3rd International Conference and Exhibition on
Nutrition & Food Sciences
September 23-25, 2014 Valencia, Spain
Cyanidin-3-O-glucoside ameliorates lipid
and glucose accumulation in C57BL/6J
mice via activation of PPAR-α and AMPK
Food Biomedical Science Lab.
Yaoyao Jia
Sep 23th, 2014
Background
 Natural organic pigment







Grapes
Berries
Cherries
Apples
Plums
Red cabbage
Red onion






Antioxidant
Anti-inflammatory effects
↓ Cardiovascular Diseases
↓ Cancer
↓ Obesity
↓ Diabetes
 To investigate the effects and molecular mechanisms of cyanidin3-O-glucoside (C3G)
Background
Exercise
(muscle stimulation)
 Berries containing C3G regulate lipid and glucose metabolisms
Nucleus
ligands
AMP:ATP ratio↑
Thr172
P
γ
–
–
Nuclear receptors
Containing 3 isoforms: PPARα, PPARγ, PPARδ/β
β
AMPK
PPRE
Peroxisome proliferator-activated receptors (PPARs):
Lipid metabolism
α
AMP-activated protein kinase (AMPK):
− An enzyme plays a role in cellular energy homeostasis
− Consists of three proteins (subunits): α, β, and γ
− Three subunits together make a functional enzyme
PPARα:
–
–
A major regulator of lipid metabolism in the liver
–
Fatty acid uptake (fatty acid transport)
–
Fatty acid utilization (fatty binding and activation)
–
Fatty acid catabolism (peroxisomal and
mitochondrial fatty acid β-oxidation)
–
Ketogenesis
–
Triglyceride turnover
Ligands:
–
Synthetic ligands include the fibrate drugs
(hyperlipidemia)
–
Endogenous ligands include fatty acids and various
fatty acid-derived compounds
* RXR: retinoid X receptor
PPRE: peroxisome proliferator hormone response elements
Glucose metabolism
AMPK:
− Stimulate:
− Fatty acid oxidation
− Ketogenesis
− Inhibit:
− Lipogenesis
− Triglyceride synthesis
− Gluconeogenesis
Experimental design
 Molecular targets of C3G
 BIAcore Surface plasmon resonance (SPR)
 Time resolution-fluorescence resonance energy transfer (TR-FRET) coactivator assay
 AMPK activity assay
 Animal/cell experimental design
Lipid-loading for 24 h
•
•
•
•
Treatment with C3G for 24 h
HepG2 cells
•
•
Sacrifice
Sample (plasma &
organs) collection



 Physiological relevance & molecular mechanisms of C3G








Body & organ weight measurement
Plasma lipid, glucose, insulin & hormone measurement
Liver & adipose tissue histology & analysis
Liver lipid concentration measurement
Oral glucose tolerance test (OGTT)
Insulin tolerance test (ITT)
Autophagy pathway analysis
qPCR & immunobloting
Lipid contents
Fatty acit oxidation/synthesis
Autophagy analysis
Gluconeogenesis
HFD: High fat diet (45%)
FF: 100 mg/kg body weight of
fenofibrate (FF)
C3G: 100 mg/kg body weight of
cyanidin-3-O-glucoside (C3G)
C3G induces PPARα coactivator activity via direct binding to PPARα
150
Concentration (M):
100
100
80
40
25
12.5
50
0
100
200
400
500
Time (s)
80
50
40
25
20
200
0
100
200
300
400
C3G
500
C3G
C3G
Time (s)
-200
PPAR/TT
1000
Concentration (M):
200
100
80
40
25
12.5
100
0
100
200
300
400
500
800
