glucose and mitochondrial function

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Glucose and Mitochondrial Function

Wednesday, March 11, 2015

Adam R. Wende, Ph.D.

Assistant Professor Inaugural Pittman Scholar

Division of Molecular and Cellular Pathology

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Outline

•  Define the question and model to determine the connection between metabolism and diabetic heart disease.

•  Identify the molecular mechanisms by which glucose directly alters molecular function using systems biology. •  Transcriptomics •  Proteomics •  Metabolomics •  Methylomics and epigenetics

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2.5 million years 50 years

Obesity, Metabolic Syndrome, Diabetes, and Heart Failure

From: Roger Unger - UTSW

ARW 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% 20%-24% 25%–29% !30%

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Maintaining Cardiac Function Through Metabolic Substrate Balance

Glucose Fatty Acids

giphy.com

ARW Ungar … Bing 1955 Am J Med 18(3):385

UAB founded in 1969

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Metabolic Substrate Utilization in the Heart

Peterson and Gropler 2010 Circ Cardiovasc Imaging 3:211

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Point/Counterpoint - The Right Balance?

Taegtmeyer and Stanley 2011 J Mol Cell Cardiol 50(1):2

Heinrich Taegtmeyer, MD, DPhil

William C. Stanley, PhD

1957 - 2013

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Diabetes and Metabolomics

Metabolomics is an integral part for understanding disease processes ! information garnered in the biomarker investigations, future research should shed more light on disease pathogenesis and explore new treatment options.

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Heart failure and substrate switching

The hypertrophy, oxidative stress, and metabolic changes that occur within the heart when glucose supplants FA as a major energy source suggest that substrate switching to glucose is not entirely benign.

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Mitochondria – a Dynamic Network

Fan ! Brooks 2010 Free Radic Biol Med 49(11):1646

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

Fatty Acids

Acyl-CoA

e-

PDPs

PDKs

Glucose O

P

Glucose O

P

Glycolysis

Pyruvate

GLUT1

PDH

GLUT4

MPC1/2

PFK

HK

Glucose O

O

TF TF

GLUT4

ATP

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Facilitative Glucose Transporters: GLUTs “Solute Carrier Family, SLC2A”

Scheepers ! Schurmann 2004 J Parenter Enteral Nutr 28:364

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Changes in Human Heart GLUT Levels

Armoni ! Karnieli 2005 J Biol Chem 280(41):34786

Biopsies obtained during coronary bypass surgery HL = hyperlipidemia DM2 = diabetes mellitus type 2

Razeghi ! Taegtmeyer 2002 Cardiology 280(41):34786

RNA Human heart failure

Protein Human heart diabetes

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Glucose Utilization and Rodent Models of Type 1 Diabetes

Panagia … Clarke 2005 Am J Physiol 288:H2677

Protein Diabetic Mouse Heart

GLUT4

Glucose Uptake Diabetic Mouse Heart

Glycolysis GLOX

Belke ! Severson 2000 Am J Physiol 279:E1104

Constitutive GLUT4 Expression Prevents Development of Glucose Utilization Defects

Question: Is the change in cardiac metabolic substrate flexibility

adaptive or maladaptive?

DOX absent = OFF

MHC-rtTA rtTA !-MHC

TRE-GLUT4 mycGLUT4 TRE

DOX present = ON

MHC-rtTA rtTA !-MHC


Inducible Cardiomyocyte-Specific GLUT4 Expression (mG4H)

TRE-GLUT4 mycGLUT4

2 4 8

myc

Glut4

DOX (d)

Con

2 4 8

mG4H

0 14 0 14

Con mG4H

14

Hrt

GC

Va

s TA

S

ol

2 4 8

myc

Glut4

DOX (d)

Con

2 4 8

mG4H

0 14 0 14

Con mG4H

14

Hrt

GC

Va

s TA

S

ol

Hrt = Heart GC = Gastrocnemius Vas = Vastus lateralis

TA = Tibialis anterior Sol = Soleus

2 4 8

myc

Glut4

DOX (d)

