Mechanisms for Improved Insulin Sensitivity byBranched-chain Amino Acids Deprivation

Reporter : Fei Xiao, PhDReporter : Fei Xiao, PhD

Shanghai Institute for Nutritional Sciences, Chinese Academia of Sciences

Prevalence of diabetes

Background

Insulin signaling

Nature. 2001, 414:799-806

Insulin Receptor,

IR

Insulin Receptor

Substrate, IRS

AKT

Insulin resistance:

a physiological condition where insulin becomes less effective at lowering blood glucose

a common feature of many metabolic diseases

Background

The DPP Research Group, NEJM,2002

Background

Various strategies to treat insulin resistance

Background

Branched-chain amino acids (BCAAs)

L-leucine L-isoleucine L-valine

The role of leucine in insulin sensitivity

Increased serum leucine level:

* Improves the whole body glucose metabolism

* Does not alter susceptibility to diet-Induced obesity

J. Nutr. 139: 715–719, 2009.

* Increases insulin resistance in models of obesity

Cell Metab 9:311-326, 2009

Diabetes 54:2674-2684, 2005

Diabetes 56:1647-1654, 2007

？

Metabolic diseases(insulin sensitivity/glucose

metabolism)

Nutrient(BCAAs)

Genetic factors

sensing

Metabolism regulationX

Research interest of our work

Outline

Part The role of leucine deprivation in insulin sensitivityⅠ

Part Effects of individual BCAAs on insulin sensitivity Ⅱ and glucose metabolism in mice

Part Looking for new genes regulating insulin Ⅲ sensitivity by leucine deprivation model

The role of leucine deprivation in insulin sensitivity

Part Ⅰ

Leucine deprivation

Fat mass Lipolysis in WAT

UCP1 in BAT

CNS food intake

Serum Insulin Blood Glucose Normal

In Our Lab

Cell Metab 5:103-114, 2007 Diabetes 59:17-25

？

Part Ⅰ

HypothesisHypothesis

Leucine deprivation may improve insulin sensitivity

Part Ⅰ

Experimental DesignExperimental Design

control

85% control

(-)leu

control

0d

Collect tissuesChange different dietsMice were acclimated to control diet for 7 days

ControlControl (-)leu(-)leu Pair-fedPair-fed

7d 14d

Part Ⅰ

Blo

od

Glu

co

se

(m

g/d

l)Result 1: (-) leu improves insulin sensitivity in vivo

0

50

100

150

200

#*

Se

rum

Ins

ulin

(n

g/m

l)

0

0.2

0.4

0.6

#*

ctrl (-) leu pf

HO

MA

-IR

0.5

1

1.5

0

#*

Fasting Fasting

*: p<0.05 vs. control #: p<0.05 vs. pf

Part Ⅰ

Result 2: (-) leu improves insulin sensitivity in vivo

0 20 40 60 80 100120

Time (min)

ITT

Blo

od

Glu

cose

(m

g/d

l)

Time (min)

0

100

200

0 20 40 60 80 100120

400

500

300

0

50

100

150

200

Blo

od

Glu

cose

(m

g/d

l)GTT

**

#

# **

ctrl (-) leu pf

*#

##

**

*#

*: p<0.05 vs. control #: p<0.05 vs. pf

Part Ⅰ

Result 3: (-) leu improves insulin sensitivity in vivo

- + - +

p-IR

t-IR

p-AKT

t-AKT

ctrlIns

Liver WAT Muscle

Arb

itra

ry U

nit

s * *

100

200

00

p-IR p-AKT

100

200

p-IR p-AKT p-IR p-AKT

300

0

100

300

ctrl + Ins (-) leu + Ins

*

*200

* *

(-) leu

- + - +

ctrl (-) leu

- + - +

ctrl (-) leu

*: p<0.05 vs. control

Part Ⅰ

Results 4 :(-) leu improves insulin sensitivity under insulin-resistance conditions

C

HFD(-) leu HFD

Blo

od

Glu

cose

(m

g/d

l)

0

50

100

200

250

150

Time (min)B

loo

d G

luco

se (

mg

/dl)

0

200

400

600

0Time (min)

ctrl db/db (-) leu db/db

ctrl WT

40 80 12020 60 100

ITT

* *

**

# # #

ITT

0 40 80 12020 60 100

*

**

*

ctrl

**

*

** *

*: p<0.05 vs. HFD#: p<0.05 vs. ctrl

*: p<0.05 vs. ctrl db/db#: p<0.05 vs. ctrl WT

* *

Part Ⅰ

Summary 1

leucine deprivation improves insulin sensitivity under normal and insulin-resistant conditon

Mechanisms ?

