Pharmacologically-induced Cytoplasmic NAD(P)+/NAD(P)H

Ratio by NQO1 Activator Ameliorates

the Metabolic Syndrome

Pharmacologically-induced Cytoplasmic NAD(P)+/NAD(P)H

Ratio by NQO1 Activator Ameliorates

the Metabolic Syndrome

Inkyu Lee MD, PhDDepartment of Internal Medicine,

Kyungpook National University, School of MedicineDaegu, Republic of Korea

Number of People With Diabetes in the Adult Population Worldwide for 2000 and

2010

Amos et al. Amos et al. Diabet MedDiabet Med. 1997;14:S1-S85; Zimmet et al. . 1997;14:S1-S85; Zimmet et al. NatureNature. 2001;414:782-787.. 2001;414:782-787.

14.2 m14.2 m

17.5 m17.5 m

↑23%

WorldWorld 2000: 151 million2000: 151 million

2010: 221 million2010: 221 million

Increase: 46%

15.6 m15.6 m

22.5 m22.5 m

↑44%

26.5 m26.5 m

32.9 m32.9 m

↑24%

9.4 m9.4 m

14.1 m14.1 m

↑50%1.0 m1.0 m

1.3 m1.3 m

↑33%

84.5 m84.5 m

132.3 m132.3 m

↑57%

Preventing Type 2 DiabetesThree Levels of Opportunity

Robust B-cells

Hyperinsulinemia

Weak B-cells

Hyperglycemia

Adipose Tissue

Liver & Muscle

Energy BalanceNegative Positive

Weight Loss Fat Accumulation

“Adipokines”

Fatty Acids

Insulin Resistance

• TZD Treatment

• Role of NQO1 and AMPK activator

Calorie restriction, Exercise,

Metabolic stress 5’-AMP

AMP KinaseGlucose transport

Fatty acid synthesis

Cell proliferationProtein synthesis

Cholesterol synthesis

b-acid oxidation

ACC/FAS

HNF4a, SREBP1c

E2F

HMG-coA reductase

p53

p21

TSC2

mTOR

CDK

LKB1Ca2+

CaMKK

AMPK (AMPK-activated protein)

혈관재형성 제어 연구실

Won Gu Jang

NAD is a key regulator of cellular energy

When a metabolite is oxidized, NAD+ accepts two electrons plus a hydrogen ion (H+) and NADH results. - NAD+ is reduced to NADH (reduced form)

Conversely, NADH can also reduce a metabolite by giving up electrons. - NADH is oxidized to NAD+

NAD plays a pivotal role in bioenergetics.

ATP

OXPHOS

Cpt1

Hypothetical Model:Transient Shift of Cytoplasmic Redox State into high

NAD+/NADHNAD+/NADH

NAD+ NADHoxidation

reduction

NADH shuttleComplex I

Electrontransport

OXPHOS

ATP

AMP/ATP

cADPR

Ca++

AMPkinaseIntracellular

Lipid

glycolysis

Fatty acid oxidation

adopted from Shong MH slide

• NQO1 catalyze metabolic detoxification of quinones • NQO1 protects cells from redox cycling, oxidative

stress and neoplasia.• Functions to detoxify quinones by two-electron

reduction (hydroquinones)• Functions to stable p53, induces apoptosis

NQO1 (NADPH Quinone reductase 1)

혈관재형성 제어 연구실

Cancer Res. 2005 65;6:2054

Skin tumors that developed in NQO1-null mouse (b) and histotype of tumor (c)

NQO1 (NADPH Quinone reductase 1) II

혈관재형성 제어 연구실

Oxidative stress

NQO1

Quinone metabolism p53 stabilization

Endogenous hydroquinonesA-tocopherol-quinone

Coenzyme Q10

Antioxidant capacity

Metabolism of stressorMutation Research 2004;555:149

-Known as NAD(P)H:quinone oxidoreductase-1 (NQO1) (E.C. 1.6.99.2), a two-electron oxidoreductase.

-Induction of apoptosis in MCF-7:WS8 breast cancer cells by b-L. Cancer Res.

