9-12 (High School)
Difficulty of the Problem
C. elegans were obtained from local university, other supplies $100+ (including dissecting microscope)
Aseptic technique was used throughout to prevent contamination with E. coli, which was used as a food source.
Time Taken to Complete the Project
7 months (50+ hours)
Excessive sugar consumption has become a major issue throughout the world and is contributing to the current obesity epidemic and other diseases. By investigating the effect of sugar-like substances on life span, the current obesity epidemic and its implications, including life span reduction, can be better understood.Therefore, this investigation used wild-type and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mutant Caenorhabditis elegans (C. elegans) as model organisms to determine the effect of sucrose and glucose on lifespan.
- Wild-type C. elegans (from university)
- GAPDH mutant C. elegans (from university)
- E. Coli strain OP50 (form university)
- worm picker- made from a platinum wire and glass Pasteur pipette
- dissecting microscope (from high school)
- sucrose (from university)
- glucose (from university)
- Petri plates
- bacteria lawn spreader
Caenorhabditis elegans (C. elegans) have been used to investigate various diseases and conditions relevant to humans. Although much smaller in size and simpler than humans, C. elegans are an excellent model organism because they are eukaryotic, which provides many similarities between C. elegans and more complex organisms. Additionally, C. elegans have a multistep developmental process because they are multicellular which allows adult organisms to be identified. The ease of maintenance and relatively short natural lifespan of C. elegans make them an ideal model organism (Revyakin, 2002). These conditions allow studies to be conducted using a large quantity of C. elegans to investigate aging.
In addition to their ease of maintenance and relatively short lifespan, C. elegans’ entire genome has been mapped. In total, C. elegans have a relatively small genome with a total of 9.7 x 107 base pairs. This smaller genome allows scientists to easily manipulate its genes. Approximately 35% of C. elegans’ genes have a human homolog, a DNA or protein sequence that is very similar to something found in humans and shows common evolutionary ancestry. One gene that has a human homolog is the gene that codes for glyceraldehyde-3-phosphate dehydrogenase (GAPDH), a protein involved in a variety of biochemical processes including glycolysis, membrane fusion, cytoskeleton formation, phosphotransfer activity, and pathways that connect the endoplasmic reticulum to the Golgi apparatus. The exact mechanisms of most of these functions are still largely unknown. One function of GAPDH that is extremely controversial is its role in regulating oxidative stress and redirecting cells’ carbohydrate flux from glycolysis to the pentose phosphate pathway and the effect of this oxidative stress on the lifespan of C. elegans. Grant (2008) as well as Ralser, Wamelink, Kowald, Gerisch, & Heeren (2007) suggested that GAPDH plays a vital role in regulating the oxidative stress and redirecting the cells’ carbohydrate metabolism pathway to the pentose phosphate pathway and without this regulation, lifespan would decrease. Schulz, Zarse, Voigt, Urban, & Birringe (2007), have stated that increased oxidative stress, such as that associated with glucose and caloric restriction, can actually extend the lifespan of C. elegans by inducing mitochondrial respiration. However, this extension can only occur when normal glucose metabolism is impaired. Additionally, it has not been broadly shown that caloric restriction increases oxidative stress as Qui, Brown, Hirschey, Verdin, & Chen (2010) showed that caloric restriction reduces oxidative stress. The role of GAPDH in aging and regulating oxidative stress is still uncertain and additional information that helps better explain GAPDH’s diverse roles could be utilized to create new treatments for existing conditions caused by increased sugar consumption and GAPDH malfunction.
Because of GAPDH’s various roles, its inhibition causes adverse effects in cells. Chuang, Hough, & Senatorov (2004) showed that GAPDH inhibition plays a role in neurodegenerative diseases, which decrease lifespan. GAPDH inhibition leads to an increase in oxidative stress because GAPDH is responsible for regulating oxidative stress. However, the effect of this increased oxidative stress on lifespan is uncertain. GAPDH inhibition also leads to improper membrane fusion and cytoskeleton formation, which can have adverse effects in embryo development.
A study by Wentzel, Ejdesjo, & Eriksson (2003) showed that rat embryos grown in diabetic rats or in high glucose environments had a 40-60% reduction in GAPDH activity, resulting from an excess of reactive oxygen species. Reduction in GAPDH activity resulted in a high incident of embryo abnormality. Thus, the presence of glucose can inhibit GAPDH function in cells without mutations affecting GAPDH.This inhibition occurs because of the excess reactive oxygen species that remain in the cell as a result of GAPDH inhibition. Additionally, diabetes can inhibit GAPDH activity.
