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A Metabolomics Driven Investigation of Polyamine Metabolism During
Adipogenesis
Taylor HughesBioresource Research Thesis Defense
Linus Pauling InstituteStevens Lab
May 15, 2015
Obesity• Achieved at the point where fat accumulation begins to
exert deleterious effects on the individual• Causes by a chronic energy imbalance
• Caloric intake > expenditure• AHA: estimated 23.9 million children ages 2 to 19 as
being overweight or obese• 33% of boys and 30% of girls
• One of the leading causes of preventable death• Main cause of metabolic syndrome
Metabolic syndrome• Characterized by a cluster of risk factors
• Visceral obesity• Dislipidemia • Insulin resistance• Hypertension• Chronic inflammation
• The prevalence of these cardiometabolic disorders increases with the severity of obesity
Adipose tissue• Old thought- energy storing mass• New thought- endocrine organ
Source: http://resources.ama.uk.com/glowm_www/uploads/1223232469_Fat_Cell__v2.JPG
Adipogenesis The process of cell differentiation by which preadipocytes become mature adipocytes• Necessary for the formation of adipose tissue (energy storage)• Occurs throughout the lifetime of an organism• Adipose tissue is a dynamic endocrine organ involved in the regulation of
whole body energy homeostasis
Reproduced from nordphysicianguide.org
The endocannabinoid system•Lipid signaling system mediated by cannabinoid receptors CB1 & CB2•Facilitate different physiological effects•Endogenous cannabinoid receptor agonists = Endocannabinoids •Include various fatty acid amides (FAAs)•Endocannabinoid signaling terminated by FAAH
Cell stress, FAAH, and adipogenesis• Acute depletion of FAAs• Spermidine is proadipogenic• Effects on FAAs mediated through FAAH enzyme?
Hypotheses• We hypothesize that treatment with exogenous
spermidine induces an adaptive stress response in fat cells, producing ROS, facilitating adipogenesis
• If so, we should observe an increase in antioxidant defense and biomarkers of oxidative stress
• If endocannabinoid signaling creates ROS, using the FAAH inhibitor URB597 should inhibit its hydrolytic activity, sustaining more FAAs and ROS in the cell to facilitate adipogenesis
Metabolomics = Measurement of the products of biochemical pathways
Genotype Phenotype
> 1,000,000 peptides
30,000 proteins 3,000 to 8,000metabolites in
mammalian cells
LC-MS/MS quantitationestablished
LC-MS/MS quantitationstill difficult
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Nat Rev Mol Cell Biol. 2012 Mar 22;13(4):263-9.
Patty, Yanes and Siuzdak (2012) Nat. Rev. Mol. Cell Biol. 13, 263-9
Reproduced from Stevens Antwerpen Nov 2013.ppt
Metabolomics workflow
Siuzdak G, Patti G, Yanes O, Tautenhahn R (2010) ASMS Metabolomics Short Course
Effects of spermidine and URB597 treatment on the metabolome of differentiating 3T3-L1 cells
Cell culture and Mass Spec• 3T3-L1 cell line• Differentiated and treated• Samples ran on the Triple TOF 5600 Mass Spectrometer
Metabolomics data processing
Classifications• Glutathione Synthesis• Polyamines• Organic Acids• Fatty Acids• Amino acids & derivatives• Carbohydrates• Steroid Synthesis• Carnitine metabolism• Purine synthesis• Pyrimidine synthesis• Vitamins & cofactors
Polyamine metabolism
_x0007_C
ontrol
_x0004_D
MSO
_x0003_S
p.
_x000c_S
p. + URB597
_x0006_U
RB5970
600012000
SPERMIDINE
Treatment
Intensity
_x0007_C
ontrol
_x0004_D
MSO
_x0003_S
p.
_x000c_S
p. + URB597
_x0006_U
RB5970
200400
PUTRESCINE
Treatment
Intensity
_x0007_C
ontrol
_x0004_D
MSO
_x0003_S
p.
_x000c_S
p. + URB597
_x0006_U
RB5970
400800
SPERMINE
Treatment
Intensity
N1-acetylspermidine
spermidine
putrescine
ornithine
acetylspermine
spermine
CELLEXPORT
Polyamine metabolism continued
N1-acetylspermidine
spermidine
putrescine
ornithine
acetylspermine
spermine
CELLEXPORT
SSAT
PAOx
SSAT
PAOx
ACR
spermine
spermidine
putrescine
-1.5 -1 -0.5 0 0.5 1 1.5 2
-1.13
-0.56
1.49
Change in Polyamine Metabolitescontrol vs. spermidine
log(2) fold change
**p-value <0.01
**
Spermidine as a source of ACR
• Polyamine metabolism is a major source of ACR
• Electrophilic nature of ACR enables it to adduct to DNA and certain proteins
• Reported to play a role in development of certain cancers
• ACR can be conjugated by certain antioxidants, making them more water soluble and readily excreted in the urine
Stevens, J. F., & Maier, C. S. (2008). Acrolein: sources, metabolism, and biomolecular interactions relevant to human health and disease. Mol Nutr Food Res, 52(1), 7-25.
Glutathione• GSH is biosynthesized in the body• Defends cellular components from oxidative damage
caused by reactive oxygen species
http://commons.wikimedia.org/wiki/File:Glutathione-skeletal.png
Control Sp.0
20000
40000
60000
80000
100000
120000
Treatment
Inte
nsity
GSH-ACR conjugation
GSH
ACR
+
GSTA4-4
Glutathione metabolism
GSH
GSH-HP
Glutamic acid
Cysteine
-1 0 1 2 3 4 5 6 7
**
**
Change in GSH pathway metabolitesControl vs. Spermidine
log2 fold change
** P-Value < 0.01
1.42
5.86
-0.317
-0.029
MS/MS for GSH-ACR metabolite
Proposed fragmentation of GSH-HP
NH
HN
NH2
OS
O
O
HO
O
OH
HO
NH
NH2
NH2
OS
O
O
HO
O
OH
HO
Chemical Formula: C11H19N2O5S+Exact Mass: 291.1009
NH2
HN
NH2
O
S
O
O
HO
O
OH
HO
Chemical Formula: C5H8NO3+Exact Mass: 130.0499
H2NNH2
NH2
OS
O
O
HOO
OH
HO
Chemical Formula: C6H10O2S•+Exact Mass: 146.0396 NH2
HN
NH2
O
S
O
O
HO
O
OH
HO
Chemical Formula: C3H4NO3+Exact Mass: 102.0186
HN
S
O
O
HO
HO
Chemical Formula: C8H14NO4S+Exact Mass: 220.0638
HN
S
OO
O
Chemical Formula: C8H12NO3S+Exact Mass: 202.0532
-H2O
-H2O
Chemical Formula: C5H9OS2•+
Exact Mass: 117.0369
H+
McLafferty Rearrangement
Conclusion• Spermidine treatment results in increased breakdown of
spermine and spermidine, forming more putrescine• Creates byproducts ACR and H2O2
• Cellular stress events induce an adaptive stress response, upregulating GSH synthesis- supporting our hypothesis
• Mature adipocytes are capable of conjugating ACR with GSH
• GSH-HP is major metabolite of GSH-ACR conjugation• Detection supported by fragmentation
Acknowledgements
• J.F. Stevens, Ph. D.• Jaewoo Choi, Ph. D.• Val Miranda, Ph. D.• Jay Kirkwood, Ph. D. –Colorado State University • Wanda Crannell• Dr. Kate Field