Concentration (M):
600
100
80
50
40
25
400
200
0
100
200
-200
Time (s)
C
300
400
KD values and EC50 values of C3G and positive controls
C3G
Positive controls
PPARα PPARγ PPARδ
PPARα PPARγ PPARδ
GW7647
TT
GW0742
SPR
456 nM 1.36 μM 4.96 μM 13.2 nM 377 nM 102 nM
TR-FRET 1126 nM 10.8 µM 31.05 μM 26.9 nM 82.3 nM 10.25 nM
500
Time (s)
PPAR//GW0742
PPAR//C3G
800
Concentration (M):
100
100
80
50
40
25
50
0
200
300
400
500
Time (s)
-50
D
PPAR
1.0
EC50:
C3G = 1126 nM
GW7647 = 26.9 nM
10-2
102
Concentration (nM)
80
50
40
25
20
100
106
200
300
400
KD, the equilibrium dissociation constant ('binding constant');
EC50, Half maximal effective concentration
500
Time (s)
-200
1.5
0.5
400
0
PPAR
C3G
GW7647
2.0
Concentration (M):
200
2.0
1.5
1.0
EC50:
C3G = 10805 nM
TT = 82.3 nM
0.5
0.0
10-2
100
102
PPAR
C3G
Troglitazone (TT)
2.5
TR-FRET Ratio
100
600
104
Concentration (nM)
106
C3G
GW0742
1.5
TR-FRET Ratio
150
Response Unit (RU)
200
Response Unit (RU)
400
300
-100
TR-FRET Ratio
Concentration (M):
Response Unit (RU)
Response Unit (RU)
300
600
PPAR/C3G
0.0
10-6
Time resolution-fluorescence resonance
Surface
Plasmon
Resonance
(BIAcore assay
SPR)
energy transfer
(TR-FRET)
coactivator
800
-50
B
PPAR/GW7647
PPAR/C3G
Response Unit (RU)
Response Unit (RU)
A
1.0
0.5
0.0
10-2
EC50:
C3G = 31046 nM
GW0742 = 10.25 nM
100
102
104
Concentration (nM)
106
108
C3G induces AMPKα1 activity via direct interaction with AMPKα1
A
AMPK (1/1/1) activity
AMPK (1/1/1) activity
550000
A-769662
C3G
750000
700000
650000
600000
10 -4
10 -2
10 0
10 2
10 4
10 6
Luminescence (RLU)
Luminescence (RLU)
800000
+ A-769662 (1000 nM)
+ A-769662 (100 nM)
500000
450000
400000
350000
10 -2
10 0
AMPK
AMPK (2/1/1) activity
Luminescence (RLU)
430000
A-769662
C3G
380000
α
γ
β
A-769662
330000
280000
230000
10 -4
10 4
C3G (nM)
Concentration (nM)
B
10 2
10
-2
10
0
10
2
10
4
10
6
EC50 (nM)
A1/B1/G1
A2/B1/G1
A-769662
262
457
C3G
599
1010
Concentration (nM)
 C3G directly activates PPARα and AMPK
C3G reduces lipid accumulation in mouse livers & hepatocytes
B
Intracellular Triglyceride
Plasma triglyceride
100
1.0
**
**
***
80
b
b
60
40
20
1
C
+
1
+
10
-
+
50
-
FD
+
-
H
-
3G
0
0.0
Lipid
C3G (M)
GW7647 (M)
a
C
0.5
***
Triglyceride (mg/dL)
Fold change
1.5
FF
A
Hepatic Triglyceride
Triglyceride (mg/dL)
150
a
100
b
b
50
3G
Plasma ALT
Plasma AST
a
100
C
H
3G
bc
C
0
3G
0
FF
50
FD
100
FD
b
b
ac
H
200
150
ALT (U/L)
300
AST (U/L)
a
200
400
FF
D
C
FF
H
FD
0
AST, Aspartate Aminotransferase;
ALT, Alanine Aminotransferase
C3G induces hepatic fatty acid oxidation and ketogenesis while
decreases fatty acid synthesis via regulation of PPARα & AMPKα1
Fatty acid oxidation
Fatty acid oxidation
6
3
Liver  -oxidation
(mol/g/min)
2
***
1
*
*
H
FD
200
b
0.5
100
0.0
D
1
10
-
50
-
E
-Hydroxybutyrate
0
C3G (M)
GW7647 (M)O-
200
1
10
-
mRNA expression in liver
18
b
16
Fold change
b
50
-
FD
10
-
H
0.0
C3G (M)
FF (M) O
GW7647 (M)O-
b
3G
0.5
***
a
300
1.0
C
**
**
Malonyl CoA
