Antioxidants & Obesity in C. elegans Manhasset Science Research Madelyn Sayed.
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Transcript of Antioxidants & Obesity in C. elegans Manhasset Science Research Madelyn Sayed.
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Antioxidants & Obesity in C. elegans
Manhasset Science Research
Madelyn Sayed
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Need: Obesity
(American Heart Association, 2004)http://www.americanheart.org/downloadable/heart/1103834132259FS16OVR5.pdf
Figure 1: Prevalence of Obesity in adults ages 20-74 by sex. (1960-2004)
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http://www.oxha.org/knowledge/backgrounders/overweight_and_obesity.gif/view
Need: Obesity (2)
Figure 2: Obese and overweight men and women in India, China, Mexico, & USA.
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Need: Diabetes Statistics
Figure 2.5: Diabetes in patients over 18 by race and sex. (Non-Hispanic Whites, Non-Hispanic Blacks, and Mexican Americans).
http://www.oxha.org/knowledge/backgrounders/overweight_and_obesity.gif/view
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Knowledge Base Hyperlipidemia Phytochemicals Antioxidant Free Radical Beta-carotene. Thiol Hypodermis
Figure 3: Layers of the skin in the human body are shown. The hypodermis is the lowest layer in this diagram.
http://www.daviddarling.info/images/hypodermis.jpg
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Knowledge Base (2)
Figure 4: The lifecycle of C. elegans in hours at a constant temperature of 25°C.
http://thalamus.wustl.edu/nonetlab/ResearchF/elegans.html
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Knowledge Base (3)
Figure 5: C. elegans at various stages in its lifecycle in hours at a constant temperature of 25°C.
http://thalamus.wustl.edu/nonetlab/ResearchF/elegans.html
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Seymour et al., 1997
Rats fed low and high fat diets with and without blueberries
Rats that received the blueberry-enriched powder ‘healthier’
Also true with grapes and cherries in other experiments
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Jones et al., 2009 crucial regulators of
lipid homeostasis in mammals serve similar functions in C. elegans.
fat metabolism, & feeding/food-related behaviors
intestinal and hypodermal cells in C. elegans (red)
Systems transport fats between cells and tissues (green).
Figure 6: C. elegans fat transport and storage areas.
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Jones et al., 2009 (2)
(A) Nile Red(B) Sudan Black B (C) LipidTOX neutral lipid staining(D) Nile Red and fatty acid lipid conjugated BODIPY staining(E) fatty acid lipid conjugated BODIPY staining(F) Both Nile red and BODIPY florescence
Figure 7: C. elegans fat deposits shown-stained with Nile Red, Sudan Black B, and other stains.
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Hildebrandt, 2004 Effect of thiol antioxidant on body fat and insulin
reactivity 2 studies: obese & hyperlipidemic/normolipidemic treated with antioxidants such as x-lipoic acid and NAC Their studies showed that the effect of NAC was
statistically significant, but the effect of creatine was not.
Figure 8
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Ashrafi et al., 2003 & 2007 (1)
Figure 9:
C. elegans stained with Nile Red in C, D, and E, with Sudan Black B in F and G. Fat appears fluorescent red (N.R.) and blue-black (S.B.)
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Ashrafi et al., 2003 & 2007 (2)
10
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Halliwell et al., 2009
Pine bark extract on metabolism and lifespan of C. elegans
Figure 11:
Antioxidant balance in body, affect on metabolism
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Purpose / Hypothesis
Purpose: to test the affect of antioxidants on fat deposition in C. elegans.
Null Hypothesis: the blueberry polyphenol compounds will not reduce fat deposition in C. elegans.
Alternate Hypothesis: the blueberry polyphenol compounds will reduce fat deposition.