Con

2 4 8

mG4H

0 14 0 14

Con mG4H

14

Hrt

GC

Va

s TA

S

ol

mG4H Mice Exhibit Inducible Cardiac-Specific Expression of GLUT4

Insulin-induced GLUT4 Vesicle Fusion and Exofacial Myc-Epitope Exposure

Ariel Contreras-Ferrat

Cardiac Myocytes

2-DG Uptake

Renata O. Pereira

GLUT4 Induction Increases Basal and Insulin-Stimulated Glucose Uptake

Basal 0.1 nM Ins

n = 3 – 4 a P < 0.01 vs. Con-Basal b P < 0.001 vs. All

0

50

100

150

200

250

Con mG4H

pmol

! m

g-1 !

min

-1

b a

a a

Transgene Induction

Streptozotocin (STZ)-Induced Hyperglycemia is Not Altered by Transgene Induction

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0

300

600

900

1200

1500

1800

2100

Con mG4H

nmol

! m

in-1

! g

dhw

-1

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

§ §

Joseph Tuinei Wende ! Abel in prep

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0

50

100

150

200

250

300

350

Con mG4H

nmol

! m

in-1

! g

dhw

-1

GLUT4 Induction Increases GLOX but Accelerates Diabetic Cardiac GLOX Defects

n = 6 – 10 ‡ P < 0.001 vs. All * P < 0.01 vs. Veh

Vehicle

STZ


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

*

‡

Joseph Tuinei Wende ! Abel in prep

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GLUT4 Induction Prevents Increased Cardiac POX in Diabetes

Vehicle

STZ


Palmitate Oxidation

(POX)

Joseph Tuinei n = 5 – 13 ‡ P < 0.001 vs. All

0

100

200

300

400

500

600

Con mG4H

µm

ol !

min

-1 !

gdh

w-1

0

100

200

300

400

500

600

Con mG4H

µm

ol !

min

-1 !

gdh

w-1

0

100

200

300

400

500

600

Con mG4H

µm

ol !

min

-1 !

gdh

w-1

‡

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Oxidative Phosphorylation

www.genome.jp/kegg/pathway.html

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GLUT4 Induction Accelerates Development of Mitochondrial Dysfunction

n = 3 – 4 * P < 0.05

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

)

* ** *

* *

Oleh Khalimonchuk Wende ! Abel in prep

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In the context of diabetes, enhancing glucose delivery by

expression of GLUT4 accelerates the progression of

mitochondrial dysfunction.

Conclusion – Part 1

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Diabetic Cardiomyopathy “Death by a Thousand Cuts!”

ER stress

Adapted from Wende, Symons, and Abel 2012 Curr Hypertens Rep 14(6):517

Inflammation

Mitochondrial dysfunction

REDOX Imbalance

Lipotoxicity

Glucotoxicity

Insulin resistance

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

Systems Biology !"#$%&"'()*+,- .,$*+,-/

0('1,$*+,-23,$*+,-

2"*),*+,- 4,)#5*6*+,-

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.

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Transcriptomic Analysis Using the Agilent SurePrint G3 60K Microarray

181.9 MB

mG4H-Veh

Microarray and Bioinformatic cores – Brian Dalley and Brett Milash Wende ! Abel in prep

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Pathway Analysis of Microarray

Wende ! Abel in prep

-log(p-value)

Metabolic disease

Amino acid metabolism

Lipid metabolism

Nucleic acid metabolism

Carbohydrate metabolism

Skeletal and muscular disorders

Energy production

Cardiovascular system development & function

Post-translational modification

Endocrine system development & function

Inflammatory response

Endocrine system disorders

Cell death

Cardiovascular disease

Gene expression

Nutritional disease

Protein degradation

Threshold 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

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Glucose Regulated Gene Expression

Wende, unpublished

0 = up-regulated 0 = contra-regulated 0 = down-regulated

Mouse STZ

Mouse mG4H

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Species Conservation of Gene Expression Changes in Diabetes

Drakos ! Wende, unpublished

Human T1D

Mouse T1D

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Species Conservation of Gene Expression Changes in Diabetes

Drakos ! Wende, unpublished

0.5 1.0 1.5 2.0 0.0 2.5

-log (P-value)

3.0 3.5 4.0

Threshold

Mitochondrial dysfunction

Calcium signaling

3-phosphoinositide degradation

Oxidative phosphorylation

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Oxidative Phosphorylation

GeneSifter using KEGG

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Ndufa9 Gene Promoter Structure

