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The Effects of PUGNAc on O-GlcNAC Levels and Dauer Formation in C. elegans

The Effects of PUGNAc on O-GlcNAC Levels and Dauer Formation in C. elegans. By Nicole Kiess. Need. Picture 1 : Shows the rate of Type 2 Diabetes in relationship to obesity over 10 years. http://www.ndep.nih.gov/diabetes/WTMD/images/figure2.gif.

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The Effects of PUGNAc on O-GlcNAC Levels and Dauer Formation in C. elegans

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  1. The Effects of PUGNAc on O-GlcNAC Levels and Dauer Formation in C. elegans By Nicole Kiess

  2. Need Picture 1: Shows the rate of Type 2 Diabetes in relationship to obesity over 10 years. http://www.ndep.nih.gov/diabetes/WTMD/images/figure2.gif

  3. Estimated prevalence of diagnosed and undiagnosed diabetes in people aged 20 years or older, by age group, United States, 2007 Need Picture 2: Shows percent of people in different age groups with Type 2 Diabetes. http://www.cdc.gov/diabetes/pubs/estimates07.htm

  4. Knowledge Base Picture 3: Is a model of what happends in the body of a Type 2 Diabetic http://savvyhealthfitness.com/get-healthy/diabetes/type-2-diabetes-lifestyle-diabetes

  5. Knowledge Base Picture 4a: Shows the pathway that regulate aging in C. Elegans. Picture4 b: Shows the model for the regulation of life span and sensory perception. http://www.nature.com/nature/journal/v408/n6809/fig_tab/408255a0_F3.html

  6. Knowledge Base Picture 5:C. Elegnas life cycle. http://www2.niddk.nih.gov/NR/rdonlyres/6B1E037B-3958-485C-8CFE-A448E43814DD/0/dauer2.jpg

  7. Literature Review • Paradis, et al,(1998) showed that mg144 does not activate AGE-1 P13K and that Akt/PBK is not dependent on AGE-1/P13K Picture 6a: Shows normal growth conditions Picture 6b: Shows dauer formation conditions

  8. Literature Review • Arias, et al, 2004 showed that PUGNAc increases O-GlcNAc levels in rat skeletal muscle Picture 7: Rate of 3-MG transport with or without PUGNAc. Rats were given different concentrations of insulin .

  9. Literature Review • Hanover, et al, 2005, findings show that OGT modulates macronutrient storage and dauer formation in C. Elegans Picture 8: Shows percentages of dauers in different strains

  10. Literature Review • Forsythe, et al, 2006 demonstrated that enzymes of O-GlcNAc cycling perfect insulin-like signaling in response to nutrient flux Picture 9: Dauer formation in different strains with temperature as a variable.

  11. Purpose • Therefore the purpose of this experiment is to observe the effects of PUGNAc on O-GlcNAc levels and dauer formation levels

  12. Hypothesis • Null- There will be no change in O-GlcNAc levels and no change in dauer formation • Alternative- The levels of O-GlcNAc will rise and dauer formation will rise

  13. Method The effects of PUGNAc on O-GlcNAC levels and dauer fomation in the insulin-like signaling pathway of C. elegans Control Wild Type N2 Ogt-1 (ok430) Oga-1 (ok1207) Daf-2 (e1370) 50um 100um 50um 100um 50um 100um 50um 100um • C. Elegans will be cultured at 16 degrees C • O-GlcNAC levels will be observed by treating C. elegans with RL1 and RL2 antibodies • Pictures will be taken with camera under immunofluorescent microscope and photoshop will measure percentages • Adults of each strain lay embryos overnight at 15 degrees C. Cultures shifted to 22.5 degrees C and are scored 3 days later as either dauer or non dauer Statistical Analysis will be run with SPSS and an ANOVA

  14. Protocol • C. eleganswill be cultured in petri dishes with the Nematode Growth Medium (NGM) and will be fed Escherichia coli (E. coli) Picture 10: How the C. elegans will be cultured Drawn by Victoria Wei

  15. Protocol • RL1 and RL2 dilated 1:400 and given to C. Elegans Picture 11: Life cycle of C. elegans http://www.sfu.ca/biology/faculty/hutter/hutterlab/research/pictures/Ce_life_cycle.gif

