Caloric Restriction and Resveratrol Reverse Caloric Restriction and Resveratrol Reverse a High-fat Diet Induced Diabetes and a High-fat Diet Induced Diabetes and

Improve Islet Improve Islet β β cell Dysfunction in Micecell Dysfunction in Mice

Jiaoyue Zhang Ph.DJiaoyue Zhang Ph.D

Endocrinology Endocrinology

Department,Union Department,Union

hospital, Huazhong hospital, Huazhong

University of Science & University of Science &

Technology, Wuhan 430022, Technology, Wuhan 430022,

ChinaChina

Caloric Restriction and ResveratrolCaloric Restriction and Resveratrol Calorie restriction (CR), Calorie restriction (CR), based on low calorie intake, is basic for therapy of is basic for therapy of

diabetesdiabetes

SIRT1 is the NADSIRT1 is the NAD++-dependent deacetylase, and controls the activities of genes -dependent deacetylase, and controls the activities of genes

that regulate circadian rhythm, apoptosis.that regulate circadian rhythm, apoptosis.

Resveratrol (Res), a type of natural phenol, is the potent natural SIRT1 Resveratrol (Res), a type of natural phenol, is the potent natural SIRT1

activator. It has been found to lower plasma glucose in streptozotocin-induced activator. It has been found to lower plasma glucose in streptozotocin-induced

diabetic rats.diabetic rats.

SIRT1 is involved in the activities of SIRT1 is involved in the activities of ββ cellcell

SIRT1 was preferentially expressed in pancreatic beta cells SIRT1 was preferentially expressed in pancreatic beta cells while not in exocrine cellswhile not in exocrine cells

In beta cell-specific Sirt1-overexpressing (BESTO) transgenic In beta cell-specific Sirt1-overexpressing (BESTO) transgenic micemice ，， GSIS GSIS ↑↑；；

SIRT1 reduced by siRNA in beta cel lines (INS, SIRT1 reduced by siRNA in beta cel lines (INS, MIN6) MIN6) ，， GSISGSIS↓↓

Our hypothesisOur hypothesis ：： SIRT1 could possibly play an important SIRT1 could possibly play an important role in the effect of CR and Res on beta cellrole in the effect of CR and Res on beta cell

Bordone L, et al. PLoS Biol, 2006,4:e31Moynihan KA, et al. Cell Metab, 2005,2:105-17

Aim of the researchAim of the research

Explore the anti-hyperglycaemic role and Explore the anti-hyperglycaemic role and

mechanisms of CRmechanisms of CR and Res on beta cell protectionand Res on beta cell protection

male C57BL/6J micemale C57BL/6J mice

control

model

standard chow ×24w

Normal controlNormal control

High fat diet High fat diet

Large dosage of ResLarge dosage of Res

Low dosage of Res Low dosage of Res

Caloric restrictionCaloric restriction

NC

HF

CR

R400

R50

15

65 High fat diet×8w

standard chow +60%CR

a high fat diet ×16w

a high fat diet +Res400mg/kg/d

a high fat diet +Res50mg/kg/d

IPGTT Caloric intake 、 BW 、 lipid

IPGTT 、 insulin 、 ITT

15只

Obesity index＝（ total fat weight ） /body weight×100

Compared to NC, *P<0.05 ， #P<0.01 ； compared to HF, △ P<0.05，▲ P<0.01

05

1015202530354045

0 2 4 6 8 10 12 14 16 18 20 22 24 (w)

Bo

dy

weig

ht

(g)

NC HF CR R400 R50

0

5

10

15

20

25

30

1 3 5 7 9 11 13 15 17 19 21 23 (w)

Kca

l co

nsu

med

NC HF CR R400 R50

0

0.5

1

1.5

2

2.5

NC HF CR R400 R50

FF

A(m

eq

/L)

.

Caloric intake Body weight

0

4

8

12

NC HF CR R400 R50

Ad

ipo

sity

In

dex

.

