SHAPE Society

1. Secretion and Regulation of ApoB48 by
Primary Hamster Intestinal EnterocytesIntestinal Enterocytes
B C
LabeledApoB48(%control)
LabeledApoB48(%control)
0
20
40
60
80
100
120
Total Cells Media
Control
MG132
P < 0.05
0
20
40
60
80
100
120
Total Cells Media
Control
Oleate
P = 0.01
Cells Media
0 45 90 45 90
A
Chase Time (min)

2. 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
LabeledApoB48(%of0Time)
LabeledApoB48(%of0Time)
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

3. In Vivo Production of TG & Intestinal ApoB48
in the Fasting State
Time (min)
55 60 65 70 75 80 85
Sf>400TGconcentration
(mmol/l)
0
1
2
3
4
Sf >400 after Triton WR1339
Time (min)
55 60 65 70 75 80 85
Sf>400ApoB48concentration
(µg/ml)
140
160
180
200
220
240
260
280
Time (min)
55 60 65 70 75 80 85
Sf100-400TGconcentration
(mmol/l)
0
1
2
3
Sf 100-400 after Triton WR1339
Time (min)
55 60 65 70 75 80 85
Sf100-400ApoB48concentration
(µg/ml)
90
100
110
120
130
140
150
160
170
Triglyceridesecretion
0.0
0.2
0.4
0.6
0 1 2 3
ApoB48secretion
0
4
8
12
16
Triglyceridesecretion
0.0
0.1
0.2
0.3
ApoB48secretion
0
2
4
6p = 0.002 p = 0.01
p = 0.14
p = 0.59
A B
DC

4. In Vivo Production of TG & Intestinal ApoB48
in the Postprandial State
Time (min)
55 60 65 70 75 80 85
Triglyceridelevel(mmol/l)
0.0
0.1
0.2
0.3
0.4
0.5
0.6
Time (min)
55 60 65 70 75 80 85
ApoB48level(µg/ml)
80
90
100
110
120
130
140
150
Time (min)
55 60 65 70 75 80 85
Triglyceridelevel(mmol/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
ApoB48level(µg/ml)
50
60
70
80
90
100
Triglyceridesecretion(µmol/min)
0.00
0.01
0.02
0.03
0.04
0.05
0.06
0.07
Col 48: 0.07
Col 48: 0.06
Triglyceridesecretion(µmol/min)
0.00
0.02
0.04
0.06
0.08
0.10
Col 50: 0.08
Col 50: 0.09
0 1 2 3
ApoB48secretion(µg/min)
0
1
2
3
4
5
6
7
8
Col 52: 2.65
Col 52: 7.46
ApoB48secretion(µg/min)
0
1
2
3
4
5
6
7
8
Col 54: 3.85
Col 54: 7.26
Sf 100-400 after Triton WR1339
Sf >400 after Triton WR1339
A B
C D
p = 0.3 p = 0.003
p = 0.8 p = 0.007

5. 0
20
40
60
80
100
120
140
160
Fructose-Fed
MTPProteinMass
(%ofControl)
Chow-Fed Fructose-Fed
0
20
40
60
80
100
120
0 0.5 1 1.5 2 2.5 3 3.5
MTP inhibitor (µM)
ApoB48Secretion(%ofcontrol)
Fructose-fed
Chow-fed
∗
∗ ∗ ∗
Chow-Fed
p=0.006D
E
LabeledCholesterylEster
(%ofControl)
p= 0.002
0
50
100
150
200
250
Cellular
p=0.001
Media
B
LabeledCholesterol
(%ofControl)
Chow-Fed
Fructose-Fed
0
100
200
300
400
500
600
700
800
Cellular
p=0.0018
p=0.0013A
Media
LabeledTriglyceride
(%ofControl)
Cellular
0
20
40
60
80
100
120
140
160
180
p=0.04
p=0.003
Media
C
Ex Vivo Evidence for Intestinal Overproduction of
Lipoprotein Lipids and Increase MTP mass/activity

