150615 uab metabolomics...150615 uab metabolomics.pptx author: adam r wende created date: 6/15/2015...
TRANSCRIPT
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