New cellular/physiologic roles of G6PD

趙 崇 義 特聘教授兼研發長Dr. Daniel Tsun-Yee Chiu, Distinguished Professor

Graduate Institute of Medical Biotechnology,

Dean of Research & Development,

Chang Gung University(長庚大學), Taiwan

Research Prof., Dept. of Pediatric Hematology,

Chang Gung Memorial Hospital (Linkou), Taiwan

Past President, Society Free Radical Research-Asia(2014, 2015)

Email: dtychiu@mail.cgu.edu.tw

July 14, 2016

2

A Brief History of Chang Gung University

(長庚大學 , CGU) of Taiwan- Established in April 1987 as Chang Gung Medical College

associated with the largest medical facilities (Chang Gung Memorial Hospitals) in Taiwan(perhaps the largest in the world, with 7 hospitals in Taiwan and 2 hospitals in China).

- Changed our name to “Chang Gung College of Medicine and

Technology” in 1993.

- Upgraded the College to “Chang Gung University” in July, 1997

consisting the Colleges of Medicine, College of Engineering and

College of Management .

- Currently has approximately 614 full-time faculty members and

7,145 students (including undergraduates 5,063 and graduates 2,082).

- Ranked 5th in Taiwan and approximately 400th world-wide.

Redox Homeostasis (Balanced Redox Status)

(A balance between Yin & Yan, 陰陽協調?)

Antioxidants = oxidants,

Oxidative stress

Antioxidant

Oxidant

Antioxidants < Oxidants

In contrast, then Reductive Stress

The Classical roles of G6PD:

to regenerate NADPH as antioxidant to produce Ribose

Glucose

G6PD

•O2-

(superoxide anion) HK

Glucose-6-phosphateNADP

NADPH

2GSH

GSSG

SOD

H2O2

H2O

CAT

H2O + O21

2

Glutathione reductaseGPx

6-phosphoglucono-δ-lactone

6PGL

6-phosphogluconate

6PGD

Ribulose-5-phosphate

Ru5PI

Ribose-5-phosphate

NADP

NADPH

Glutathione reductase

2GSH

GSSG

GPxH2O2

H2O

NADPHIsocitrate dehydrogenase (ICDH)

Malic enzyme (ME)NADP

4

Pentose phosphate pathway, PPP

5

G6PD deficiency (Also known as Favism)

• Glucose-6-phosphate dehydrogenase (G6PD) deficiency, a most

common enzyme deficiency affecting over 400 million people

worldwide, causes a spectrum of diseases including, acute and

chronic hemolytic anemia, neonatal jaundice and etc.

J Med Screen 19:103-104, 2012

Lancet 371: 64-74, 2008

G6PD deficiency

in Taiwan:Male 3%

Female 0.9%

Redox Rep 12: 109-18, 2007

Free Rad Res 48: 1028-48, 2014

Free Rad Res (in press), 2016

G6PDNADPH

NOS

Nitric oxide

SuperoxideNOX

NADP

NOS：Nitric oxide synthase

NOX：NADPH oxidase

Pro-oxidant role of G6PD :

Provides substrate to generate free radicals

(% of resting cells)

Decreased NO & Superoxide production FEBS Lett . 436:411-4, 1998

But Effective Neutrophil Extra-cellular Trap Formation Free Rad Res 47:699-709, 2013

Oxidants

6

Th

resh

old

Dual roles of G6PD in redox homeostasis affecting cellular function

cyto-regulatory cyto-toxic

Approximate concentration of oxidants

Level of

Oxidative Stress

oxidant

G6PD Deficiency

(Redox Report 12: 109, 2007; Free Rad Res 48: 1028, 2014; Free Rad Res in press, 2016)

Decreased NADPH ProductionDecreased NO &

Superoxide production

FEBS Letters 436:411-414, 1998,

But Effective Neutrophil Extra-cellular Trap Formation Free Rad Res 47:699, 2013 Redox Imbalance

Accelerated SenescenceFree Rad Biol Med 29: 156-169, 2000;

FEBS Letters 475: 257-262, 2000

Retarded Cell GrowthFree Rad Biol Med 29: 156-169, 2000

Increased susceptibilityto certain diseases

Jpn J Canc Res 92: 576-581, 2001

Endocrine 19: 191-196, 2002

[Ophthalmic Epid. 13: 109-114, 2006]

Increased susceptibility to:

1.Corona Virus infection.

