Date post: | 15-Feb-2017 |
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Secretion and Regulation of ApoB48 by Primary Hamster Intestinal EnterocytesIntestinal Enterocytes
B C
Labe
led
Apo
B48
(% c
ontro
l)
Labe
led
Apo
B48
(% c
ontro
l)
0
20
40
60
80
100
120
Total Cells Media
ControlMG132
P < 0.05
0
20
40
60
80
100
120
Total Cells Media
ControlOleate
P = 0.01
Cells Media
0 45 90 45 90
A
Chase Time (min)
Cells Media
Fructose-Fed
Chow-Fed
Chase Time (Min) 0 45 90 45 90
D
Chase Time (Min) Chase Time (Min)
100
100
0
10
20
30
40
50
60
70
80
90
0 20 40 60 80
Total apoB 48
Labe
led
Apo
B 4
8( %
of
0 Ti
me)
Labe
led
Apo
B 4
8( %
of
0 Ti
me)
p= 0.003 p= 0.001
Secreted apoB 48
0
10
20
30
40
50
60
70
80
90
100
0 20 40 60 80 100
Chow-Fed
Fructose-Fed
E F
Ex vivo evidence for oversecretion of intestinal apoB48 & Enhanced intracellular apoB48 stability in fructose-
fed hamster enterocytes
In Vivo Production of TG & Intestinal ApoB48 in the Fasting State
Time (min)
55 60 65 70 75 80 85
S f >40
0 TG
con
cent
ratio
n (m
mol
/l)
0
1
2
3
4Sf >400 after Triton WR1339
Time (min)
55 60 65 70 75 80 85
S f >40
0 A
poB
48 c
once
ntra
tion
(g/
ml)
140
160
180
200
220
240
260
280
Time (min)
55 60 65 70 75 80 85
S f 100
-400
TG
con
cent
ratio
n (m
mol
/l)
0
1
2
3Sf 100-400 after Triton WR1339
Time (min)
55 60 65 70 75 80 85S f 100
-400
Apo
B48
con
cent
ratio
n(
g/m
l)
90
100
110
120
130
140
150
160
170
Trig
lyce
ride
secr
etio
n
0.0
0.2
0.4
0.6
0 1 2 3
ApoB
48 s
ecre
tion
0
4
8
12
16
Trig
lyce
ride
secr
etio
n
0.0
0.1
0.2
0.3
ApoB
48 s
ecre
tion
0
2
4
6p = 0.002 p = 0.01
p = 0.14 p = 0.59
A B
DC
In Vivo Production of TG & Intestinal ApoB48 in the Postprandial State
Time (min)55 60 65 70 75 80 85
Trig
lyce
ride
leve
l (m
mol
/l)
0.0
0.1
0.2
0.3
0.4
0.5
0.6
Time (min)55 60 65 70 75 80 85
Apo
B48
leve
l ( g
/ml)
80
90
100
110
120
130
140
150
Time (min)55 60 65 70 75 80 85
Trig
lyce
ride
leve
l (m
mol
/l)
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
Time (min)55 60 65 70 75 80 85
Apo
B48
leve
l ( g
/ml)
50
60
70
80
90
100Tr
igly
cerid
e se
cret
ion
( mol
/min
)
0.000.010.020.030.040.050.060.07
Col 48: 0.07 Col 48: 0.06
Trig
lyce
ride
secr
etio
n ( m
ol/m
in)
0.00
0.02
0.04
0.06
0.08
0.10
Col 50: 0.08 Col 50: 0.09
0 1 2 3
ApoB
48 s
ecre
tion
( g/m
in)
012345678
Col 52: 2.65 Col 52: 7.46
ApoB
48 s
ecre
tion
( g/m
in)
012345678
Col 54: 3.85 Col 54: 7.26
Sf 100-400 after Triton WR1339
Sf >400 after Triton WR1339
A B
C Dp = 0.3 p = 0.003
p = 0.8 p = 0.007
020406080
100120140160
Fructose-Fed
MTP
Pro
tein
Mas
s (%
of C
ontro
l)
Chow-Fed Fructose-Fed
0
20
40
60
80
100
120
0 0.5 1 1.5 2 2.5 3 3.5MTP inhibitor (M)
Apo
B 4
8 Se
cret
ion
(% o
f con
trol) Fructose-fed
Chow-fed
Chow-Fed
p=0.006D
E
Labe
led
Cho
lest
eryl
Est
er
(% o
f Con
trol)
p= 0.002
0
50
100
150
200
250
Cellular
p=0.001
Media
B
Labe
led
Cho
lest
erol
(% o
f Con
trol)
Chow-FedFructose-Fed