Blood FFA Obesity index#

#▲#▲

*▲

#▲

#▲# #

0

4

8

12

NC HF

FB

G (

mm

ol/L

)

0

4

8

12

NC HF CR R400 R50

FB

G (

mm

ol/

L)

0

1000

2000

3000

NC HF

AU

C (

Mm

*m

in)

0

5

10

15

20

25

0 15 30 45 60 75 90 105 120 (min)

glu

cose

(m

mo

l/L

)

NC HF

0

5

10

15

20

25

0 15 30 45 60 75 90 105 120(min)

glu

cose

(m

mol/L

)

NC HF CR R400 R50

# *

0

1000

2000

3000

NC HF CR R400 R50

AU

C (

Mm

*m

in)

▲

#▲

#▲

8w

24w

IPGTT curveFBG AUC

#

▲ *△▲

-0.2

-0.1

0

NC HF CR R400 R50

Ki

0

0.5

1

1.5

2

NC HF CR R400 R50

HO

MA

-IR

2

4

6

8

10

12

0 15 30 (min)

Glu

co

se (

mm

ol/

L)

Fasting blood insulin HOMA-IR

Insulin tolerance (ITT) Ki

0

0.5

1

NC HF CR R400 R50

insu

lin

(n

g/m

l)

▲

#▲

#▲#

#

#▲

#▲#▲

#

▲

#▲

#▲

Islet morphologyIslet morphology

NC R400CR R50HF

NC R400CR R50HFInsulin protein expressions by immunohistochemistry (×400)

HE stainig (×200)

0

4

8

12

16

20

NC HF CR R400 R50

Pan

creati

c in

sulin

con

ten

t (n

g/m

g)

.

0

0.5

1

1.5

2

2.5

3

NC HF CR R400 R50

β-c

ell

mass

(m

g)

Β cell mass Insulin content in pancreas

▲

#△

*▲

#

△△

ΔI30/ΔG30 Insulin stimulatory index

00.5

11.5

22.5

33.5

NC HF CR R400 R50

Sti

mu

lato

ry

In

dex

.

0

50

100

NC HF CR R400 R50

ΔI3

0/Δ

G3

0 (

ng

/mm

ol)

.

*

△

#▲

#▲

#

#▲*▲▲

A B C

NC HF

CR R400 R50

Apoptosis by TUNEL (×200)

SIRT1 mRNA SIRT1 protein

Marker NC HF CR R400 R50

0

0.5

1

1.5

2

2.5

NC HF CR R400 R50

Rel

ati

ve

am

ou

nt

of

SIR

T1 p

rote

in .

0

0.2

0.4

0.6

NC HF CR R400 R50

Rel

ati

ve

am

ou

nt

of

SIR

T1

mR

NA

0

1

2

3

4

NC HF CR R400 R50

Rel

ati

ve

am

ou

nt

of

UC

P2 m

RN

A . UCP2 mRNA

SIRT1

Marker NC HF CR R400 R50

NC HF CR R400 R50

#

#▲

*

△

#

#▲

*

△

#

▲ ▲

#▲

UCP2SIRT1

0

1

2

3

4

5

6

NC HF CR R400 R50

Rela

tiv

e a

mou

nt

of

Ba

x m

RN

A

0

0.5

1

1.5

2

NC HF CR R400 R50

Rela

tive a

mou

nt

of

PD

X-1

mR

NA

.

PDX-1 mRNA Bax mRNA Bcl-2 mRNA

Marker NC HF CR R400 R50

PDX-1

Bcl-2

Marker NC HF CR R400 R50Marker NC HF CR R400 R50

GAPDH

GAPDH GAPDH

Bax

0

0.2

0.4

0.6

0.8

NC HF CR R400 R50Rela

tive a

mou

nt

of

Bcl-

2 m

RN

A .

#

#▲# #▲▲ ▲

▲ ▲

0

4

8

12

NC HF CR R400 R50

Pan

creati

c M

DA

con

ten

t .