J Infect Dis. 197:812-6, 2008

2. Enterovirus infection .

J Gen. Virol.89:2080-9, 2008

3. Protection by EGCGJ Agr Food Chem 57:6140-7, 2009

Increased Susceptibility to Oxidative Insult

Free Rad Biol Med 36: 580, 2004; Cytometry Part A 69: 1054, 2006

J Agr Food Chem 54: 1638, 2006; Free Radic Res. 41:571, 2007

Free Rad Biol Med 47: 529, 2009

G6PD deficiency-induced cellular abnormalities &

related phenotypes (beyond RBCs’ abnormalities)

Alterations in 1. Signal transduction(MAPK)

Free Rad Biol Med 49: 361, 2010

2. Proteomic J Proteom Res 12: 3434, 2013

3.Metabonomic Free Rad Biol Med 54: 71, 2013

4. C. elegans model. Cell Death Disease 4, e616, 2013

Inability to maintain GSH pool in G6PD-deficient

red cells causes futile AMPK activation and

irreversible metabolic disturbance

Tang Hsiang Yu et al(唐湘瑜)

(Antioxid Redox Sign 22: 744-759, 2015;

IF=7.407)

9

This work is, in part, based on the publication of

Ho HY, Cheng ML, Shiao MS and Chiu DTY.

Characterization of global metabolic responses

of G6PD-deficient hepatoma cells to diamide-

induced oxidative stress.

Free Radic Biol Med 54: 71-84, 2013; IF=5.736

(Applying New Technology to tackle traditional problems)

Typical workflow for MS-based metabolic

profiling TOF

Sample

collection and

pretreatment

Data analysis

(Big data)Data collection

Statistical analysis / Bioinformatics

Volcano plot

Heat map (ANOVA)

Principal

component analysis

(PCA)

Condition tree

(Clustering)

Metabolome: Metabolic

pathways and interactionFunction and

dysfunction

Targeted Metabolite

identification

10

Principal component analysis (PCA) in

G6PD deficient and control RBCs

with or without diamide-treatment

N

N_1 diamide

P_1 mM

diamide

P

Principal component analysis (PCA) of metabolomes in control and G6PDdeficient RBCs with or without diamide treatment. Both groups were un- or treatedwith 1mM of diamide for various time period. Features were acquired in ESI positiveion mode. Antiox. Redox Signaling 22: 744-759, 2015

27.31 %

37.78 %

11

Cysteine

Ophthalmic acid

Altered glutathione metabolism in G6PD

deficient RBCs leading to the formation of

ophthalmic acid upon diamide treatment

γ-glutamylcysteine

synthetase

Normal GSH Synthetic Pathway

Altered GSH Synthetic

Pathway

Methionine

Cycle

Ophthalmic acid has never been reported in human RBCs before

Antiox. Redox Signaling 22: 744-759, 2015

2-aminobutyrate

12

Antiox. Redox Signaling 22: 744-759, 2015

Cysteine

Ophthalmic acid

Alterations are mainly due to the reprogramming

from GSH regeneration via the glutathione

reductase system to GSH synthesis via

γ-glutamylcysteine synthetase

γ-glutamylcysteine

synthetase

Normal GSH Synthetic Pathway

Altered GSH Synthetic

Pathway

Methionine

Cycle

GR

NADPH NADP

G6PD

Reprogramming from GSH

regeneration to synthesisX

X

2-aminobutyrate

13

Blockade

of GSH

regeneration

1. Diamide treatment induces major alterations in GSH related metabolites in G6PD

deficient RBCs including the appearance of unusual metabolites such as

opthalmic acid which has never been reported in human RBCs before.

2. Such impairment in GSH related metabolism is mainly due to the reprogramming

from GSH regeneration to GSH synthesis and is accompanied by exhaustive

ATP consumption and enhanced glycolytic activities in G6PD deficient RBCs.

Unfortunately, the last step in glycolysis catalyzed by pyruvate kinase(PK) to

produce ATP is blocked in G6PD-deficient RBCs due to the inactivation of PK

by oxidative stress.

3. Changes in metabolic activities cause functional defects such as membrane

protein aggregation and decreased in RBC deformability of G6PD-deficient

RBCs and these new findings provide additional explanation concerning acute

hemolytic anemia in G6PD-deficient patients upon encountering oxidative stress

such as favism and infection.

In conclusion, this metabolomic study shows that G6PD-deficient RBCs

desperately struggle to maintain redox homeostasis upon oxidant challenge to

avoid cell death but without success.

Tang et al. Antiox. Redox Signaling 22: 744-759, 2015

Summary & Conclusion from our metabonomic study

14

Th

resh

old

Dual roles of G6PD in redox homeostasis affecting cellular function

cyto-regulatory cyto-toxic

Approximate concentration of oxidants

Level of

Oxidative Stress

oxidant

(One major

aspect of

research

is to prove

New Concepts)

A novel protective role for G6PD in

aflatoxin B1-mediated cytotoxicity

revealed by proteomic study

Hsin-Ru Lin(林欣如), Chih-Ching Wu(吳治慶)et al.

