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The Effect of Glucose and Sucrose as Dietary Additives on the Lifespan of Wild-type and GAPDH Mutant Caenorhabditis Elegans (page 2)

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Author: Megan S.

Terms and Concepts for Background Research

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.

Research Questions

Will excess sucrose and glucose decrease lifespan of wild-type and GAPDH mutant C. elegans?

Experimental Procedure

Standard Conditions
  1. C. elegans were maintained according to standard procedure at 20°C (Sulston, & Hodgkin, 1988).  
  2. C. elegans were obtained from a local university. GADPH mutant C. elegans were obtained from the C. elegans Knockout Consortium.

Lifespan establishment-no additives

  1. Wild-type and GAPDH mutant C. elegans were grown according to standard procedure without additives to establish lifespan.
  2. 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.
  3. After 2 hours, the adults were removed from the cultures. The eggs laid during this time were allowed to develop to maturity.
  4. 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.
  5. The development of C. elegans was monitored daily and the number of live C. elegans was counted to establish a survivorship rate.  
  6. 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

  1. 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.
  2. To ensure that the glucose or sucrose diffused throughout the cultures, the cultures were allowed to sit overnight.
  3. C. elegans were synchronized as described in steps 2-3 above, but in cultures with either glucose or sucrose additive.
  4. 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.
  5. To prevent contamination with future generations, these 20 adult C. elegans were transferred to new cultures every two days until they stopped producing progeny.
  6. The number of live C. elegans was counted daily until no live C. elegans remained.

Results

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.

Conclusions

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. 

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