Part Ⅰ

Nature 431:200-205, 2004

Role of mTOR/S6K1 signaling in insulin sensitivity

mTOR ： mammalian target of rapamycin S6K1 ： ribosomal protein S6 kinase 1

Part Ⅰ

Results 5 :(-) leu increases insulin sensitivity by decreasing mTOR/S6K1 signaling in vivo

Liver

p-S6K1

t-S6K1

p-S6

t-S6

p-mTOR

t-mTOR

ctrl (-) leu

0

50

100

150

200

Blo

od

Glu

cose

(m

g/d

l)

0 40 80 120

Time (min)

ITT

20 60 100

+ Ad-CA-S6K1

**

*

- Ad-CA-S6K1

*: p<0.05 vs. control

On a leucine-deficient dietA B

Part Ⅰ

GCN2

General control nonderepressible (GCN)2

A serine protein kinase

Function as a sensor for amino acid deprivation

●

●

●

Leucine deprivation

p-IR/p-IRS/p-AKT

p-mTOR /p-S6K1 GCN2

?

Part Ⅰ

Results 6:(-) leu increases insulin sensitivity by activation of GCN2

p-GCN2

t-GCN2

Liver

Arb

itra

ry U

nit

s

0

100

200 *

ctrl (-) leu

0 40 80 120

Time (min)

20 60 1000

50

100

150

Blo

od

Glu

cose

(m

g/d

l) Gcn2-/-

ITT

Gcn2+/+

* **

*: p<0.05 vs. control

Part Ⅰ

AMPK

AMP-activated protein kinase

Energy sensor

●

●

Target of many drugs●

Leucine deprivation

p-IR/p-IRS/p-AKT

p-mTOR /p-S6K1

GCN2

AMPK

?

Part Ⅰ

Results 7 :(-)leu improves insulin sensitivity via activation of AMPK

DN-AMPKIns

p-ACC

- -- + - +

+ +

p-IR

t-AMPK

t-IR

t-ACC

t-IRS1

p-IRS1(Tyr612)

HepG2

p-AKT

t-AKT0

+ DN-AMPK + Ins

100

Arb

itra

ry U

nit

s

50

150

**

*

p-IR p-IRS1 p-Akt

HepG2

+leu -leu

*

p-AMPK

t-AMPK

Liver

Arb

itra

ry U

nit

s

0

100

200

*

ctrl (-) leu - DN-AMPK + Ins

*: p<0.05 vs. control

Part Ⅰ

Summary2

Leucine deprivation increases hepatic insulin sensitivity via GCN2/mTOR/S6K1 and AMPK pathways

model

Leucine deprivation

p-IR/p-IRS/p-AKT

p-mTOR/p-S6K1

p-GCN2 p-AMPK

Part Ⅰ

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

●

●

Part Ⅰ

The paper titled “leucine deprivation increases hepatic insulin sensitivity via GCN2/mTOR/S6K1And AMPK pathways” was published in Diabetes, 60:746-756,2011.

L-isoleucine (ile)

L-valine (val)

How about the other two branched-chain amino acids ?

Effects of individual branched-chain amino acids on insulin sensitivity and glucose metabolism in mice

Part Ⅱ

Results 8: (-)val and (-)ile improves insulin sensitivity in vivo

200

150

100

50

Time (min)

0Blo

od

Glu

cose

(m

g/d

l)

0 40 80 120

ITT

**

* *

A Bctrl (-) val ctrl (-) ile

Blo

od

Glu

cose

(m

g/d

l)

Time (min)

0

100

200

0 40 80 120

400

500

300

GTT

*

***

0

100

200

400

500

300

Blo

od

Glu

cose

(m

g/d

l)

Time (min)

0 40 80 120

GTT

*

***

0

50

100

150

200

Blo

od

Glu

cose

(m

g/d

l)

Time (min)

