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Antioxidants & Obesity in C. elegans
Manhasset Science Research
Madelyn Sayed
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)
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.
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
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
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
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
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
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.
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.
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
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.)
Ashrafi et al., 2003 & 2007 (2)
10
Halliwell et al., 2009
Pine bark extract on metabolism and lifespan of C. elegans
Figure 11:
Antioxidant balance in body, affect on metabolism
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.
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
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)
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
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
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
Bibliography
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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:
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(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/>.