3rd Annual Workshop on Metabolomics

Integra(on  of  Biology  and  the  Metabolome:  Breaking  Barriers  Across  Disciplines

Monday, June 15, 2015

Adam R. Wende, Ph.D.

Assistant Professor Molecular and Cellular Pathology

Department of Pathology

From: Roger Unger - UTSW

Defining the Problem

2.5 million years 50 years

www.cdc.gov/diabetes/statistics and www.cdc.gov/mmwr

2010 – Obesity

2010 – Diabetes 2010 – Heart Disease

2010 – Physical Inactivity

≤19% 20%–23% 24%–27% 28%-30% ≥31%

Peterson and Gropler 2010 Circ Cardiovasc Imaging 3:211

Cardiac Metabolic Substrate Utilization

Ungar … Bing 1955 Am J Med 18(3):385

Cardiac Metabolic Substrate Utilization

UAB founded in 1969

ARW Ask for image credits

Fatty Acids

CAC

CPT1

CPT2

Acyl- Carnitine

FAO

Acetyl- CoA

ADP

ATP e- e- e-

I II III IV

Metabolic Substrate Utilization

H+ H+ H+

Glycogen

Glycogen synthase

Hexosamine biosynthetic pathway

UDP-GlcNAc

ACS Acyl-CoA

e-

PDPs

PDKs

Glucose O

P

Glucose O

P

Glycolysis

Pyruvate

GLUT1

PDH Acetyl-

CoA

GLUT4

MPC1/2

PFK

HK

Glucose O

O

TF TF

GLUT4

Defining the Mechanism

Barallobre-Barreiro … Mayr 2013 Rev Esp Cardiol 66:657

Model Development

DOX absent = OFF

MHC-rtTA rtTA α-MHC

TRE-GLUT4 mycGLUT4 TRE

DOX present = ON

MHC-rtTA rtTA α-MHC

TRE-GLUT4 mycGLUT4 TRE

GLUT4 Induction Increases Glycolysis and Rescues Diabetic Cardiac Glycolytic Defects

n = 6 – 10 § P < 0.01 vs. Con

Vehicle

STZ

Isolated Working Hearts

Glycolysis

0

300

600

900

1200

1500

1800

2100

Con mG4H

nmol

! m

in-1

! g

dhw

-1

0

300

600

900

1200

1500

1800

2100

Con mG4H

nmol

! m

in-1

! g

dhw

-1

§ §

GLUT4 Induction Increases GLOX but Accelerates Diabetic Cardiac GLOX Defects

n = 6 – 10 § P < 0.01 vs. Con

Vehicle

STZ

Isolated Working Hearts

Glucose Oxidation (GLOX)

0

50

100

150

200

250

300

350

Con mG4H

nmol

! m

in-1

! g

dhw

-1

0

50

100

150

200

250

300

350

Con mG4H

nmol

! m

in-1

! g

dhw

-1

*

‡

Oxidative Phosphorylation

www.genome.jp/kegg/pathway.html

GLUT4 Induction Accelerates Development of Mitochondrial Dysfunction

0

20

40

60

80

100

120

Con mG4H

Com

plex

I ac

tivity

(%

of C

on-V

eh)

0

20

40

60

80

100

120

Con mG4H C

ompl

ex II

I act

ivity

(%

of C

on-V

eh)

0

20

40

60

80

100

Con mG4H

Com

plex

II a

ctiv

ity

(% o

f Con

-Veh

)

0

20

40

60

80

100

Con mG4H

Com

plex

IV a

ctiv

ity

(% o

f Con

-Veh

)

* ** *

* *

n = 3 – 4 * P < 0.05

Oleh Khalimonchuk Wende … Abel in prep

Systems Biology of the Diabetic Heart

Adapted from Lewis and Abdel-Haleem 2013 Front Physiol 4:237

Genome  /Epigenome  

Phenome  

Proteome   Metabolome  

Transcriptome   Phenome Obesity, diabetes, heart failure, BHI, etc.

Transcriptome Northerns, qPCR, microarray RNA-seq, miR, lncRNA, etc.

Proteome

Mass spec, western blot, Co-IP, IHC, PTMs, etc.

Metabolome Glucometer, ELISA, GC-MS, HPLC, NMR, fluxomics, etc.

Genome / Epigenome Southerns, sequencing, GenBank, ENCODE, ChIP-seq, bsDNA-seq, etc.

Metabolism, Bringing the System Together

Hart … Lagerlof 2011 Annu Rev Biochem 80:825

Metabolite Modification of the Proteome

2D-PAGE

15%

SD

S-P

AG

E Con-STZ mG4H-Veh

pH 10 pH 3 IEF pH 10 pH 3 IEF Isolated Mitochondria

2D-PAGE Pro-Q

Emerald

mG4H-Veh

Metabolite Modification of the Transcriptome

181.9 MB

mG4H-Veh

pathway analysis of Microarray

Microarray 2-way

ANOVA

From Human to Mouse and Back Again…

Broad Institute Communications

Epigenetics - Memory EDIC: Epidemiology of Diabetes Interventions Trial

Pirola … El-Osta 2010 Nat Rev Endocrinol 6(12):665

Metabolite Signaling to Epigenetics

Gut and Verdin 2013 Nature 502:489

How does GlcNAc fit in?

Mariappa … Aalten 2013 EMBO J 32:612

Metabolite Modification of the Methylome

Abat

Con mG4H Veh STZ Veh STZ

Bdh1

Con mG4H Veh STZ Veh STZ

Legend: 0% 100%

RNA

DNA Abat

Con mG4H Veh STZ Veh STZ

Bdh1

Con mG4H Veh STZ Veh STZ

Legend: 0% 100%

RNA

DNA

Human/Mouse Comparisons

Irvin … Arnett 2014 Circulation 130:565

d  

Human/Mouse Comparisons

Wende, unpublished

Mouse Gene Expression

Con  Veh  

Con  STZ  

mG4H  Veh  

mG4H  STZ   GENE  

Mouse DNA Methylation

Metabolite Modification of the Metabolome

-20

-10

0

10

20

-20 -10 0 10 20

t[2]

t[1]

Class 1Class 2Class 3Class 4

Con-VehCon-Veh

Con-Veh

Con-Veh Con-Veh

Con-Veh

Con-STZ

Con-STZCon-STZ

Con-STZ

Con-STZCon-STZ

mG4H-VehmG4H-Veh

mG4H-VehmG4H-VehmG4H-Veh

mG4H-STZmG4H-STZmG4H-STZ

mG4H-STZmG4H-STZmG4H-STZ

SIMCA-P 11 - 1/31/2013 11:54:55 AM

Metabolite Modification of the Metabolome

U of U Cores James Cox – Metabolomics

Brett Milash – Genomics/Informatics Krishna Parsawar – Proteomics Hansjörg Schwertz – GU, Germany

Acknowledgements

E. Dale Abel – Utah to Iowa John C. Schell – MD/PhD student

Joseph Tuinei – Industry

Wende Lab Manoja Brahma – Postdoc

Mark C. McCrory – Manager Brenna G. Nye – Undergrad

Mark Pepin – MSTP Lamario J Williams – Undergrad

R00 HL111322 JDRF 51002608

Molecular & Cellular Pathology Redox Biology Diabetes Center Cardiovascular Center

Zymo Research Keith Booher – DNA methylation

Hunter Chung –Informatics

UAB Collaborators Steve Barnes – Metabolomics John C. Chatham – GlcNAc

David Crossman – Genomics/Informatics Farah D. Lubin – Epigenetics