  16. Protocol • Fluorescent microscope and camera used for picture • Photoshop used for percentages Picture 12: A) Fluorescent picture to show O-GlcNAc levels with RL-2 B) Fluorescent picture to show O-GlcNAc levels with RL-2 Hanover, et al, 2005

  17. Do-Ability • Need to Purchase: • RL-1 RL-2 • C. elegans strains • E. coli • PUGNAc • Already Obtained • Fluorescent microscope • Camera • Photoshop • Petri Dishes

  18. Budget

  19. Bibliography • Biggs, Alton, Whitney Crispen Hagins, Chris Kapicka, Linda Lundgren, Peter Rillero, and Kathleen G. Tallman. Biology The Dinamics of Life. Columbus, Ohio: The McGraw-Hill Companies Inc., 2004 • Cauter, Eve V., Rachel Leproult, Esra Tasali, and Karine Spiegel. "Effects of poor and short sleep on glucose metabolism and obesity risk." Nature Reviews Endocrinology 5 (2009): 253-61. • Chang, Annette M., and Jeffrey B. Halter. "Aging and Insulin secretion." AJP-Endocrinol Metab 284 (2003). • Clevenger, Christopher M., Pamela Parker Jones, Hirofumi Tanaka, Douglas R. Seals, and Christopher A. DeSouza. "Decline in insulin action with age in endurance-trained humans." Journal of APplied Physiology 93 (2002): 2105-111. • "Introduction to C. Elegans." Genes, Genomes, and Human. 07 May 2009 <http://avery.rutgers.edu/WSSP/StudentScholars/project/introduction/worms.html>. • Kao, G., C. Nordenson, M. Still, A. Ronnlund, S. Tuck, and P. Naredi. "ASNA-1 positively regulates insulin secretion in C. elegans and mammalian cells." Cell 128 (2007): 577-87. • Kimura K.D., Tissenbaum H.A., Liu Y., Ruvkun G. (1997) daf-2, an insulin receptor-like gene that regulates longevity and diapause in Caenorhabditis elegans.Science277:942–946. • Kodama, Eiji, Atsushi Kuhara, Akiko Mohri-Shiomi, Koutarou D. Kimura, Masatoshi Okumura, Masahiro Tomioka, Yuichi Iino, and Ikue Mori. "Insulin-like signaling and the neural circuit for integrative behavior in C. Elgans." Genes and Development 20 (2006): 2955-960. • Manzella, Debra. "Obesity and Type 2 Diabetes - Why Being Overweight Can Damage Health." Diabetes - Diabetes Management - Signs of Diabetes - Symptoms of Diabetes. 04 May 2009 <http://diabetes.about.com/od/symptomsdiagnosis/a/obesity.htm>. • Muzumadar, Radhika, Xiaohui Ma, Gil Atzmon, Patricia Vuguin, Xiaoman Yang, and Nir Barzilai. "Decrease in Glucose-Stimulated Insulin Secretion With Agine Is Independent of Insulin Action." Diabetes 53 (2004): 441-46. • Norman, James. "Introduction to Insulin: insulin discovery and actions." Endocrine Diseases: thyroid, parathyroid adrenal and diabetes. 07 May 2009 <http://www.endocrineweb.com/diabetes/2insulin.html>. • Paradis, Suzanne, and Gary Ruvkun. "Caenorhabditis elegans Akt/PKB transduces insulin receptor-like signals from AGE-1 P13 kinase to the DAF-16 transcription factor." Genes and Development 12 (1998): 2488-498. • "Type 2 Diabetes: Conditions, Treatments, Resources - American Diabetes Association." American Diabetes Association Home Page. 04 May 2009 <http://www.diabetes.org/type-2-diabetes.jsp>. • Twyman, Richard. "Model organisms: The nematode worm." The Human Genome. 07 May 2009 <http://genome.wellcome.ac.uk/doc_WTD020809.html>. • Waksman Student Scholars. "Introduction to C. Elegans." Genes, Genomes, and Human Genetics. 12 May 2009 <http://avery.rutgers.edu/WSSP/StudentScholars/project/introduction/worms.html>.

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