KEY TSS = Transcription start site

= CpG island

= Sp1 RE

http://ecrbrowser.dcode.org

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-2 kb +1 kb

Luc

-0.5 kb

-0.3 kb

TSS -0.5 kb TSS

Ndufa9

Ndufa9 Gene Promoter Mapping

C2C12 Myotubes n = 9 * P < 0.05

5.5 mM 25 mM

Glucose

0

20

40

60

80

100

-2 kb -0.5 kb -0.3 kb

Nor

mal

ized

RLU

* * *


Transient Transfection

Promoter Activity

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0

20

40

60

80

100

-2 kb WT -2 kb M3

Nor

mal

ized

RLU


C2C12 Myotubes n = 9 * P < 0.05

5.5 mM 25 mM

Glucose

*

-2 kb +1 kb

Luc

TSS

Ndufa9 Sp1-M3



Promoter Activity

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0

20

40

60

80

100

-2 kb + (-) -2 kb + Sp1

Nor

mal

ized

RLU


C2C12 Myotubes n = 9 * P < 0.05

5.5 mM 25 mM

Glucose

*

*

*

*

-2 kb +1 kb

Luc

TSS

Ndufa9

TSS Sp1



Promoter Activity

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O-GlcNAcylation O-GlcNAcylation

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Metabolic Integration: Protein O-GlcNAcylation

Hart ! Lagerlof 2011 Annu Rev Biochem 80:825

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O-GlcNAc Cycling

Hanover ! Love 2012 Nat Rev Mol Cell Biol 13(5):312

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GlcNAc Regulation of Sp1

Vosseller ! Hart 2002 Curr Opin Chem Biol 6(6):851

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GlcNAcylation Regulates Ndufa9 Gene Expression

0

20

40

60

80

100

120

None GFP OGA

Nor

mal

ized

RLU

C2C12 Myotubes n = 3 * P < 0.05

5.5 mM 25 mM

Glucose

Li Wang Wende ! Abel in prep


Promoter Activity

0

20

40

60

80

100

120

None GFP

Nor

mal

ized

RLU

0

20

40

60

80

100

120

None GFP OGA

Nor

mal

ized

RLU

*

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Enhanced glucose delivery regulates oxidative capacity

via transcriptional mechanisms including GlcNAcylation of

transcription factors.


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Mitochondrial Protein O-GlcNAcylation and Neonatal Cardiomyocyte Metabolic Function

Hu ! Dillmann 2009 J Biol Chem 284(1):547

Mitochondrial Protein O-GlcNAcylation

O-GlcNAcylation of NDUFA9

5.5 mM Glc 30 mM Glc + Adv (-)

30 mM Glc + Adv-OGA

Complex I Activity

5.5 mM Glc 30 mM Glc 30 mM Glc + Adv (-)

30 mM Glc + Adv-GCA

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NDUFA9 – Complex I

Chase A. Andrizzi

Western Blot mG4H Con

STZ Veh STZ Veh

NDUFA9

VDAC

0.0

0.2

0.4

0.6

0.8

1.0

Con mG4H

CI -

ND

UFA

9/VD

AC

(N

orm

aliz

ed a

.u.) *

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

Con mG4H

Com

plex

II a

ctiv

ity

(% o

f Con

-Veh

)

* ** *

* *

OXPHOS Activity

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Western Blot

IB: O

-Glc

NA

c

Veh STZ

Con

mG

4H

Con

mG

4H

Mitochondrial Protein O-GlcNAcylation

GLUT4 Induction Alters Mitochondrial Protein O-GlcNAcylation

IP Ndufa9

Con mG4

IB: O-GlcNAc Ndufa9

Veh

STZ

Veh

STZ

NDUFA9 Immunoprecipitation

NDUFA9


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2D-PAGE

15%

SD

S-P

AG

E Con-STZ mG4H-Veh

pH 10 pH 3 IEF pH 10 pH 3 IEF

GLUT4 Induction Alters the Cardiac Mitochondrial Glycoproteome

Isolated Mitochondria

2D-PAGE Pro-Q

Emerald

Hansjörg Schwertz Wende, unpublished

2D-PAGE

15%

SD

S-P

AG

E Con-STZ mG4H-Veh

pH 10 pH 3 IEF pH 10 pH 3 IEF

Con-Veh Con-STZ

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dbOGAP http://cbsb.lombardi.georgetown.edu and YinOYang www.cbs.dtu.dk

