Another Reason to Skip Dessert

Glucose Impairs the Hypertonic Stress Response of Daf-2-deficient C. elegans

David Viazmenski, New Hampshire Academy of Science, 2022

Cellular viability relies on the maintenance of normal salt concentrations (tonicity) in the cytoplasm. The loss of water and an increase in intracellular tonicity lead to protein misfolding, macromolecular crowding, and cellular death. Under hypertonic stress, cellular mechanisms are activated that prevent and repair damage. In Caenorhabditis elegans, one such mechanism is mediated by the protein DAF-16, an evolutionarily conserved homolog of the human FOXO. DAF-16 controls the expression of proteins that protect against hypertonic stress and other environmental challenges. DAF-16 expression is downregulated via the DAF-2 insulin signaling pathway. Loss-of-function daf-2(-) mutants possess a heightened ability to withstand hypertonic stress with an associated increase in DAF-16-mediated protective gene expression. Daf-2(-) mutants have also been shown to possess unique longevity, for which an intact copy of the daf-16 gene is required. In prior research, this life span was dramatically reduced by adding glucose to the worms’ diet, which was theorized to inhibit DAF-16 activity by activating the weakened DAF-2 insulin signaling pathway. As both the increased ability to withstand hypertonic stress and prolonged lifespan of daf-2(-) mutants appear to be DAF-16 dependent, we studied the effects of dietary glucose on the worms in hypertonic conditions. We compared the survival of wild type and daf-2(-) C. elegans on standard growth medium (51 mM NaCl) and hypertonic growth medium (400 mM NaCl) with and without added dietary glucose. We observed no statistically significant difference in survival between wild type worms on hypertonic plates with and without added glucose. A statistically significant decrease (p<0.05) in survival of daf-2(-) worms on hypertonic medium with added glucose was observed at 24 hours when compared to daf-2(-) on hypertonic plates without added glucose, supporting our hypothesis that both the life span prolongation and hypertonic stress response are mediated by the same insulin signaling pathway. Our study provides added insight into the hypertonic stress response in C. elegans and opens an additional avenue for investigation of its underlying molecular mechanisms.

My path to becoming an AJAS Fellow began in the woods of rural New Hampshire. As a student at Crossroads Academy in Lyme, NH, I often explored the nature around our campus with my classmates and teachers. From discoveries made in the waters of the brook behind my school as a second grader to conversations with middle school students investigating the effects of herbicides on the nematode C. elegans, I was fascinated by the way that science helps us understand our effect on the environment.

Once in middle school, I began spending one afternoon each week in the New Hampshire Academy of Science (NHAS) STEM lab learning basic laboratory techniques. After a year of this, I attended the summer research program in 2019 and created my own project studying the C. elegans. Throughout this process, I learned how to carry out an experiment from a hypothesis to the gathering and analysis of results, to putting together an oral presentation to explain to others the findings of my work.

In the summer of 2020, I again planned to attend the summer research program. However, due to the coronavirus pandemic, a change of plans was in order! Thankfully, the NHAS found creative ways to use their resources to allow us to work from home while still benefitting from their mentorship. I shifted my focus to a water quality project that allowed me to get outside, enjoy my local surroundings. This became my first project to be submitted to AJAS. Utilizing standard water quality testing measures, I wanted to find out if and how the water quality changes, from the source of a brook in my town to its output near the Connecticut River.

During the 2021 summer, the NHAS laboratory was reopened for student research and I returned to embark on a different project. I wanted to get back to using the C. elegans to look at the effects of environmental changes on small organisms. This interest connects to my previous year's work as I have always been curious about how changes in water, soil, or air might impact the surrounding biotic populations. I began to research what is known about how the nematode responds when its environmental conditions change. After preliminary research, I landed on an idea and got to work trying to understand how they are affected by hypertonic stress and what cellular pathways may be involved in their response.

I'm looking forward to all the events of this year's 2022 conference!

Slide 1: My work utilized a mutant strain of Caenorhabditis elegans to better understand research that showed their ability to withstand hypertonic stress, or the presence of a high salt concentration in their environment. In particular, I examined how adding D-glucose to the diet of daf-2-deficient C. elegans affects their HTSR (hypertonic stress response).

Slide 2: Why do we care about glucose and worms?  One reason is that studies on the C. elegans often have relevance to human health.  Using this model organism, we can try to sort out the mechanisms behind some of the greatest health challenges that humans face.

In recent years, much attention has been given to the growing problems of hypertension, heart disease, and diabetes, as shown on this illustration of the rising rates of diabetes around the world comparing the year 2000 with predicted numbers for 2030. These debilitating problems, sometimes tied to obesity, are on the rise in countries with growing prosperity. Often these countries have begun to adopt Western diets laden with simple carbohydrates, like sugar and white flour. These foods have a high glycemic index which means that when consumed they cause a large spike in insulin.