1998.

-NAD(P)H:Quinone oxidoreductase activity is the principal determinant of b-L cytotoxicity. J Biol Chem. 2000.

-b-L-induced apoptosis is associated with activation of caspase-3 and inactivation of NF-kappaB in human colon cancer HCT-116 cells. Anticancer Drugs. 2003.

MB12066 (b-L)

혈관재형성 제어 연구실

MB660

NQO1NQO1

NADHNAD+

AMP ATP

AMPKAMPK

Fatty acid oxidation

Acetyl coA

Glycolysis

Pyruvate

Acetyl coA

p53

p21

SMC proliferation

Oxidative phosphorylation

ATP ROS

Kv1.5

Apoptosis

TCA cycleTCA cycle

NAD+/NADH

AMP/ATP

Sco2

CDK

MB12660 activates AMPK

혈관재형성 제어 연구실

Enhanced cytosolic NADH oxidation by NQO1 stimulates cellular energy

metabolism.

catalytically-inactive NQO1 (NQO1 C609T) (gray)

JW Whang, 2008 Diabetes

βL activates AMPK signaling pathway & fatty acid oxidation in vivo.

JW Whang, 2008 Diabetes

βL activates AMPK signaling pathway & fatty acid oxidation in vivo.

JW Whang, 2008 Diabetes

βL treatment ameliorates the metabolic sx of DIO mice.

JW Whang, 2008 Diabetes

The effects of βL on body weight and food intake of pair-fed DIO mice

JW Whang, 2008 Diabetes

•Beta-lapachone (βL) stimulated AMP-activated protein kinase (AMPK), subsequently triggers mitochondrial fatty acid oxidation by regulating acetyl-CoA carboxylase (ACC) and carnitine palmitoyltransferase (CPT) in L6 fibroblast and NQO1 MEF.

•βL treatment in the rodent models with the metabolic syndrome dramatically ameliorates obesity, glucose intolerance, dyslipidemia, and fatty liver.

•Collectively, elevation of NADH oxidation by using NQO1 can be a new therapeutic intervention in treating metabolic diseases.

SUMMARY & CONCLUSION

Activation of NAD(P)H:quinone oxidoreductase 1 (NQO1) prevents arterial restenosis by suppressing vascular smooth

muscle cell proliferation

혈관재형성 제어 연구실

혈관평활근세포의 증식과 분화 ( 거품세포 , 석회화 )

동맥경화증의 진행

1) 혈관평활근세포 증식 , 이주

2) 분화 (거품세포 , 혈관 석회화 )

위험인자( 당뇨병 , 고혈압 ,

고콜레스테롤 )

혈관재형성 제어 연구실

Restenosis 의 주범은 혈관평활근세포의 증식

STENT

Balloon

Drug Eluting Stent 260 만원국내소비 3 만 -4 만개 /년간1000 억 규모비용 /년간 한국

현재 동맥경화증의 유일한 직접치료는 혈관 풍선확장술

Figure1

BI + L(200 mg/kg/day)Control BI

(B)

(A)

(c) BI + L (100 mg/kg/day)

(d) BI + L (200 mg/kg/day)

(a) Control (b) BI

Inti

ma

/me

dia

ra

tio

0

5

10

15

20

25

a b c d

#

*

**

SH Kim et al, 2009, Circulation Research

Figure 2

Time (h) : 0 0.5 1 2 3 6

p21

p27

P-p53

-actin

(C)L (2 M)

p53

PDGF :L (M) :

(A)

+2

+1

+0.5

+0.1

+-

--

Brd

U

inco

rpo

rati

on

(%

of

con

tro

l)

serum :L (M) :

+2

+1

+0.5

+0.1

+-

--

serum :L (M) :

+2

+1

+0.5

+0.1

+-

--

0

50

100

150

200

250

300

0

50

100

150

200

250

300C

ell n

um

ber

(O

.D a

t 45

0/65

5)

0.0

0.5

1.0

1.5

2.0

2.5

Cel

l nu

mb

er

(% o

f co

ntr

ol)