J Proteome Res. 12: 3434-3448, 2013

(IF=5.001)

16

Lin et al.

(A)

(B)

0.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

G6P

D a

ctiv

ity

(IU

/mg)

Ctrl G6PD-kd Ctrl G6PD-kd

Single clone A549 Mixed clone A549

Ctrl G6PD-kd Ctrl G6PD-kd

Single clone A549 Mixed clone A549

Actin

G6PD

1.00 0.22 0.81 0.18

Fig.1. Reduced G6PD status in G6PD-knockdown cells. (A) G6PD activities in A549 single and mixed clone cell lysates were

assayed, and expressed in IU/mg of protein. (B) Expression of G6PD protein in A549 single and mixed clone cell lysates was

analyzed by western bloting. Data are mean±SD, N=2, ** P＜0.001. (J Proteome Res. 12: 3434-3448, 2013)

* ** *

Generation of G6PD-knockdown A549 cells by shRNA

For subsequent protein analysis by proteomic technique

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Verification of Proteins Differentially Expressed in

G6PD-Knockdown A549 Cells

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The mRNA levels of EPHX1 and AKR1C3 were decreased in G6PD-knockdown A549 cells

compared with control cells, in which mRNA fold changes were positively correlated to the

iTRAQ ratios. (J Proteome Res. 12: 3434-3448, 2013)

Table -2

18

Pathway analysis of differentially expressed

proteins in G6PD-knockdown A549 cells Table.3.

J Proteome Res. 12: 3434-3448, 2013 19

Protective Role of G6PD in Aflatoxin B1 (AFB1)

Induced Cytotoxicity

Figure 4. G6PD deficiency augments AFB1-

induced cell death. (A) G6PD-kd and Ctrl

A549 cells were treated with 25, 50μM AFB1

for the indicated time intervals. Cell

viabilities were evaluated with MTT assay

and calculated as a ratio relative to cells

without AFB1 treatment. Results were

expressed as mean values ± SD from four

independent experiments (*P < 0.01; paired t-

test). (B) Cell death was monitored with

annexin V/PI staining. Either annexin V- or

PI-positive cells are classified as dead cells.

Results were expressed as mean values ± SD

from three independent experiments (*P <

0.01; paired t-test).(B)

G6PD may perform a

protective role in AFB1-induced

cytotoxicity via regulation of

EPHX1 expression.

J Proteome Res. 12: 3434-3448, 2013

20

G6PD Activity Is Required for ROS-Mediated

EPHX1 Production

Figure 6. Impairment of nuclear Nrf2 translocation by G6PD

knockdown. (A) Nuclear localization of Nrf2 was assessed immunochemically by using Nrf2-specific Ab in A549

cells treated with AFB1.(B) Superoxide level was monitored with DHE and flow cytometry in A549 cells treated

with 50 μM AFB1 for the indicated time intervals. Fold superoxide induction in the AFB1-treated cells was

calculated as a ratio relative to their own controls.(*P < 0.01; paired t-test).

J Proteome Res. 12: 3434-3448, 2013

21

Figure. AFB1 treatment induces

cytotoxicity via production of

AFB1−DNA adducts. Physiologically,

stimulus by low level of AFB1 causes

cellular ROS production via oxidation of

NADPH, the product in the pentose

phosphate pathway catalyzed by G6PD.

The induced ROS accordingly leads to

Nrf2 nuclear translocation and expression

of EPHX1, an enzyme involved in

detoxification of AFB1. G6PD knockdown

results in reduced generation of cellular

NADPH, thereby causing impairment of

AFB1-induced ROS production.

Consequently, EPHX1 expression is less

active in G6PD-depletion cells, suggesting

a protective role of G6PD upon AFB1-

inducible cytotoxicity.