Research suggests that (Barbieri et al., 2003, Guarente, 2007, Katic and Kahn, 2005, Kenyon, 2005, Piper and Bartke, 2008, Stepanyan et al., 2006), the insulin-signaling pathway that deals with these spikes might be evolutionarily conserved from C. elegans to mammals. For this reason, studying the worms’ response to glucose can improve our understanding of the human response to insulin spikes.

Like the C. elegans and other organisms that are constantly confronted with changes in their environments, humans also experience salt stress due to excessive amounts of salt in our diet. I theorize that high sugar in our diet may further exacerbate the ill effects of salt on human health via increased insulin production that may inhibit our own cellular responses to hypertonic stress.

Slide 3: As I was reading about C. elegans to get ideas for my project, I came across a few studies that intrigued me. The first was a 1993 study by Cynthia Kenyon and colleagues who discovered that fertile, active, adult hermaphrodite mutant worms with DAF-2 deficiency, missing a protein called Daf-2, live twice as long as regular worms (as illustrated in the graph from their study). Their groundbreaking discovery also showed that this lifetime extension required the activity of a second gene, DAF-16. At this time, scientists had already figured out that these same genes also regulated the formation of a long-lived larval dauer worm under conditions of crowding and starvation, but what was new was that the lab of Dr. Kenyon discovered that Daf-2 gene activity could extends the life of a worm without forcing it into the dauer state.

Slide 4: DAF-2 in C. elegans is a receptor homolog of the mammalian insulin/IGF receptor family. On the diagram, you can see insulin/insulin like growth factor, binding to the insulin receptor Daf-2. This in turn downregulates the production of the protective proteins preventing DAF-16 entry into, and promoting removal of DAF-16 out of, the nucleus by phosphorylation. This insulin/IGF-1 signaling pathway that regulates DAF-16 activity is evolutionarily conserved between C. elegans and humans (In humans the homolog of DAF-16 is FOXO). It is a key component in a complex pathway regulating many events, including nematode development, longevity, stress response, and dauer formation.

Slide 5: Continuing my literature review, I started to look into more articles about daf-2 deficient worms and their special qualities. I came across a study by Lamitina and Strange from 2005 in which they discovered that daf-2 deficient worms have striking resistance to hypertonic stress compared to their wild type counterparts. They determined that this resistance is achieved via a DAF-16 dependent mechanism. From this research, it appeared that the same DAF-16/Daf-2 pathway underlies both the life span prolongation described by Kenyon’s lab and now, hypertonic stress resistance in daf-2 deficient worms. The dramatic results from Lamitina and Strange are shown in the graph.

Slide 6: As I was reading further, I came across a study by Lee and colleagues from 2009 that demonstrated the dramatic effect of dietary glucose on the life span of BOTH wild type and daf-2(-) mutant worms.

Slides 7 - 8: They discovered that in addition to shortening the lifespan of wild type worms (as shown in their graph), dietary glucose shortens the normally prolonged life span of daf-2(-) worms as shown in purple on the second graph.

Lee’s study showed that the administration of D-glucose negated the benefit of the daf-2(-) mutation that increases lifespan.

Slide 9: Reading these studies brought up a question - would dietary glucose decrease daf-2(-) worms’ ability to resist hypertonic stress due to the inhibition of the DAF-16, similar to what was theorized to shorten their life span in the Lee study?

Slide 10: My data confirmed that daf-2(-) worms do have increased resistance to hypertonic stress as witnessed by their increased survival over the wild type, just as was described by Lamitina et al. in 2005.

When I added glucose to the hypertonic agar, this addition took away the advantage that the daf-2(-) worms had over the wild type worms in their ability to withstand hypertonic stress, supporting my hypothesis.

The loss of this advantage may be due to daf-2 upregulation that decreases the production of DAF-16 proteins that were previously shown to confer hypertonic stress resistance. In other words, the benefit of stress resistance from daf-2(-) worms’ upregulation of DAF-16 was negated by the introduction of glucose.

Given these findings, we can hypothesize that low-sugar diets might have beneficial effects on mammalian aging. As Lee showed, dietary glucose suppresses the long life span of daf-2(-)insulin/IGF-1 receptor mutants in C. elegans, which leads us to the possibility that individuals with an impaired insulin receptor might benefit from a low-sugar diet.

References

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This work was supported by a grant to The New Hampshire Academy of Science (NHAS) from the National Institute of Health’s Science Education Partnership Award (SEPA 1R25GM132946-01A1). I am grateful for the guidance of the scientist mentors at the NHAS and to my sister, Miriam Viazmenski, for assistance with statistical analysis. As well, I thank the laboratory of Dr. Seung-Jae Lee of the Korea Advanced Institute of Science and Technology for help with the protocol for adding D-glucose to the growth medium.