Background
According to the definition of the WHO, obesity is defined as an excessive fat accumulation that causes detrimental health problems. Nowadays, experts and doctors use BMI as the measurement of obesity. People with body mass index (BMI) over 25 are considered overweight, while a BMI of 30+ is count as obese. According to the WHO, 1.9 billion people were overweight and 650 million obese in 2016. What we found even more alarming than the sheer magnitude of the numbers, was that the figures tripled in percentage, to 39% obese people worldwide, between 1975 to 2016 [1]. Locally, in Taiwan, 49% of adults suffered from being overweight or obesity in 2019, one of the connections to this is that our country is the second highest ranking in type 2 diabetes, a disease closely related to obesity [2].
Graph 1: Percentage of obese adults in the World [3]
Obesity isn’t exclusively about appearance, it is a complex disease that will induce the risk of other chronic diseases such as type 2 diabetes, heart diseases, and high blood pressure [4]. Obesity is also known to cause deterioration and diseases, including the top 10 causes of deaths, such as strokes. Therefore, solving obesity is a current priority of the world’s public health [5].
The issue of obesity has greatly affected our society. In Taiwan, eight of the 10 most common causes of death are linked to overweight and obesity [6]. Moreover, as obesity is correlated to other diseases, the annual medical expenses and social costs in Taiwan increased because of the growth of obese rate. A study in 2018 showed that, people classed as obese in Taiwan have an expenditure of between 39.5% and 93.3% more than adults having normal weight in NHI claims[7]. Obesity can be seen as a problem that is rising rapidly, hence our team’s decision to get involved. During our research, we identified factors causing obesity, including imbalance in food consumption and regular physical workout, environmental stress, malfunction of metabolism. Even though people assume food consumption is the main cause of obesity, which can easily be solved by having regular diets and workout schedule, environmental-stress and metabolism casued obesity are much more complicated. Instead of focusing on how to lose weight, we focused on a different perspective: metabolism.
Project Inspiration and Design
As we started researching metabolism, we found the gene AMPK, a master regulator of energy in the human body, which is known to be related to human metabolism. Upon induction by upstream pathways, it will boost metabolism, triggering fatty acid catabolism, while suppressing anabolism for more ATP [8]. When excess fat accumulates in non-adipose cell due to excess capacity of fat storage in adipose tissues, cellular stresses such as lipotoxcicity and reactive oxygen species harmful to DNA and protein structure are induced; furthermore, suppress AMPK activities and its downstream signals, ultimately resulting in abnormal lipid metabolism and ROS accumulation [9].
Local people, solving local problems, using synthetic biology
After determining our project focus, Obesity, we investigated a few past example projects from previous teams. The project done by TAS Taipei in 2021 helped to inspire our team’s focus on the solution due to the method they used to tackle their problem, which is blood shortages, on a local scale [10]. Our team really liked the idea of trying to help the community in which we all grew up to become a healthier place, hence we continued developing our project by finding current local supports and strategies of preventing obesity. This brought us to the findings of the two main natural components of local obesity-preventing-supplements in Taiwan: brown algae extract and green tea extract.
Brown algae:
Brown algae, or seaweed, is utilized as nutritious food in Asia. Brown algae contains many compounds, such as fucoxanthin. Fucoxanthin is a marine carotenoid discovered in macroalgae and microalgae, such as seaweed [11]. Due to a unique unusual structure in fucoxanthin, it contributes as an antioxidant, anti-inflammatory, antiobesity, and anticancer supplement. Several researchers have shown that fucoxanthin supplement intake can significantly reduce triglycerides and cholesterol, and mitigate the mis-regulation of several enzymes related to fatty acid synthesis in patients with obesity [12].
Green tea:
Green tea contains many compounds, such as caffeine, free amino acids, and catechins, a type of polyphenol, which have shown high biological activities to stimulate metabolism processes and reduce adipogenesis (reduce fatty acid accumulates in organs). Taiwan has around 20,000 hectares of tea plantarions, which produce around 20,000 tons of tea each year.[13]
Figure 2: Tea plantations in central Taiwan
These nutrient supplements all claim to boost metabolism to aid consumers to lose weight, however, it is not clear whether the supplements mitigate the stresses in the cell environment, since stresses can cause obesity and other chronic disorders. Thus, our research focus develops into further investigation to clarify the effectiveness of these natural extract supplements for stress-caused obesity in Taiwan.
