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Secretion and Regulation of ApoB48 by Primary Hamster Intestinal Enterocytes - PowerPoint PPT Presentation


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

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120

120

100

100

80

80

60

60

40

40

20

20

0

0

Secretion and Regulation of ApoB48 by

Primary Hamster Intestinal Enterocytes

Media

A

Cells

Chase Time (min)

45

90

0

45

90

B

C

P < 0.05

P = 0.01

Control

Control

MG132

Oleate

Labeled ApoB48 (% control)

Labeled ApoB48 (% control)

Media

Total

Cells

Total

Cells

Media


100

90

80

70

60

50

40

30

20

10

0

Ex vivo evidence for oversecretion of intestinal apoB48

& Enhanced intracellular apoB48 stability in fructose-fed hamster enterocytes

Media

Cells

D

Fructose-Fed

Chow-Fed

Chase Time (Min)

0

45

90

45

90

E

F

Total apoB 48

Secreted apoB 48

100

90

80

Labeled ApoB 48( % of 0 Time)

70

p= 0.003

Labeled ApoB 48( % of 0 Time)

p= 0.001

60

Chow-Fed

50

Fructose-Fed

40

30

20

10

0

0

20

40

60

80

0

20

40

60

80

100

100

Chase Time (Min)

Chase Time (Min)




180

160

140

120

120

100

100

80

80

60

60

40

40

20

20

0

0

Ex Vivo Evidence for Intestinal Overproduction of Lipoprotein Lipids and Increase MTP mass/activity

p=0.04

p=0.0013

Labeled Cholesterol

(% of Control)

A

C

B

p=0.003

800

Labeled Triglyceride

(% of Control)

Labeled Cholesteryl Ester (% of Control)

p= 0.002

250

p=0.001

700

Chow-Fed

200

600

Fructose-Fed

500

p=0.0018

150

400

300

100

200

50

100

0

0

Media

Media

Media

Cellular

Cellular

Cellular

E

Fructose-fed

Chow-fed

Chow-Fed

Fructose-Fed

D

p=0.006

160

ApoB 48 Secretion (% of control)

140

120

100

MTP Protein Mass

(% of Control)

80

60

40

20

0

0.5

1

1.5

2

2.5

3

3.5

0

MTP inhibitor (M)

Chow-Fed

Fructose-Fed


80

70

60

50

40

120

30

20

100

10

80

0

60

20

Density/Size Distribution of Intestinal Lipoproteins

Evidence for Increased Number & Size

A

B

Chow-fed

p= 0.06

80

p<0.026

Fructose-Fed

70

60

50

p<0.024

% of Total Labeled APoB48

40

% of Total Labeled ApoB48

p<0.05

30

20

p<0.03

10

0

Large

CM

Small

CM

VLDL

LDL

HDL

Particles< 1.006

Particles > 1.006

140

Fasting

Postprandial

C

D

p<0.01

120

p<0.0007

P=0.0001

P=0.008

100

80

Labeled ApoB48

(% of Fructose-fed)

Labeled ApoB48

(% of Fructose-fed)

60

40

40

20

0

0

Total apoB48

CM apoB48

Total-apoB48

CM apoB48


110

100

90

110

80

100

70

90

60

80

50

70

40

60

30

50

20

40

10

30

0

20

10

0

350

300

250

200

150

100

50

0

Increased De Novo Lipogenesis in FF Enterocytes

Evidence that ApoB48 Secretion is Linked to

the Rate of De Novo Lipogenesis

A

p= 0.002

Fatty Acid

Synthesis

Labeled Fatty Acid (% as control)

Labeled Lipid (% as control)

Sensitivity of TG & FA Secretion

to Cerulenin

Cholesterol

Fatty Acid

Triglyceride

0

2

4

6

8

10

12

14

16

Chow-fed

Fructose-fed

Cerulenin g/ml

C

Labeled Secreted ApoB-48 (% as control)

Sensitivity of ApoB48 Secretion

to Cerulenin

0

2

4

6

8

10

12

14

16

Cerulenin g/ml


1000

800

600

400

200

0

Acute Fructose Feeding or Exposure Does NOT Affect Intestinal ApoB48 Secretion

Two-day Fructose Feeding

In Vitro Incubation of Enterocytes with Fructose

120

120

100

100

80

80

Labeled ApoB-48 (% of control)

Labeled ApoB-48 (% of control)

60

60

40

40

20

20

0

0

Chow-fed

Fructose-fed

(2 days)

Control

Fructose

(3mM )

450

P < 0.01

P < 0.0001

400

350

[14C] Fructose

Incorporation

into TG & Cholesterol

300

Labeled cholesterol

Labeled Triglyceride

250

200

150

100

50

0

hepatocytes

enterocytes

hepatocytes

enterocytes


p85

b

b

IR

a

a

Mechanisms of Intestinal Lipoprotein

Overproduction in Insulin Resistant/

Hyperinsulinemic States

TG dietary

monoglyceride

Apical Surface

Intestinal

Enterocyte

FA

De Novo (FA)

FABP

TG

Glucose

+

FABP-FA

FA

ER

FAS & ACC

TG

TG

SREBP1c

MTP

PL

Golgi

PKB (Akt)

TG

CE

PI3-K

SOS

ApoB48

IRS

Proteasome

p110

Grb-2

Degradation

SHP-2

NCK

Basolateral Surface

PTK

PTK

ApoB48 Lipoprotein

Particles

Insulin (??)


Acknowledgements
Acknowledgements

Collaborators:Gary Lewis- TorontoAndre Carpentier- SherbrookeSven Olof Olofsson - Sweden

Janet Sparks - Rochester, NY

Raphael Cheung – Windsor

Michel Tremblay – Montreal

Denny Trinh - Toronto

Laboratory Group:

Changiz Taghibiglou Mehran Haidari Steven Van Iderstine

Wei QuiTaryne Chong

Farhana Mahboob

Biao ChenLeyla MangalogluLouisa Pontrelli

Fariborz Rashid

Rita Kohen

Debbie Rudy

Funding:

Heart & Stroke Foundation of Ontario

Canadian Institutes of Health Research

NSERC Hospital for Sick Children Merck-Frosst pfizer

GlaxoSmithKline

BBDC, University of Toronto


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