400
FF
**
a
2
+
50
-
ng/mL
fold change
1.0
+
10
-
C
1.5
1.5
Fold change
+
1
Fatty acid synthesis
Fatty acid synthesis
c
4
0
0
Lipid
C3G (M)
GW7647 (M)O-
B
b
14
2.0
 C3G reduces lipid accumulation via increases
fatty acid oxidation, ketogenesis, whereas
inhibits fatty acid synthesis
b
100
a
50
b
b b
1.5
a
b
b
a
b
a
a
a
1.0
0.5
PPAR
CPT1
LPL
FD
F
C F
3G
H
FD
F
C F
3G
H
FD
F
C F
3G
H
H
3G
C
FF
FD
FD
F
C F
3G
0.0
0
H
M
150
HMGCS2
ACC, acetyl-CoA carboxylase
CPT1, carnitine palmitoyltransferase 1;
LPL, lipoprotein lipase;
HMGCS2, 3-hydroxy-3-methylglutaryl-CoA synthase 2
C
3G
Fold change
***
FF
A
C3G induces phosphorylation of AMPK thus blocks the mTOR-S6K1
axis
HFD
FF
C3G
p-AMPK/AMPK
p-AMPKT172
4
Fold change
AMPK
p-mTORT2446
mTOR
p-S6K1T389
b
3
b
2
a
1
S6K1
p-mTOR/mTOR
p-S6K1/S6K1
1.5
4
b
3
2
a
1
0
Fold change
b
a
1.0
b
b
0.5
mTOR, mammalian target of rapamycin; S6K1, P70-S6 Kinase 1
3G
C
FF
FD
H
3G
C
FF
FD
0.0
H
Fold change
5
3G
C
FD
H
FF
0
β-actin
C3G induces hepatic autophagy pathway
A
GW7647
C
1
B
C3G (µM)
10
HFD FF
50
LC3I
LC3II
LC3I
LC3II
α-tubulin
β-actin
LC3 II
LC3 II
Autophagy
2.0
2.0
8000
**
*
1.0
0.5
0.0
b
a
1.0
0.5
0.0
6000
c
a
2000
3G
0
D
-
10
-
10
No. of Autolysosomes/Cell
Number of Autolysosomes
20
***
15
***
10
**
5
 C3G reduces lipid accumulation via
activates hepatic autophagy pathway
STF, STF-62247
d
4000
Tamoxifen (M)
C3G (M)
C
50
-
FF
10
-
FD
1
H
C3G (M)
GW7647 (M)O-
b
b
1.5
Fluorescence
(Relative Units)
**
1.5
Fold change
Fold change
C
C3G
0
STF62247 (M)
C3G (M)
-
5
-
10
50
50
C3G reduces plasma glucose & insulin concentrations and improves
insulin sensitivity
a
0.3
AUC
C
3G
FF
FD
H
ac
a
0.2
10
ac
bc
5
0.1
0
HOMA-IR, Homeostatic Model Assessment - Insulin Resistance;
AUC, Area under the curve
3G
0.0
C
0.0
120
15
bc
FF
Time(min)
**
90
120
FD
60
90
3G
30
60
C
15
30
FF
*
15
Time(min)
FD
**
b
0.0
0
H
*
0.5
**
3G
*
C
***
150
0
b
AUC
*
b
0.5
*
100
HOMA-IR
b
FD
***
**
0.4
a
H
200
*
200
D
AUC of ITT
1.0
**
a
1.0
H
H
FD
3G
C
H
250
1.5
0
HFD
FF 1.5
C3G
300
Plasma glucose level
(mg/dL)
0.0
300
3G
0
FF
0.2
ITT
b
0.4
50
C
Plasma glucose level
(mg/dL)
0.6
AUC of OGTT
HFD
FF
C3G
C
100
a
FF
ng/mL
150
a
0.8
bc
FD
Glucose (mg/dL)
ac
200
OGTT
400
1.0
Insulin-sensitive Index
a
250
B
Plasma insulin
Plasma glucose
FF
A
C3G reduces gluconeogenesis
A
B
Gluconeogenesis
250
b
200
150
ab ab
a
a a a
a a a
100
50
0
CE-TOF & QqQMS：Selected component analysis
cAMP -
+
-
-
-
-
+
-
-
-
Metformin -
-
+
-
-
-
-
+
-
-
Compound C -
-
-
-
-
+
+
+
+
+
C3G -
-
-
10 50 -
-
- 10 50
C3G reduces gluconeogenesis via increases plasma adiponectin
concentration & inhibits FOXO and CREB activity
FF C3G
p-AMPK/AMPK
2
a
1
b
bc
a
2
a
1
3G
C
FF
H
b
b
20
b
0.5
3
Adiponectin
30
ac
1.0
3G
FD
H
C
a
4
0
g/mL
***
***
50
a
a
400
150
* *** ***
3
C
FD
H
1.5
***
600
100
mRNA expression in liver
CRE-Luc
activity
Fold change
Luciferase activity (%)
FOXO
activity
4
0
FF
0
β-actin
800
b
2
X
HDAC5
B
3
PEPCKa