0100200300400500600700800
Cellular
p=0.0018
p=0.0013A
Media
Labe
led
Trig
lyce
ride
(% o
f Con
trol)
Cellular 020406080
100120140160180
p=0.04 p=0.003
Media
C
Ex Vivo Evidence for Intestinal Overproduction of Lipoprotein Lipids and Increase MTP mass/activity
Fructose-Fed Chow-fed
0
10
20
30
40
50
60
70
80
LargeCM
SmallCM
VLDL LDL HDL
p<0.05
p= 0.06
p<0.03
% o
f Tot
al L
abel
ed A
poB
48A
0
20
40
60
80
100
120
140
CM apoB48 Total-apoB48
Labe
led
Apo
B48
(%
of
Fruc
tose
-fed
)
p<0.0007p<0.01C
0
10
20
30
40
50
60
70
80
Particles< 1.006 Particles > 1.006
% o
f Tot
al L
abel
ed A
PoB
48
p<0.024
p<0.026B
0
40
20
60
80
100
120
CM apoB48 Total apoB48
Labe
led
Apo
B48
(%
of F
ruct
ose-
fed)
P=0.008 P=0.0001
DFasting Postprandial
Density/Size Distribution of Intestinal LipoproteinsEvidence for Increased Number & Size
Increased De Novo Lipogenesis in FF EnterocytesEvidence that ApoB48 Secretion is Linked to
the Rate of De Novo Lipogenesis
0102030405060708090
100110
0 2 4 6 8 10 12 14 16
CholesterolFatty AcidTriglyceride
Labe
led
Lipi
d (%
as c
ontro
l)
Cerulenin g/ml
0
50
100
150
200
250
300
350
Chow-fed Fructose-fed
Labe
led
Fatty
Aci
d (%
as c
ontro
l)
0102030405060708090
100110
0 2 4 6 8 10 12 14 16Cerulenin g/ml
Labe
led
Secr
eted
Apo
B-4
8 (%
as c
ontro
l)
A
C
p= 0.002
Sensitivity of ApoB48 Secretion to Cerulenin
Fatty AcidSynthesis
Sensitivity of TG & FA Secretion to Cerulenin
20
60
80
100
0
40
120
Chow-fed Fructose-fed(2 days)
0
20
40
60
80
100
120
Control Fructose(3mM )
0
200
400
600
800
1000
enterocytes hepatocytes
P < 0.01
Labe
led
Apo
B-4
8 (%
of c
ontro
l)
Labe
led
Apo
B-4
8 (%
of c
ontro
l)
Labe
led
chol
este
rol
050
100150200250300350400450
enterocytes hepatocytes
Labe
led
Trig
lyce
ride
P < 0.0001
Acute Fructose Feeding or Exposure Does NOT Affect Intestinal ApoB48 Secretion
[14C] FructoseIncorporation
into TG & Cholesterol
Two-day Fructose Feeding In Vitro Incubation
of Enterocytes with Fructose
Mechanisms of Intestinal Lipoprotein Overproduction in Insulin Resistant/
Hyperinsulinemic States
Apical Surface
Basolateral Surface
FA
monoglyceride
FABP-FAFABP
TG
TG
TG
ApoB48
PLCE
De Novo (FA)
Insulin (??)
TG dietary
SOS
Grb-2IRS
PI3-Kp110
SHP-2
NCK
PTKPTK
IR
PKB (Akt)
SREBP1c
FAS & ACC
Glucose+
FA
MTP
ERER
GolgiGolgi
Intestinal Enterocyte
TG
ApoB48 Lipoprotein Particles
p85 ProteasomeDegradation
AcknowledgementsLaboratory Group:Changiz Taghibiglou Mehran Haidari Steven Van IderstineWei QuiTaryne ChongFarhana MahboobBiao ChenLeyla MangalogluLouisa PontrelliFariborz RashidRita KohenDebbie Rudy
Collaborators:Gary Lewis- TorontoAndre Carpentier- SherbrookeSven Olof Olofsson - SwedenJanet Sparks - Rochester, NYRaphael Cheung – WindsorMichel Tremblay – MontrealDenny Trinh - Toronto
Funding:Heart & Stroke Foundation of OntarioCanadian Institutes of Health ResearchNSERC Hospital for Sick Children Merck-Frosst pfizerGlaxoSmithKline BBDC, University of Toronto