Mind of Overeating. Data for the video was collected from March 23, 2017, to April 20, 2017, while production was completed near the end of April 2017. Tools used to make the documentary include Google Images and the iMovie program standard to Mac computers.