1
G6Pase
p-HDAC5
b
5
b
C
CRTC2
5
b
Fold change
CRTC2
Fold change
HDAC5
p-CRTC2
p-HDAC5/HDAC5
FD
4
AMPK
p-CRTC2/CRTC2
Fold change
p-AMPK
FF
HFD
3G
A
a
10
***
0
0.0
0
PEPCK
3G
C
FF
FD
H
3G
C
FD
FF
H
3G
C
- - 100 - - 100 - - - - - 10 50
H
- 100 - - - - - - 10 50
FD
FF
 C3G reduces glucose accumulation via
inhibits hepatic gluconeogenesis
A769662 (M)
Foskolin (M)
C3G (M)
G6Pase
FOXO1, Forkhead box protein O1; CREB, cAMP response element-binding protein; HDAC5, Histone deacetylase 5; CRTC2, CREB regulated transcription coactivator 2;
PEPCK, Phosphoenolpyruvate carboxykinas; G6Pase, Glucose 6-phosphatase
C3G reduces body weight, visceral fat weight & adipocyte size
A
Body weight
HFD
FF
C3G
Body weight (g)
50
B
* **
40
* ** ******
30
20
0 1 2 3 4 5 6 7 8 9 10 11 12 13
Weeks
C
a
2000
Epididymal Fat (g)
Visceral Fat (g)
Perirental Fat (g)
Total White Adipose Tissue (WAT, g)
Brown Adipose Tissue (BAT, g)
WAT/BAT
Liver (g)
Liver/Body weight
FF
0
2.45
1.67
1.52
5.63
C3
G
Organ weight of mice
Skeletal Muscle (g)
WAT/Skeletal Muscle
b
b
4000
HF
D
Adipocyte size (m2)
Adipocytes
6000
HFD
± 0.16a
± 0.11a
± 0.10a
± 0.20a
2.43
0.71
1.02
4.16
FF
±
±
±
±
0.26a
0.09bc
0.09bc
0.42bc
2.41
0.98
1.19
4.58
C3G
± 0.19a
± 0.19c
± 0.15ac
± 0.52ac
0.29
20.81
±
±
0.03ab
2.07a
0.22
19.90
±
±
0.03a
1.52a
0.36
12.90
±
±
0.04b
0.87b
0.68
8.43
±
±
0.04a
0.49a
0.55
7.98
±
±
0.08a
0.58ab
0.76
5.85
±
±
0.06a
0.80b
1.59
0.036
±
±
0.13a
0.002a
1.46
0.040
±
±
0.05a
0.001a
1.37
0.034
±
±
0.17a
0.003a
C3G increases energy expenditure via induces thermogenesis gene
expressions in brown adipose tissue (BAT)
CON
C3G
1.0
RQ(Respiratory Quotient)
VO2 (ml/min/kg)
*
20
10
Total
Light
0.6
0.4
0.2
CON
C3G
*
Light
10
0
1
6
12
CON
C3G
0.8
0.6
0.4
0.2
17
50
Total
200
Light
Dark
CON
C3G
*
**
150
100
50
0
0.0
19
100
Dark
1.0
20
150
0
Total
RQ(Respiratory Quotient)
VO2 (ml/min/kg)
30
*
C3G
0.8
Dark
CON
C3G
200
0.0
0
19
1
6
12
17
19
1
6
12
17
mRNA expression in BAT
B4
Fold change
CON
Energy Expenditure
(kcal/day/kg)
30
Energy Expenditure
(kcal/day/kg)
A
3
b
b b
b
b
b
 C3G reduces body weight via increases
energy expenditure and thermogenesis in
brown adipose tissue
2
a
a
1
a
PPAR
PGC-1
3G
C
FD
FF
H
3G
C
FD
FF
H
3G
C
H
FD
FF
0
UCP1
PGC-1α, Peroxisome proliferator-activated receptor gamma coactivator 1-alpha;
UCP1, uncoupling protein 1
Conclusion
C3G
C3G
Fatty acid
oxidation
Energy
expenditure
Fatty acid
synthesis
Autophagy
Insulin
sensitivity
Gluconeogenesis
 ↓ Body weight, visceral fat weight & adipocyte size
 ↓ Lipid accumulation in liver
 ↓ Glucose & insulin concentrations in plasma
 Improves insulin sensitivity
↓Atherosclerosis
Acknowledgement
Food Biomedical Science Lab.
Ewha Women’s University
•
•
Supervisor
Prof. Young-Suk Kim
Prof. Sung-Joon Lee
 Minyoung So
•
FBS lab. Members
 Ji Hae Lee
 Chunyan Wu
 Bobae kim
 Ji Ah Kim
 Soyoung Kim
 Boram Mok
•
Rural Development Administration of Korea
Thank you for your attention!