Wende, unpublished

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GLUT4 Induction Alters the Cardiac Mitochondrial Glycoproteome Cardiac Mitochondrial Glycoproteome

Con mG4H Veh STZ Veh STZ

UQCRFS1

DLAT

ATP5A1

SUCLA2

ATP5O

MDH2

Control-VDAC

Chase Andrizzi

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Lamario Williams, Manoja Brahma, and Chase Andrizzi

25 kDa

50 kDa

37 kDa Actin

42 kDa

UQCRFS1 25 kDa

IB: UQCRFS1

IB: Actin

Inpu

t

Unb

ound

Elut

e 1

(200

uL)

Elut

e 2

(200

uL)

Elut

e 1&

2 (1

:1)

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Metabolomics

-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

Wende, unpublished

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Metabolomics

Wende, unpublished

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Manoja Brahma

'()*+(&,-+).(/0/&

BHB

Acetoacetate

BDH1/Bdh1

AcAc-CoA

SCOT/Oxct1

m-Thiolase/Acaa2

Acetyl-CoA

$12&3-34(&

'()*+(&560789*+&RNA - Microarray -1 1 -1 1

AcAc-CoA

Acetyl-CoA

HMG-CoA

Acetoacetate

BHB

HMGCS2/Hmgcs2

Fatty acids

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GLUT4 Induction Alters the Cardiac Mitochondrial Glycoproteome Cardiac Mitochondrial Glycoproteome


UQCRFS1

DLAT

ATP5A1

SUCLA2

ATP5O

MDH2

Control-VDAC

SCOT1

Control-GAPDH

Lamario Williams, Manoja Brahma, and Chase Andrizzi

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GLUT4 Induction Alters Mitochondrial Protein O-GlcNAcylation

:;<&5=!43>23&

,15$&0??@+*AB(30A0)89*+&CB*?&"D&*C&E%.&C8/)(7&?03(&

FG&:+

A@)&

#+H

*@+7

&

:I&

1*+)

B*4&

:;<&,15$&

:I<&,15$&

:;<&!43>23=,15$&

Manoja Brahma

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Enhanced cardiac glucose delivery alters metabolic flux through other pathways and regulates the mitochondrial

proteome via O-GlcNAcylation.


ARW Broad Institute Communications

From Human to Mouse and Back Again

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Epigenetics - Programming DCCT: Diabetes Control and Complications Trial

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Epigenetics - Memory EDIC: Epidemiology of Diabetes Interventions Trial

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

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Epigenetics: Transgenerational and Drift

Gut and Verdin 2013 Nature 502:489

ARW Fischer 2014 EMBO J 33(9):945:489

Epigenetic Code

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Metabolite Signaling to Chromatin

Gut and Verdin 2013 Nature 502:489

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How does GlcNAc fit in?

Gut and Verdin 2013 Nature 502(7472):489

ARW Gräff and Tsai 2013 Nat Rev Neurosci 14(2):97

Chromatin Regulation

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Arnaudo ! Garcia 2013 Epigenetics Chromatin 6(1):24

ARW ucsf.edu

DNA Methylation 101

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Exercise Alters DNA Methylation of Key Metabolic Genes

Low = 40% VO2peak High = 80% VO2peak

Subjects fasted overnight and then consumed a high carbohydrate diet 4 hr prior to exercise.

Barres and Zierath 2012 Cell Metab 15:405

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Diabetes Regulated Cardiac DNA Methylation

Pdk4

= C, = 5mC CpG :

Ndufa9

Con STZ

-552 bp -301 bp

Con STZ *

| |

-523 bp -259 bp | |

Wende, unpublished

Pdk4

= C, = 5mC CpG :

Ndufa9

Con STZ

-552 bp -301 bp

Con STZ *

| |

-523 bp -259 bp | |

Pdk4

= C, = 5mC CpG :

Ndufa9

Con STZ

-552 bp -301 bp

Con STZ *

| |

-523 bp -259 bp | |

Heart, LV n = 10 * P < 0.05

Targeted bsDNA-seq

5-mCpG

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Methylation and Expression

GeneSifter and Zymo/UCSC Genome Browser

RNA – microarray

Methylation – genome sequencing Legend:

0% 100%

Legend:

PDK4

VDAC

Protein – western blot Protein – western blot


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Other Human/Mouse Comparisons

Irvin ! Arnett 2014 Circulation 130:565

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Other Human/Mouse Comparisons Mouse

Gene Expression 7*$-8,3-

7*$-9!:-

+.;<-8,3-

+.;<-9!:- !J>J&

Mouse DNA Methylation

Wende, unpublished

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DOX present = ON OFF

Where Does Glycemic Memory Fit In?