# ##

*** ** * * *

** * *

(B)serum + Lcontrol serum

0

20

40

60

80

100

% o

f to

tal

cells

G1SG2/M

G0

contro

l

seru

m

seru

m +

-Lap

#

*

*#

Time : 6 12 240serum serum + L

P-pRB

cyclin E

cyclin D

-actin

(D)

6 12 240

pRB

SH Kim et al, 2009, Circulation Research

Figure 3(A)

Time (h) :

P-ACC (Ser79)

P-AMPK (Thr172)

AMPK

L (2 M)

0.5 1 2 30 6 6 2DM

SO

AICAR

Total ACC

-actin

AICAR

L (M) :

Time (2 h)

0 0.1 0.5 1 2

P-ACC (Ser79)

P-AMPK (Thr172)

AMPK

Total ACC

-actin

AICAR :

L :

HeLaVSMC-

-

+

-

-

+

-

-

+

-

-

+

(B)

P-ACC(Ser-79)

P-AMPK(Thr172)

LKB1

NQO1

AMPK

P-LKB1

Total ACC

-actin

(C)

0 10 20 30 600

25

50

75

To

tal

NA

D+/N

AD

H

rati

o

0 10 20 30 6040 500.0

0.1

0.2

0.3

AM

P/A

TP

ra

tio

(min)

(min)

*

DMSOβL

DMSOβL

*

*

SH Kim et al, 2009, Circulation Research

NADH

NQO1

P-AMPK

p53

p21

CDK

VSMC proliferation

βL βL-H2

NAD+

[NAD]/[NADH]

[AMP]/[ATP]LKB1

CAMKK ?

SH Kim et al, 2009, Circulation Research

Ad-DN-LKB1Ad-Null

P-AMPK

LKB1

NQO1

P-ACC

AMPK

ACC

L : - + - +P-ACC(Ser-79)

P-AMPK(Thr172)

-actin

AMPK

CaMKK

L : - + - +

NQO1

RASMC + Mock

RASMC + STO609

Total ACC

SH Kim et al, 2009, Circulation Research

Figure 4

-actin

P-P53

P21

- + - +L :Comp C

(C)

P53

- + - +

P-ACC(Ser79)

P-AMPK(Thr172)

-actin

L :

AMPK

(B)Comp C

Total ACC

serum :L :

Comp C :

---

+--

++-

+++

+-+

0.0

0.2

0.4

0.6

0.8

1.0(A)

Brd

U

inc

orp

ora

tio

n

(% o

f c

on

tro

l)

Ce

ll n

um

be

r (

% o

f c

on

tro

l)

0

50

100

150

200

250

serum :L :

Comp C :

---

+--

++-

+++

+-+

#

**

## ##

#

SH Kim et al, 2009, Circulation Research

(D)Comp C

L : - + - +-

serum

P-pRb

pRb

-actin

Ad-DN-AMPKL : - + - +-

serum

P-pRb

pRb

-actin

(E)

Serum:L:

--

+-

++

Ad-Null

Cel

l n

um

ber

(%

of

con

tro

l)

--

+-

++

Ad-DN-AMPK

0

50

100

150

200

250

#

*

##

(F)(a) CONTROL

(d) BI + L +Ad-DN-AMPK

(b) BI

(c) BI + L + Ad-LacZ

Inti

ma/

med

ia

rati

o

024681012141618

a b c d

#

**

#*

Figure 4

SH Kim et al, 2009, Circulation Research

Figure 5

L :

P-ACC(Ser-79)

P-AMPK(Thr172)

NQO1

-actin

- + - +

AMPK

HEK293VSMC

(A)

Total ACC

- + - +

HEK293 + Ad-Null

(B)HEK293 + Ad-NQO1

(D) dicoumarolAICAR :

L :P-ACC(Ser 79)

P-AMPK(Thr172)

NQO1

-actin

AMPK

Total ACC

--

+-

-+

--

+-

-+

ES936--

+-

-+

--

+-

-+

--

+-

-+

--

+-

-+

RASMC + Ad-si-NQO1

RASMC + Ad-Null

(C)AICAR :

