Mechanisms%20for%20Improved%20Insulin%20Sensitivity%20by

1
Mechanisms for Improved Insulin Sensitivity
by Branched-chain Amino Acids Deprivation
Reporter Fei Xiao, PhD
Shanghai Institute for Nutritional Sciences,
Chinese Academia of Sciences
2
Background
Prevalence of diabetes
3
Insulin signaling
Insulin Receptor, IR
Insulin Receptor Substrate, IRS
AKT
Nature. 2001, 414799-806
4
Background
Insulin resistance a physiological condition
where insulin becomes less effective at
lowering blood glucose a common feature of many
metabolic diseases
5
Background
Various strategies to treat insulin resistance
The DPP Research Group, NEJM,2002
6
Background
Branched-chain amino acids (BCAAs)
L-leucine
L-isoleucine
L-valine
7
The role of leucine in insulin sensitivity
Increased serum leucine level Improves the
whole body glucose metabolism
Diabetes 561647-1654, 2007
Does not alter susceptibility to diet-Induced
obesity
J. Nutr. 139 715719, 2009.
Increases insulin resistance in models of
obesity
?
Cell Metab 9311-326, 2009
Diabetes 542674-2684, 2005
8
Research interest of our work
Genetic factors
Nutrient (BCAAs)
X
sensing
Metabolism regulation
Metabolic diseases (insulin sensitivity/glucose
metabolism)
9
Outline
Part ? The role of leucine deprivation in insulin
sensitivity
Part ? Effects of individual BCAAs on insulin
sensitivity and glucose
metabolism in mice
Part ? Looking for new genes regulating insulin
sensitivity by leucine
deprivation model
10
Part ?
The role of leucine deprivation in insulin
sensitivity
11
Part ?
In Our Lab
Fat mass
Lipolysis in WAT
UCP1 in BAT
Leucine deprivation
?
CNS food intake
Serum Insulin Blood Glucose Normal
Cell Metab 5103-114, 2007
Diabetes 5917-25
12
Part ?
Hypothesis
Leucine deprivation may improve insulin
sensitivity
13
Part ?
Experimental Design

• Control
• (-)leu
• Pair-fed

control
control
(-)leu
85 control
14d
0d
7d
Change different diets
Mice were acclimated to control diet for 7 days
Collect tissues
14
Part ?
Result 1 (-) leu improves insulin sensitivity
in vivo
pf
ctrl
(-) leu
1.5
200
0.6
Fasting
Fasting
Serum Insulin (ng/ml)
150

1
0.4
HOMA-IR

Blood Glucose (mg/dl)

100



0.5
0.2
50
0
0
0
plt0.05 vs. control plt0.05 vs. pf
15
Part ?
Result 2 (-) leu improves insulin sensitivity
in vivo
ctrl
(-) leu
pf
ITT
GTT
500
200
400
150


300



Blood Glucose (mg/dl)
100

Blood Glucose (mg/dl)


200





50

100
0
0
0
20
40
60
80
100
120
0
20
40
60
80
100
120
Time (min)
Time (min)
plt0.05 vs. control plt0.05 vs. pf
16
Part ?
Result 3 (-) leu improves insulin sensitivity
in vivo
Liver
WAT
Muscle
ctrl
(-) leu
ctrl
(-) leu
ctrl
(-) leu
Ins
- -
- -
- -
p-IR
t-IR
p-AKT
t-AKT
ctrl Ins
(-) leu Ins
200

300
300





200
200
Arbitrary Units
100
100
100
0
0
0
p-AKT
p-AKT
p-AKT
p-IR
p-IR
p-IR
plt0.05 vs. control
17
Part ?
Results 4 (-) leu improves insulin sensitivity
under insulin-resistance
conditions
HFD
ctrl db/db
(-) leu HFD
(-) leu db/db
ctrl
C
ctrl WT
250
600
ITT
ITT
200
Blood Glucose (mg/dl)
Blood Glucose (mg/dl)
400
150