Humans consume mass quantities of sugar and sugar-like substances. Increased sugar consumption has been associated with detrimental health conditions such as obesity, diabetes, and higher rates of disease. However, the effect of a high sugar diet on lifespan is uncertain. Lee, Murphy, & Kenyon (2009) showed that C. elegans grown in the presence of glucose have a shorter lifespan than counterparts grown on regular medium. The effects of other sugar-like substances, such as sucrose have not yet been investigated. Investigating the effect of sugar-like substances on the lifespan of C. elegans provides preliminary data that can be used to conduct future studies to investigate the specific biochemical processes related to lifespan regulation and GAPDH function.
In order to complete background research, I had to research C. elegans, GAPDH and its role in various essential biochemical processes, human sugar consumption, and oxidative stress.
Will excess sucrose and glucose decrease lifespan of wild-type and GAPDH mutant C. elegans?
- C. elegans were maintained according to standard procedure at 20°C (Sulston, & Hodgkin, 1988).
- C. elegans were obtained from a local university. GADPH mutant C. elegans were obtained from the C. elegans Knockout Consortium.
Lifespan establishment-no additives
- Wild-type and GAPDH mutant C. elegans were grown according to standard procedure without additives to establish lifespan.
- C. elegans’ developmental stages were synchronized by placing 2-3 adult wild-type C. elegans on a standard culture. GAPDH mutants were synchronized using the same procedure.
- After 2 hours, the adults were removed from the cultures. The eggs laid during this time were allowed to develop to maturity.
- After these original offspring reached maturity, ten adults were transferred to new cultures every 2-3 days to prevent contamination with progeny. C. elegans were kept at 10 per culture.
- The development of C. elegans was monitored daily and the number of live C. elegans was counted to establish a survivorship rate.
- This process was repeated to gain additional data. The life spans established from these trials were used as a comparison to the life spans of the C. elegans grown in the presence of sugar-like substance.
Lifespan establishment-sucrose and glucose additive
- To establish life spans for the experimental group of wild-type and GAPDH mutant C. elegans grown in the presence of sugar-like substances, 40mM glucose and sucrose, respectively, were added to two separate cultures.
- To ensure that the glucose or sucrose diffused throughout the cultures, the cultures were allowed to sit overnight.
- C. elegans were synchronized as described in steps 2-3 above, but in cultures with either glucose or sucrose additive.
- Upon reaching maturity, 20 adult offspring were transferred to a new culture. Twenty C. elegans were used in place of the ten used in the control group (no additives) to increase the validity of the study by gathering more data.
- To prevent contamination with future generations, these 20 adult C. elegans were transferred to new cultures every two days until they stopped producing progeny.
- The number of live C. elegans was counted daily until no live C. elegans remained.
Wild-type C. elegans grown without sugar-like additives had a mean lifespan of 19.1±1.8 days (with 0.01 confidence level). When grown in the presence of 40 mM glucose, the mean lifespan for wild-type C. elegans decreased to 14.8±0.6 days. In the presence of 40 mM sucrose, the mean lifespan decreased to14.6±0.4 days (see Figure 1, Appendix A).
GAPDH mutant C. elegans grown without sugar-like additives had a mean lifespan of 18.8±1.5 days (with 0.01 confidence level). Grown in the presence of 40 mM glucose, the mean lifespan of GAPDH mutant C. elegans decreased to14.6±0.4 days. When grown in the presence of 40 mM sucrose, mean lifespan decreased to 15.1±0.5 days (see Figure 2, Appendix A). The reductions in lifespan for wild-type and GAPDH mutant C. elegans grown on cultures of glucose or sucrose were statistically significant to the 0.01 confidence level (see Statistical Significance).
The resulting reduction in life spans was evaluated using the Mann-Whitney U test. Adding sucrose or glucose to the C. elegans’ medium had a statistically significant impact on reducing lifespan to at least the 99% level, meaning that there is less than a 1% chance that the decrease in lifespan happened by chance.Therefore, the null hypothesis was rejected and the alternative hypothesis was accepted.
The null hypothesis: a diet supplemented with sugar-like substances, glucose or sucrose, will not significantly decrease the life span of wild-type and GAPDH mutant C. elegans., is rejected while the alternative hypothesis: a diet supplemented with sugar-like substances, glucose or sucrose, will significantly decrease the lifespan of wild-type and GAPDH mutant C. elegans is accepted. This decrease in lifespan in the presence of glucose agreed with the findings of Schulz, Zarse, Voigt, Urban, & Birringe (2007) as well as Lee, Murphy, & Kenyon (2009).
