Post on 29-Dec-2015
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