100






200



50







0
0
0
40
80
120
20
60
100
0
40
80
120
20
60
100
Time (min)
Time (min)
plt0.05 vs. HFD plt0.05 vs. ctrl
plt0.05 vs. ctrl db/db plt0.05 vs. ctrl WT
18
Part ?
Summary 1
leucine deprivation improves insulin sensitivity
under normal and insulin-resistant conditon
Mechanisms ?
19
Part ?
Role of mTOR/S6K1 signaling in insulin
sensitivity
mTOR mammalian target of rapamycin
S6K1ribosomal protein S6 kinase 1
Nature 431200-205, 2004
20
Part ?
Results 5 (-) leu increases insulin sensitivity
by decreasing mTOR/S6K1
signaling in vivo
On a leucine-deficient diet
A
B
Liver
- Ad-CA-S6K1
Ad-CA-S6K1
ctrl
(-) leu
p-mTOR
200
ITT
150
Blood Glucose (mg/dl)
t-mTOR


100

p-S6K1
50
t-S6K1
0
p-S6
0
40
80
120
20
60
100
Time (min)
t-S6
plt0.05 vs. control
21
Part ?
GCN2
General control nonderepressible (GCN)2
?
A serine protein kinase
?
Function as a sensor for amino acid deprivation
?
22
Part ?
Results 6(-) leu increases insulin sensitivity
by activation of GCN2
Liver
Gcn2/
Gcn2-/-
(-) leu
ctrl
150
ITT
p-GCN2
100
Blood Glucose (mg/dl)
t-GCN2
50
0
0
40
80
120
20
60
100
Time (min)

200
Arbitrary Units
100
0
plt0.05 vs. control
23
Part ?
AMPK
AMP-activated protein kinase
?
Energy sensor
?
Target of many drugs
?
Leucine deprivation
?
GCN2
AMPK
p-mTOR /p-S6K1
p-IR/p-IRS/p-AKT
24
Part ?
Results 7 (-)leu improves insulin sensitivity
via activation of AMPK
HepG2
HepG2
Liver
Ins
- -


leu -leu
ctrl
(-) leu
- -
DN-AMPK
- DN-AMPK Ins
p-AMPK
p-IR
DN-AMPK Ins
t-AMPK
t-IR
150
p-IRS1 (Tyr612)
100


Arbitrary Units
t-IRS1

50

200
p-AKT

0
Arbitrary Units
100
t-AKT
p-IR p-IRS1 p-Akt
t-AMPK
0

p-ACC
t-ACC
plt0.05 vs. control
25
Part ?
Summary2
Leucine deprivation increases hepatic insulin
sensitivity via GCN2/mTOR/S6K1 and AMPK pathways
model
Leucine deprivation
p-AMPK
p-GCN2
p-mTOR /p-S6K1
p-IR/p-IRS/p-AKT
26
Part ?
Conclusion One
Elucidate the mechanisms underlying increased
insulin sensitivity by leucine deprivation
?
GCN2/mTOR/S6K1 and AMPK pathways
Demonstrate a novel function for GCN2 in the
regulation of insulin sensitivity
?
The paper titled leucine deprivation increases
hepatic insulin sensitivity via
GCN2/mTOR/S6K1 And AMPK pathways was published
in Diabetes, 60746-756,2011.
27
How about the other two branched-chain amino
acids ?
L-isoleucine (ile)
L-valine (val)
28
Part ?
Effects of individual branched-chain amino acids
on insulin sensitivity and glucose metabolism in
mice
29
Part ?
Results 8 (-)val and (-)ile improves insulin
sensitivity in vivo
B
A
(-) val
(-) ile
ctrl
ctrl
500
200
500
200
ITT
GTT
ITT
GTT
400
400
150
150

300
300
100
Blood Glucose (mg/dl)
Blood Glucose (mg/dl)
Blood Glucose (mg/dl)
Blood Glucose (mg/dl)

