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