DOX absent = OFF

MHC-rtTA rtTA !-MHC


MHC-rtTA rtTA !-MHC


TRE-GLUT4 mycGLUT4

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Glucose Cycling Alters Epigenetic Programming

Legend:

0% 100%

Legend:

100%

Heart, LV Zymo Research Wende, unpublished

Genomewide bsDNA-seq

5-mCpG

mG4H-ON

mG4H-OFF

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Background

http://chemistry.uchicago.edu/faculty/faculty/person/member/chuan-he.html

5-hmC Wyatt and Cohen 1952 Nature 170(4338):1072 Kriaucioni and Heintz 2009 Science 324(5929):929 Tahiliani ! Rao 2009 Science 324(5929):930

ARW

!"!!#

!"!$#

!"%!#

!"%$#

!"&!#

!"&$#

!"#$%&'( !"#$)*+( ,-./$%&'(

,-./$011(

23$',

!(

Glucose Cycling Alters Epigenetic Programming

5-hmCpG ELISA

Heart, LV Zymo Research Wende, unpublished

ARW


Mariappa … Aalten 2013 EMBO J 32:612

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Tissue Specific Promoter Utilization

Heflin Center for Genomic Sciences, UAB Nye ! Wende, unpublished

RNA-seq Transcripts

Bdh1


RNA Legend: -1 1

Legend:

Con-Veh

Con-STZ

mG4H-Veh

mG4H-STZ

403

241

127

144

510 531

440

466

60 97 45

165

146

139

106

109

187

157

5

5

187

198 185

151

329

Heart, LV n = 3

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Combined Transcriptome/Methylome

Zymo Research and UCSC Genome Browser Wende, unpublished

Genomewide bsDNA-seq

5-mCpG

Heart, LV

Bdh1


RNA Legend: -1 1

Legend:

Legend: 0% 100%

Legend:

5-mCpG

ARW

Combined Transcriptome/Methylome

Zymo Research and UCSC Genome Browser Nye ! Wende, unpublished

Genomewide RRHP

5-hmCpG

Heart, LV

Bdh1


RNA Legend: -1 1

Legend:

Legend: 0% 100%

Legend:

5-mCpG 5-mCpG

5-hmC

5-hmC

5-hmC

5-hmC

5-hmC

ARW

Cellular glucose fluctuations regulates the epigenome via

histone modifications and controlling the machinery for

DNA methylation.


ARW

Sugar Gumming Up the Works

giphy.com

ARW

Using combined methylomics, transcriptomics, proteomics, and metabolomics we have

begun to define the mechanism of glucotoxicity.

Overall Summary

ARW

Acknowledgements

!"#$%&'(&)*+,*)$-%%).+,"(&)/#01)$#1%')

234)

234)

5-6-7-#")#8)9#%&.0%,1),"'):&%%0%,1);,*+#%#(/)

Thomas J Bailey – Undergrad; Cox6a2 Manoja K. Brahma – Postdoc; Bdh1/Oxct1 Mark C. McCrory – Lab Manager; Hmgcs2 Brenna G. Nye – Undergrad; Pcx Mark Pepin – MSTP; Abat Lamario J Williams – Undergrad; UQCRFS1/ROS

Wende Lab

K99R00 HL111322 R00 HL111322-S1

U24 DK076169 AHA 0725064Y JDRF 51002608

Other Colleagues & Mentors past and present

Oleh Khalimonchuk – UNL

Hansjörg Schwertz

E. Dale Abel John C. Schell Joseph Tuinei many others!

UAB Collaborators Steve Barnes

John C. Chatham David K. Crossman Steve M. Pogwizd Martin E. Young

Stavros G. Drakos Nikos A. Diakos

glucose and mitochondrial function

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