L :P-ACC(Ser-79)

P-AMPK(Thr172)

NQO1

-actin

AMPK

Total ACC

LKB1

P-LKB1

SH Kim et al, 2009, Circulation Research

Figure 6

(B)control serum

serum + L serum + L + dicoumarol

(A)

050100150200250300350

Ad-Null

Ce

ll n

um

be

r (%

of

co

ntr

ol)

Ad-si-NQO1

Serum:L:

--

+-

++

--

+-

++

0.00.20.40.60.81.01.2

serum :L :

dicoumarol :

---

+--

++-

+++

+-+

Brd

U

inc

orp

ora

tio

n

(% o

f c

on

tro

l)

serum :L :

dicoumarol :

---

+--

++-

+++

+-+

0

50

100

150

200

250

Ce

ll n

um

be

r (%

of

co

ntr

ol)

- + - +-Lap :

P21

-actin

P-p53

dicoumarol

p53

dicoumarol-Lap : - + - +-

serum

P-pRb

pRb

(C)

# **

##

*

#

*

#

##

# ## #

LKB1

NADH

NQO1

P-AMPK

p53

p21

CDK

VSMC proliferation

βL βL-H2

NAD+

[NAD]/[NADH]

[AMP]/[ATP]

-actin

(D)

SH Kim et al, 2009, Circulation Research

Calorie restriction, Exercise,

Metabolic stress 5’-AMP

AMP KinaseGlucose transport

Fatty acid synthesis

Cell proliferationProtein synthesis

Cholesterol synthesis

b-acid oxidation

ACC/FAS

HNF4a, SREBP1c

E2F

HMG-coA reductase

p53

p21

TSC2

mTOR

CDK

LKB1Ca2+

CaMKK

AMPK (AMPK-activated protein)

혈관재형성 제어 연구실

MB12660 prevents atheroscelrosis induced by diabetes in

Apo E-deficient mice fed high-Fat diet

혈관재형성 제어 연구실

MB12660 prevents the increase in atheroscelrosis induced by diabetes in

Apo E-deficient mice fed high-Fat diet

0 10 20 30 40 50 6024

25

26

27

28

29

30

31

32

day

Bo

dy

wei

gh

t (g

)

STZ ControlSTZ MB660 25mg/kgSTZ MB660 25mg/kg pair fedSTZ MB660 50mg/kgSTZ MB660 50mg/kg pair fed

-4 -3 -2 -1 0 1 2 3 4 5 6 7 8100

150

200

250

300

350

week

Body weight Glucose level

STZ 투여

MB660투여

STZ control

MB660 25 mg/ml

MB660 50 mg/ml

STZ control

MB660 25 mg/ml

MB660 50 mg/mlPla

sma

glu

cose

(m

g/d

l)

혈관재형성 제어 연구실

Control MB660 25 mg/kg MB660 50 mg/kg

Heart aorticvalves

Control MB660 25 mg/kg MB660 50 mg/kg

Aorta

(A)

(B)

MB12660 prevents the increase in atheroscelrosis induced by diabetes in

Apo E-deficient mice fed high-Fat diet

혈관재형성 제어 연구실

Conclusion MB660

NADHNAD+

AMP ATP

AMPKp53

p21

CDK

SMC proliferation

MB660-H

NQO1

ACC/FAS

Fatty acid synthesis

•βL-induced NADH oxidation by NQO1 and by LKB1, at least in part, upregulation of AMPK reduced VSMC proliferation in vitro and in vivo.

•βL-induced NADH oxidation by NQO1 reduced atheroma formation.

This study provides the regulation of NAD+/NADH redox potential may be novel therapeutic target for the prevention of metabolic syndrome

Summary & Conclusion

Sun-Yee Kim

Acknowledgments

Chang-Joo Oh

충남대학교 의과대학

-송민호 MD., PhD.

- 황정환

울산대학교 의과대학

- 이기업 MD., PhD.

전남대학교 호르몬센터- 최흥식 PhD.

MD BioAlpha

Young-Keun Choi