While growth in glucose or sucrose decreased lifespan, GAPDH mutants did not have a significantly shorter lifespan than wild-type C. elegans when grown with glucose or sucrose. This suggests glucose and sucrose can possibly inhibit GAPDH function in normally functioning cells, as suggested by Wentzel, Ejdesjo, & Eriksson (2003). GAPDH plays vital roles in regulating oxidative stress, redirecting cells’ carbohydrate flux from glycolysis to alternative pathways, preventing neurodegenetive diseases, and regulating cytoskeleton formation and membrane fusion in embryo development (Chuang, Hough, & Senatorov, 2004). Thus, the inhibition of GAPDH, as possibly occurs when glucose or sucrose is consumed, can have adverse effects on cells’ function and can decrease an organism’s lifespan.
Increased oxidative stress as a result of GAPDH inhibition or glucose metabolism is a likely cause of lifespan reduction when C. elegans are grown on cultures with glucose or sucrose. The results of this experiment agree with the findings of Ralser, Wamelink, Kowald, Gerisch, & Heeren (2007) as well as Grant (2008) stated GAPDH’s role as a regulator of oxidative stress is vital for cell and organism survival. In wild-type C. elegans where GAPDH is inhibited by consumption of glucose or sucrose, oxidative stress increases because cells no longer have GAPDH to regulate this stress. In the presence of oxidative stress, GAPDH helps switch metabolism from glycolysis to alternative metabolic pathways such as the pentose phosphate pathway. However, when GAPDH is inhibited, as occurs in GAPDH mutants or possibly in wild-type C. elegans in the presence of glucose or sucrose, cells cannot switch to alternative metabolic pathways. These metabolic pathways have been hypothesized to more effectively deal with increased oxidative stress and prevent the cells from getting harmed. Without GAPDH to regulate this switch of metabolic pathways, the increased oxidative stress that occurs from the glucose and sucrose begins to adversely affect the cells, decreasing the organism’s lifespan.
Because increased caloric consumption can cause a decrease in lifespan (Schulz, Zarse, Voigt, Urban, & Birringe, 2007), the increased caloric consumption of C. elegans grown on cultures with glucose or sucrose could have contributed to the decrease in lifespan. These extra calories would have increased oxidative stress, especially since the metabolism of these glucose based substances resulted in by-products of glycolysis that cause oxidative stress. Thus, increased caloric consumption from glucose or sucrose would have also increased oxidative stress. This increased stress would have become a larger problem if GAPDH was inhibited from the glucose and sucrose.
Based on the results of this experiment, a diet high in sugar-like substances can also shorten human lifespan. Because there was little difference in lifespan reduction between C. elegans grown on cultures with glucose and C. elegans grown on cultures with sucrose, it is likely that the effect of these sugar-like substances on lifespan are comparable.
In summary, sucrose and glucose significantly reduced the lifespan of wild-type and GAPDH mutant C. elegans, possibly by inhibiting GAPDH and increasing oxidative stress. Understanding the effects of consuming a diet high in sugar-like substances will help combat the current obesity epidemic and increase human longevity.
The next step will be quantifying the possible GAPDH inhibition resulting from added sugar in the C. elegans’ diet. This could be done using either real-time polymerase chain reaction (PCR) to use gene expression to quantify the GAPDH inhibition or using zymography, which would utilize GAPDH’s substrate to quantify this possible inhibition. While this experiment indicated that glucose and sucrose cause a decrease in C. elegans’ lifespan, the data were solely quantitative and focused on the length of life. Because other factors besides length of life, such as quality of life and strength of bodily functions, are important when studying longevity, future
experiments should investigate both the quality and length of life. Future experiments could also investigate the effect of other sugar-like substances such as fructose, corn syrup, and artificial sweeteners on C. elegans’ lifespan and GAPDH inhibition. This experiment utilized wild-type C. elegans as well as GAPDH mutant C. elegans; future experiments could investigate the effect of sugar on the lifespan of C. elegans with other mutations. These results could help scientists better understand the effect of certain genes on aging and the metabolism of sugar-like substances. The effectiveness of antioxidants in preventing aging has been challenged in the controversy over the role of oxidative stress in aging. Thus, future experiments could use C. elegans grown in cultures with antioxidant additives to investigate antioxidants effect on aging.
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