0 40 80 120

****

ITT

*

*

Part Ⅱ

Result 9: (-)val and (-)ile decrease mTOR/S6K1 and increase AMPK signaling

Liverctrl (-) Val

p-S6K1

t-S6K1

p-S6

t-S6

p-mTOR

t-mTOR

200

100

0

Liverctrl (-) Ile

p-S6K1

t-S6K1

p-S6

t-S6

p-mTOR

t-mTOR

Arb

itra

ry U

nit

s

Arb

itra

ry U

nit

s

* ** * * *

200

100

0

p-AMPK

t-AMPK

p-AMPK

t-AMPK

*

p-s6k

1

p-mTOR

p-s6

p-AM

PK

*

p-s6k

1

p-mTOR

p-s6

p-AM

PK

Part Ⅱ

150

100

50

0

* *

Blo

od

Glu

cose

(m

g/d

l)

0.4

0.2

0Ser

um

Insu

lin (

ng

/ml)

* *0.5

1

HO

MA

-IR

0

* *

ctrl (-) val (-) ile

Fasting Fasting

Results 10: Effects of individual BCAA deficiency on glucose metabolism

*: p<0.05 vs. control

Part Ⅱ

200

150

100

50

0

* *

Blo

od

Glu

cose

(m

g/d

l) 1.5

1

0.5

Ser

um

Insu

lin (

ng

/ml)

0

* *

g6pase0

100

200

Rel

ativ

e m

RN

A (

%)

Fed Fed

pepck

**

**

Results 11: Effects of individual BCAA deficiency on glucose metabolism

ctrl (-) val (-) ile（ - ） leu

*: p<0.05 vs. control

Part Ⅱ

Results 12: BCAA deprivation for 1 day improves whole body insulin sensitivity

200

Blo

od

Glu

cose

(m

g/d

l)

100

0

** *

*

200B

loo

d G

luco

se (

mg

/dl)

100

0

*

** *

200

Blo

od

Glu

cose

(m

g/d

l)

100

0

*

**

*

Time (min)0 40 80 120

Time (min)0 40 80 120

Time (min)0 40 80 120

(-) leu (-) val (-) ile

ctrl (-) BCAA

*: p<0.05 vs. control

Part Ⅱ

Conclusion Two

Leucine deprivation represents a general effect of BCAAs on regulation of insulin sensitivity

●

● The effect of BCAAs deprivation differs in glucose metabolism

Part Ⅱ

The paper titled “Effects of individual branched-chain amino acids deprivation on insulin sensitivity and glucose metabolism in mice ” was published in Metabolism, 2014.

Looking for new genes regulating insulin sensitivity by leucine deprivation model (gene chip)

Part Ⅲ

Hepatic Gene Chip of lecine-deprived male mice

0200400

600

800

Prl

R m

RN

A le

vel

*

liver

con (-) leu

（（ --）） leu/conleu/con Gene Gene numbernumber

Upregulate more than Upregulate more than twice twice

984984

Downregulate more than Downregulate more than twicetwice

11631163

Prolactin receptor (PRLR)

Part Ⅲ

Prolactin Receptor (PRLR)

Prolactin ： A hormone best known for its role in lactation.

PRLR: Present in nearly all organs and tissues. Numerous biological functions of PRLR have been identified. (Regulates glucose levels by modulating the secretion of insulin）

a direct effect on insulin sensitivity

？

Part Ⅲ

working model

PRLR

p-STAT5

p-IR / p-AKT

db/db

Insulin sensitivity

Normal condition

Insulin resistance Insulin sensitivity

Leucine deprivation

GCN2/ mTOR/S6K

Part Ⅲ

Conclusion ThreeIdentify a novel function for hepatic PRLR in the regulation of insulin sensitivity

Provide important insights in the nutritional regulation of PRLR expression

●

●

Part Ⅲ

The paper titled “PRLR Regulates Hepatic Insulin Sensitivity in Mice via STAT5” was published in Diabetes, 62:3103-3113,2013.

Our lab:Feifan Guo, Ph.DChunxia Wang, Ph.DJunjie Yu , Ph.DShanghai ChenKai LiHao LiuYajie GuoJiali DengYuzhong XiaoYalan Deng

Acknowledgments

Previous members: Ying Cheng, PhDQian Zhang, Ph.DYing Du , Ph.DTingting Xia, Ph.DQingshu MengZhiying HuangBin Liu

Douglas cavener (Penn State Univ) Brad lowell (Harvard Med School)Xiang Gao (Nanjing Univ)Yong Liu (INS, CAS) Hongguang Sheng (CAS clinical center)

Houkai Li, PhD Ziquan Li, Ph.D