The test
Next, our problem emerged: how can we combine our research into AMPK and the efficacy of obesity supplements into a project? Then we found further research about cancer drug development, where they used viability assays in cancer drug research.
As we continued with our research, as we found cancer drug research papers, we were inspired by their assays used for drug testing. In cancer drug efficacy research, researchers utilize similar methods of survival plate assay to test the target cell viability after interacting with specific cytolytic factors, which would demonstrate the drug’s cytolytic efficacy. For instance, in a cancer research paper investigating “Generation of an artificial human B cell line test system to evaluate the therapeutic efficacy of novel antigen-specific fusion proteins”, they also employed the cell viability assay to test the TTC (Transitional cell carcinoma)-based proteins against the hybridoma cell line and human lymphocytic TTC-reactive REH cells, which is an acute lymphocytic leukemia cell line [14].
After being inspired by the TAS project and cancer drug test method, we came up with our brilliant project: We aim to clarify the long-standing question of whether supplement pills can aid in solving the problem of obesity by mitigating stress, a significant factor that causes an imbalanced metabolism using a survival plate assay. This approach allows people to identify the efficiency of the supplements, providing a clear test for whether supplements aid in solving the root cause of obesity.
SNF1
After deciding on our initial design the next problem we encountered was that we are unable to use human cell lines in P1 laboratories in our school. Therefore, SNF1 became our main focus in the project. SNF1, which is sucrose non-fermenting 1, is a homology gene of AMPK in yeast. Therefore, by discovering SNF1, we can use it as an alternative to AMPK to solve the problem. To understand the characteristics and mechanisms of SNF1 genes, we overexpressed SNF1 genes through cloning processes in yeast to investigate its significance in boosting metabolic activity in the human body and efficiency in mediating stress, and further ensuring the supplements benefit the obesity patients.
Within our SNF1 gene cloning, we also included 2 sets of truncation in order to observe which parts of the gene play more of a significant role in its metabolic functions. There are two truncations: N truncate and C truncate. The N terminal is responsible for the catalytic activities of SNF1. Therefore, the N truncate removes its catalytic activity. C terminal is responsible for interaction with other proteins, so truncate removes its ability to interact with other proteins. Comparing the results of the two truncation would allow a reasonable indication of which part of genetic expression plays more of a crucial role.
Additionally, the experimental design altered characteristics of the SNF1 genes to amplify SNF1 gene expression. Originally in the plasmid, there was an SPT5 gene. The team designed the primers with the flank enzyme cut site to amplify SNF1 using PCR.
To manipulate SNF1 induction, the team will clone SNF1 gene into pGal1,10 promoter plasmid. The galactose will be used to induce SNF1 gene driven by pGal1,10 promoter. For the control, the pGal1,10-eGFP(green fluorescent protein) in BY4741 yeast strain is used since the SNF1 protein will not be able to express under the presence of galactose. For the dominant negative proteins, the snf1Δ2-306 and snf1Δ381-633, which interfere with the function of SNF1, are used to determine the domain that caused severe defective phenotypes.
The final product
The stress will then be added to the designed plasmid with the overexpressed SNF1 combined with the green tea and brown algae extracts to investigate if the two obesity products can mitigate stress respectively. Since glucose is the food source of yeast, the carbon deprivation(galactose) and 37-degree heat shock stress will act as the stress on the survival plate for the growth of yeast. Furthermore, we aimed to investigate if the overexpressed SNF1 gene combining with these two nutrients supplements respectively could show synergistic effects in mitigating stress on cell viability plates. With the additional steps of the stress test, we envisioned our project will improve and strengthen the knowledge of how supplements can help the mechanisms behind a cause of obesity, further contributing to the finding of solutions to control metabolism, and solving the root cause of obesity, and helping consumers and producers of supplements to know if the products are helpful.
Figure 3: The cell viability experiment is set up with 2 different stresses, such as carbon deprivation by adding galactose instead of glucose along with heat shock stress by putting the plates at 37 degree v.s 30 degree (those stresses in the environment can change).
We aimed to test the effectiveness of our product with the supplements from local products in Taiwan proven to have anti-obesity and metabolism-boosting properties such as green tea and brown algae, as discussed before, containing Catechins and Fucoxanthin respectively. With our design, possible synergistic effects between overexpression SNF1 and nutrient samples could mitigate the growth of defective phenotypes more significantly, allowing us to effectively test the possible mitigating properties of supplements.