100
200
200



50


50

100



100







0
0
0
0
0
40
80
120
0
40
80
120
0
40
80
120
0
40
80
120
Time (min)
Time (min)
Time (min)
Time (min)
30
Part ?
Result 9 (-)val and (-)ile decrease mTOR/S6K1
and increase AMPK signaling
Liver
Liver
ctrl
(-) Val
ctrl
(-) Ile
p-mTOR
p-mTOR
t-mTOR
t-mTOR
p-S6K1
p-S6K1
t-S6K1
t-S6K1
p-S6
p-S6
t-S6
t-S6
p-AMPK
p-AMPK
t-AMPK
t-AMPK

200
200

Arbitrary Units
Arbitrary Units

100
100





0
0
p-s6
p-s6
p-s6k1
p-s6k1
p-mTOR
p-mTOR
p-AMPK
p-AMPK
31
Part ?
Results 10 Effects of individual BCAA
deficiency on glucose
metabolism
(-) ile
(-) val
ctrl
Fasting
Fasting
0.4
150
1



100



HOMA-IR
Blood Glucose (mg/dl)
Serum Insulin (ng/ml)
0.5
0.2
50
0
0
0
plt0.05 vs. control
32
Part ?
Results 11 Effects of individual BCAA
deficiency on glucose
metabolism
ctrl
(-) val
(-) ile
(-)leu
Fed
Fed
1.5
200
200




150
1
Relative mRNA ()
Blood Glucose (mg/dl)
Serum Insulin (ng/ml)
100
100

0.5



50
0
0
0
g6pase
pepck
plt0.05 vs. control
33
Part ?
Results 12 BCAA deprivation for 1 day improves
whole body insulin
sensitivity
ctrl
(-) BCAA
(-) leu
(-) val
(-) ile
200
200
200


Blood Glucose (mg/dl)
Blood Glucose (mg/dl)
Blood Glucose (mg/dl)
100
100
100










0
0
0
0
40
80
120
0
40
80
120
0
40
80
120
Time (min)
Time (min)
Time (min)
plt0.05 vs. control
34
Part ?
Conclusion Two
Leucine deprivation represents a general effect
of BCAAs on regulation of insulin sensitivity
?
The effect of BCAAs deprivation differs in
glucose metabolism
?
The paper titled Effects of individual
branched-chain amino acids deprivation on insulin
sensitivity and glucose metabolism in mice was
published in Metabolism, 2014.
35
Part ?
Looking for new genes regulating insulin
sensitivity by leucine deprivation model (gene
chip)
36
Part ?
Hepatic Gene Chip of lecine-deprived male mice
(-)leu/con Gene number
Upregulate more than twice 984
Downregulate more than twice 1163
liver

800
600
Prolactin receptor (PRLR)
PrlR mRNA level
400
200
0
con
(-) leu
37
Part ?
Prolactin Receptor (PRLR)
38
Part ?
working model
Insulin resistance
Insulin sensitivity
Leucine deprivation
db/db


PRLR

GCN2/
mTOR/S6K
p-STAT5
p-IR / p-AKT
Insulin sensitivity
Normal condition
39
Part ?
Conclusion Three
Identify a novel function for hepatic PRLR in
the regulation of insulin sensitivity
?
Provide important insights in the nutritional
regulation of PRLR expression
?
The paper titled PRLR Regulates Hepatic Insulin
Sensitivity in Mice via STAT5 was published in
Diabetes, 623103-3113,2013.
40
Acknowledgments
Our lab Feifan Guo, Ph.D Chunxia Wang,
Ph.D Junjie Yu , Ph.D Shanghai Chen Kai Li Hao
Liu Yajie Guo Jiali Deng Yuzhong Xiao Yalan Deng
Previous members Ying Cheng, PhD Qian Zhang,
Ph.D Ying Du , Ph.D Tingting Xia, Ph.D Qingshu
Meng Zhiying Huang Bin Liu
Houkai Li, PhD Ziquan Li, Ph.D
Douglas cavener (Penn State Univ) Brad lowell
(Harvard Med School) Xiang Gao (Nanjing
Univ) Yong Liu (INS, CAS) Hongguang Sheng (CAS
clinical center)