Post on 30-Dec-2015
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
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
•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
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