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Methodology
Cultured axenically Grown under monoxenic conditions Pine Bark Extract E. coli strain OP50 Stain: Sudan Black B
http://individual.utoronto.ca/proy/worms.htm
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Flow ChartCaenorhabditis
elegansLifespan: (2-3 weeks) Rapid generation time Experimental flexibility
Control GroupSample Size N = ? Not fed Pine Bark Extract
Experimental GroupSample Size N = ?Fed Pine Bark Extract
Measurement:Sudan Black B fat staining
Motic Plus camera with StereomicroscopeAmount of fat (using Adobe Photoshop) deposited in the worm (cm3)
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Do Ability
Fluorescent Dissection microscope is available in the lab
Pycnogenol® easily obtainable C. elegans easily cultured Sudan Black B reliable fat stain Motic plus camera with stereomicroscope for
photography Adobe Photoshop available for fat measurement
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Protocols for Measurement
C. elegans will be fed E.coli OP50 Pycnogenol® 50 mg capsules Sudan Black: 0.7 gm Propylene Glycol: 100.0 ml
Figure: E. Coli bacteria
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Budget
Item Vendor Catalogue # Pg. Unit Price Qty Total Price
C. elegans N2 (wild type)
Carolina Science 2009
NP-17-3500 248 $10.95
(per culture)
1
NGM Carolina Science 2009
NP-17-3520 248 $6.25
(per 135 mL bottle)
2
Sudan Black III(Fatstain Herxheimer Lab Grade)
Carolina Science 2009
NP-89-2963 821 $6.65
(120 mL bottle)
2
Pycnogenol®
(Pine Bark Extract)
Vitacosthttp://www.vitacost.com/Schiff-Pycnogenol?csrc=PPCADWLT-pycnogenol&s_kwcid=TC|3001|pycnogenol||S|p|2989626851
Item #:
SCF 5127419or020525127419
$16.53
(30 capsules) (50 mg/ capsule)
1
$40.38
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Bibliography
"Antioxidant Definition." WebContent. 07 May 2009 <http://www.webcontent.com/articles/113/1/Antioxidant-Definition/Page1.html>.Ashrafi, Kaveh. "Genome-wide RNAi analysis of Caenorhabditis elegans fat regulatory genes." Nature (2003): 268-72. "Blueberries Health". US Highbush Blueberry Council. 07 May 2009 <http://www.blueberry.org/health.htm>.Childers, Norman F. "Blueberry." Encyclopedia Americana. 2009. Grolier Online. 4 May 2009 <http://ea.grolier.com/cgi-bin/article?assetid=0051620-00>.Clarkson, Priscilla M. "Antioxidants: what role do they play in physical activity and health?" American Journal of Clinical Nutrition 72 (2000). The American
Journal of Clinical Nutrition. 15 May 2009 <http://www.ajcn.org/cgi/content/full/72/2/637S>. Diabetes Information. American Diabetes Association. <http://www.diabetes.org/about-diabetes.jsp>.Eisenmann, D. M., Wnt signaling (June 25, 2005), WormBook, ed. The C. elegans Research Community, WormBook, doi/10.1895/wormbook.1.7.1,
http://www.wormbook.org.Halliwell, Barry. Free Radicals in Biology and Medicine. 3rd ed. New York: Oxford University, 1999.Hope, Ian A. C. elegans A Practical Approach. New York: Oxford UP, USA, 1999.Knutson, Mitchell D. "Resveratrol and novel potent activators of SIRT1: effects on aging and age-related diseases." Nutrition Reviews 66 (2008): 591-96.
Wiley InterScience.<http://www3.interscience.wiley.com/journal/121421590/abstract?CRETRY=1&SRETRY=0>."Maintenance of C. elegans." WormBook. 06 May 2009 <http://www.wormbook.org/chapters/www_strainmaintain/strainmaintain.html>McKay, R.M., McKay, J.P., Avery, L., and Graff, J.M. (2003). C. elegans: a model for exploring the genetics of fat storage. Dev. Cell 4, 131–142. Neto, Catherine C. "Cranberry and blueberry: Evidence for protective effects against cancer and vascular diseases." Molecular Nutrition & Food Research 51:
652-64. Wiley InerScience. <http://www3.interscience.wiley.com/journal/114274034/abstract?CRETRY=1&SRETRY=0.>"Obesity and Overweight: Defining Overweight and Obesity | DNPAO | CDC." Centers for Disease Control and Prevention. 13 May 2009
<http://www.cdc.gov/nccdphp/dnpa/obesity/defining.htm>.“Obesity”. Companies, Mcgraw-Hill. McGraw-Hill Multimedia Encyclopedia of Science and Technology: Version 2.0. New York: Mcgraw-hill Companies, 1997.
(278-280)"Obesity Statistics: Number of Obese Americans Increasing." American Sports Data - Sports Participation Marketing Research. 03 May 2009
<http://www.americansportsdata.com/obesitystats.asp>."Phytochemicals and Cardiovascular Disease." American Heart Association. 03 May 2009 <http://www.americanheart.org/presenter.jhtml?identifier=4722>.Riddle, Donald L. Introduction to C.elegans. 1997. <http://www.ncbi.nlm.nih.gov/b ooks/bv.fcgi? rid=ce2.chapter.19>Rizza, Robert A. "Diabetes Mellitus." Encyclopedia Americana. 2009. Grolier Online. 4 May 2009 <http://ea.grolier.com/cgi-bin/article?assetid=0126230-00>.The Obesity Society. 13 May 2009 <http://www.obesity.org/>.University of Michigan. "Blueberries May Help Reduce Belly Fat, Diabetes Risk." ScienceDaily 20 April 2009. 3 May 2009 <http://www.sciencedaily.com
/releases/2009/04/090419170112.htm>."Welcome to the Institute for Diabetes, Obesity and Metabolism." University of Pennsylvania || School of Medicine. 13 May 2009
<http://www.med.upenn.edu/idom/>.