A Metabolomics Driven Investigation of
Polyamine Metabolism During
Adipogenesis
Taylor Hughes
Bioresource Research Thesis Defense
Linus Pauling Institute
Stevens 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
Phenotype
Genotype
Patty, Yanes and Siuzdak (2012)
Nat. Rev. Mol. Cell Biol. 13, 263-9
30,000 proteins
3,000 to 8,000
metabolites in
mammalian cells
> 1,000,000 peptides
LC-MS/MS quantitation
still difficult
Reproduced from Stevens Antwerpen Nov 2013.ppt
LC-MS/MS quantitation
established
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
Intensity
PUTRESCINE
ornithine
400
300
200
100
0
CELL
EXPORT
putrescine
Treatment
Intensity
SPERMIDINE
N1-acetylspermidine
15000
10000
5000
0
spermidine
Treatment
Intensity
SPERMINE
acetylspermine
800
600
400
200
0
spermine
Treatment
Polyamine metabolism continued
ornithine
Change in Polyamine Metabolites
control vs. spermidine
ACR
CELL
EXPORT
putrescine
PAOx
**
putrescine
1.49
N1-acetylspermidine
-0.56
spermidine
spermidine
SSAT
PAOx
**p-value <0.01
acetylspermine
spermine
-1.13
spermine
-1.5
-1
-0.5
0
0.5
log(2) fold change
1
1.5
2
SSAT
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
120000
100000
Intensity
80000
60000
40000
20000
0
Control
Sp.
Treatment
http://commons.wikimedia.org/wiki/File:Glutathione-skeletal.png
GSH-ACR conjugation
ACR
+
GSH
GSTA4-4
Glutathione metabolism
Change in GSH pathway metabolites
Control vs. Spermidine
** P-Value < 0.01
Cysteine
-0.317
Glutamic acid
-0.029
**
GSH-HP
**
GSH
-1
0
1
5.86
1.42
2
3
log2 fold change
4
5
6
7
MS/MS for GSH-ACR metabolite
Proposed fragmentation of GSH-HP
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