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