MDA GSH-Px

0

4

8

12

NC HF CR R400 R50

Pa

ncrea

tic G

SH

-Px

co

nte

nt

.

#

▲▲

*▲#

▲#▲ #▲

Mitochondrial changes in isletsMitochondrial changes in islets（（ electron microscopy electron microscopy ））

NC HF

CR R400 R50

CR and Res administration rendered the animals resistant to CR and Res administration rendered the animals resistant to diet-induced obesity and insulin resistance, which was diet-induced obesity and insulin resistance, which was supported by previous studies. Importantly, the functions of supported by previous studies. Importantly, the functions of GSIS in vivo and in vitro were improved robustly in HR group GSIS in vivo and in vitro were improved robustly in HR group compared with NC and HF group in our experiment. compared with NC and HF group in our experiment.

Compared with CRCompared with CR ，， Res does not affect caloric intake and Res does not affect caloric intake and sorts of food, less weight loss, lower blood glucose and less sorts of food, less weight loss, lower blood glucose and less insulin sensitivity.insulin sensitivity.

The role of beta cell protectionThe role of beta cell protection Long term CR intervetion can inhibit compensary Long term CR intervetion can inhibit compensary

changes of islet changes of islet β β cell, and improve increased basal cell, and improve increased basal

insulin secretion and impaired GSIS. It might be insulin secretion and impaired GSIS. It might be

related to improved lipotoxicity and apoptosis.related to improved lipotoxicity and apoptosis. Res also has β cell protection, especially in the capacity of Res also has β cell protection, especially in the capacity of

insulin secretion. Res400mg/kg/dinsulin secretion. Res400mg/kg/d is more effective than is more effective than

50mg/kg/d.50mg/kg/d.

SIRT1 as a mediator of CRSIRT1 as a mediator of CR

NAD+/NADH SIRT1

热卡限制

NAD+/NADH

Visceral fat, liver, kidney and brain

SIRT1

Anti-aging

islet

UCP2 ATP

GSIS凋亡

Res increased SIRT1 expression on β cell only in higher Res increased SIRT1 expression on β cell only in higher dosage, but inhibited UCP2 in both groups. dosage, but inhibited UCP2 in both groups.

The above role is still unclear especially in different tissues.The above role is still unclear especially in different tissues. The impact on UCP2 of Res might induce increased insulin The impact on UCP2 of Res might induce increased insulin

secretion, and the role is independent on the expression of secretion, and the role is independent on the expression of SIRT1.SIRT1.

ResRes impact on SIRT1 pathwayimpact on SIRT1 pathway

Baur JA, et al. Nature, 2006, 444(7117):337-42

Lagouge M, et al.Cell, 2006, 127(6):1109-22

Both CR and Res can reverse diabetes induced by a high-Both CR and Res can reverse diabetes induced by a high-

fat diet. CR is more effective on insulin resistance. Res is fat diet. CR is more effective on insulin resistance. Res is

more potent on blood glucose while not affecting caloric more potent on blood glucose while not affecting caloric

intake and body weight.intake and body weight.

Both can improve beta cell dysfunction in secretion and Both can improve beta cell dysfunction in secretion and

morphology, and Res is more potent than CR.morphology, and Res is more potent than CR.

ConclusionsConclusions

SIRT1 pathway might mediate the protective roleSIRT1 pathway might mediate the protective role of CR and Res of CR and Res

on β cellon β cell ，， but the effects on SIRT1but the effects on SIRT1 and UCP2 expressions are and UCP2 expressions are

different.different.

CRCR and Res do not affect PDX-1 mRNAand Res do not affect PDX-1 mRNA expressionexpression ，， but might but might

improve Bcl-2/Baximprove Bcl-2/Bax imbalance and oxidative damageimbalance and oxidative damage ，， which which

induce improved mitochondrial damage and apoptosisinduce improved mitochondrial damage and apoptosis

ConclusionsConclusions