6. Fructose-Fed
Chow-fed
0
10
20
30
40
50
60
70
80
Large
CM
Small
CM
VLDL LDL HDL
p<0.05
p= 0.06
p<0.03
%ofTotalLabeledApoB48 A
0
20
40
60
80
100
120
140
CM apoB48 Total-apoB48
LabeledApoB48
(%ofFructose-fed)
p<0.0007
p<0.01C
0
10
20
30
40
50
60
70
80
Particles< 1.006 Particles > 1.006
%ofTotalLabeledAPoB48
p<0.024
p<0.026
B
0
40
20
60
80
100
120
CM apoB48 Total apoB48
LabeledApoB48
(%ofFructose-fed)
P=0.008 P=0.0001
DFasting Postprandial
Density/Size Distribution of Intestinal Lipoproteins
Evidence for Increased Number & Size

7. Increased De Novo Lipogenesis in FF Enterocytes
Evidence that ApoB48 Secretion is Linked to
the Rate of De Novo Lipogenesis
0
10
20
30
40
50
60
70
80
90
100
110
0 2 4 6 8 10 12 14 16
Cholesterol
Fatty Acid
Triglyceride
LabeledLipid(%ascontrol)
Cerulenin µg/ml
0
50
100
150
200
250
300
350
Chow-fed Fructose-fed
LabeledFattyAcid(%ascontrol)
0
10
20
30
40
50
60
70
80
90
100
110
0 2 4 6 8 10 12 14 16
Cerulenin µg/ml
LabeledSecretedApoB-48(%ascontrol)
A
C
p= 0.002
Sensitivity of ApoB48 Secretion
to Cerulenin
Fatty Acid
Synthesis
Sensitivity of TG & FA Secretion
to Cerulenin

8. 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
LabeledApoB-48(%ofcontrol)
LabeledApoB-48(%ofcontrol)
Labeledcholesterol
0
50
100
150
200
250
300
350
400
450
enterocytes hepatocytes
LabeledTriglyceride
P < 0.0001
Acute Fructose Feeding or Exposure Does NOT Affect
Intestinal ApoB48 Secretion
[14
C] Fructose
Incorporation
into TG &
Cholesterol
Two-day Fructose
Feeding In Vitro Incubation
of Enterocytes with
Fructose

9. Mechanisms of Intestinal Lipoprotein
Overproduction in Insulin Resistant/
Hyperinsulinemic States
Apical Surface
Basolateral Surface
FA
monoglyceride
FABP-FA
FABP
TG
TG
TG
ApoB48
PL
CE
De Novo (FA)
Insulin (??)
TGdietary
SOS
Grb-2
IRS
PI3-K
p110
SHP-2
NCK
PTKPTK
β β
α α
IR
PKB (Akt)
SREBP1c
FAS & ACC
Glucose
+
FA
MTP
ERER
GolgiGolgi
Intestinal
Enterocyte
TG
ApoB48 Lipoprotein
Particles
p85Proteasome
Degradation

10. Acknowledgements
Laboratory Group:
Changiz Taghibiglou
Mehran Haidari
Steven Van Iderstine
Wei Qui
Taryne Chong
Farhana Mahboob
Biao Chen
Leyla Mangaloglu
Louisa Pontrelli
Fariborz Rashid
Rita Kohen
Debbie Rudy
Collaborators:
Gary Lewis- Toronto
Andre Carpentier- Sherbrooke
Sven Olof Olofsson - Sweden
Janet Sparks - Rochester, NY
Raphael Cheung – Windsor
Michel Tremblay – Montreal
Denny Trinh - Toronto
Funding:
Heart & Stroke Foundation of Ontario
Canadian Institutes of Health Research
NSERC Hospital for Sick Children
Merck-Frosst pfizer
GlaxoSmithKline
BBDC, University of Toronto