Model depicting the protective role of G6PD in

AFB1- mediated cytotoxicity

Lin HR el. al , J. Proteome Res. 2013, 12, 3434−3448 22

Glucose-6-Phosphate Dehydrogenase Enhances Antiviral

Response through Downregulation of NADPH Sensor

HSCARG and Upregulation of NF- B Signaling

Dr. Yi-Hsuan Wu, 吳依璇

23

Viruses 7: 6689-6706, 2015

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Summary: Glucose-6-Phosphate Dehydrogenase

Enhances Antiviral Response through Downregulation

of NADPH Sensor HSCARG and Upregulation of NF-B

Signaling

Viruses 7: 6689-6706, 2015

Diminished COX-2/PGE2-mediated antiviral

response due to impaired NOX/MAPK signaling in

G6PD-knockdown lung epithelial cells

Lin HR, Wu YH, Yen WC,

Yang CM, Chiu DTY

Plos ONE 11: e0153462, 2016

Dr. Yi-Hsuan Wu

(吳依璇)

Dr. Hsin-Ru Lin

(林欣如)

Diminished COX-2/PGE2-Mediated Antiviral Response

Due to Impaired NOX/MAPK Signaling

in G6PD-Knockdown Lung Epithelial Cells

(G)

PLoS One. 2016 Apr 20;11(4):e0153462

Glucose 6-phosphate dehydrogenase knockdown

enhances IL-8 expression in HepG2 cells via

oxidative stress and NF-kB signaling pathway

Hung-Chi Yang, Mei-Ling Cheng, Yi-Syuan Hua,

Yi-Hsuan Wu, Hsin-Ru Lin, Hui-Ya Liu, Hung-Yao Ho,

Daniel Tsun-Yee Chiu

Yang, et al., Journal of Inflammation 12:34, 2015

Dr. Hung Chi Yang

(楊宏基)

27

G6PD deficiency up-regulates IL-8 through

oxidative stress and NF-κB pathway

28

NF-kB

knockdown

suppresses IL-8

antioxidants

suppress IL-8

Yang, et al., Journal of Inflammation 12:34, 2015

29

Glucose 6-phosphate dehydrogenase

deficiency enhances germ cell apoptosis

and causes defective embryogenesis in

Caenorhabditis elegans

Dr. Hung Chi Yang

(楊宏基)

Cell Death & Disease 4: e616, 2013

(IF=5.014)

30

G6PD knockdown enhances germ cell apoptosis, impairs egg production and

induces severe hatching defect (A)

(C)

(B)

Germ cell apoptosis

Egg production

Hatching

1. G6PD-knockdown C. elegans displays enhanced germ cell

apoptosis and reduced egg production as well as a severe

defect in hatching.

2. In contrast to its toxic effect in egg production, ROS may play

an important role as signal molecules to mediate certain

pathway(s) that in turn can affect hatching in C. elegans.

3. This G6PD-knockdown animal model allows us to delineate

the chronic effects of G6PD-deficiency at the multicellular

organism level.

31

Summary from G6PD-deficient

C. elegans study

Cell Death & Disease 4: e616, 2013

g6pd knockdown in zebrafish embryos

produces pericardial edema

Wu. et al. Unpublished data

G6PD knockdown induces morphological

changes and cadherin switch in A549 cells

Wu. et al. Unpublished data

LS LG

A549

G6PD

E-cadherin

-actin

N-cadherin

LS

LG

Scale bar, 50 µm

AcknowledgementLong time colleagues and collaborators

Dr. Mei-Ling Cheng, Associate Prof., Chang Gung Univ.

Dr. Hung-Yao Ho, Associate Prof., Chang Gung Univ.

Other Collaborators of Chang Gung

Dr. Ming-Shi Shiao, Prof. , CGU

Dr. Chih-Ching Wu, Associate Prof., CGU

Dr. Sze-Cheng John Lo, Prof., CGU

Collaborators of other Institutes

Dr. Li-Ping Gao (Lanzhou Univ., China )

Dr. Chang-Jun Lin (Lanzhou Univ., China )

Prof. Arnold Stern (NYU, USA)

Prof. Frans Kuypers(CHORI, Oakland/

UC Berkeley, USA)

Dr. Hung-Chi Yang

Dr. Hsin-Ru Lin Dr. Hsiang-Yu Tang

Dr. Yi-Hsuan Wu

34

Thank you very much for your attention!

此行是拋磚引玉Email: dtychiu @mail.cgu.edu.tw A professor is just as good as his/her

graduate students and/or postdoctoral fellows

ULK1/2 Constitute a Bifurcate Node Controlling

Glucose Metabolic Fluxes in Addition to Autophagy

Authors

Terytty Yang Li, Yu Sun, Yu Liang, ...,

Shu-Yong Lin, Daniel Tsun-yee Chiu,

Sheng-Cai Lin

Correspondence

linsc@xmu.edu.cn

In Brief

Metabolic reprogramming underlies

adaptive responses to various stresses.

Li et al. discover that the autophagy-initiating

kinases ULK1/2 also phosphorylate multiple

glycolytic enzymes to sustain glycolysis and

increase the proportion of glucose flux to

the pentose phosphate pathway during

nutritional stresses.

Molecular Cell. 62:359-70, 2016 (SCI IF=13.958)