VLDL

ApoB mRNA Translation

Degradation

ER

M

embr

ane

5' 3'ApoB mRNA

VLDL Assembly

Degradation

Secretion

MTP

Proteasome

Hepatic Synthesis and Secretion of VLDLHepatic Synthesis and Secretion of VLDL

Lipid Poor State

Lipid Rich State

Lipid Poor State

ApoB Gene Expression

VLDL

PlasmaHepatocyte

CEPL

TGC

Mechanisms of VLDL Overproduction in Mechanisms of VLDL Overproduction in Insulin Resistance (Recent Progress)Insulin Resistance (Recent Progress)

• Development of a Fructose-Fed Hamster Model of Insulin Resistance

• Investigations into Mechanisms of Hepatic VLDL Overproduction

• Investigations into Mechanisms of Intestinal Lipoprotein Overproduction

• Assessment of the Efficacy of hypolipidemic agents and insulin sensitizers in ameliorating metabolic dyslipidemia

Insulin Resistance ModelFructose-Fed Syrian Golden Hamster

• Lipoprotein metabolism closely resembles that in humans

• Hamster liver secretes VLDL containing only apoB100 with a density close to that of human VLDL

• Hamsters develop hyperTG, hyperCHOL, & atherosclerosis in response to a modest increase in dietary cholesterol & saturated fat

• Hamster can be made Obese, Hypertriglyceridemic, Hyperinsulinemic, and Insulin Resistant by carbohydrate feeding (particularly Fructose)

Male Syrian Golden Hamsters (80-100 grams)Male Syrian Golden Hamsters (80-100 grams)

60% Fructose Diet60% Fructose Diet

(2 weeks)(2 weeks)

Control HamstersControl Hamsters

Control DietControl Diet

(2 weeks)(2 weeks)

Fructose-fed HamstersFructose-fed Hamsters

Plasma Analysis: Glucose, TG, Chol, InsulinPlasma Analysis: Glucose, TG, Chol, Insulin

Liver Perfusions >>>>>>Primary HepatocytesLiver Perfusions >>>>>>Primary HepatocytesIntestinal Fragments >>>>>>Primary EnterocytesIntestinal Fragments >>>>>>Primary Enterocytes

Experiments on Hepatic & Intestinal LipoproteinsExperiments on Hepatic & Intestinal Lipoproteins

Plasma Glucose, TG, Chol, InsulinPlasma Glucose, TG, Chol, Insulin

Insulin Resistance ModelFructose-Fed Syrian Golden Hamster

Evidence for Development of Insulin Resistance: Evidence for Development of Insulin Resistance: • Increased Plasma Insulin, FFA, TriglycerideIncreased Plasma Insulin, FFA, Triglyceride• Reduced whole body insulin sensitivity (based on Euglycemic-Reduced whole body insulin sensitivity (based on Euglycemic-

Hyperinsulinemic Clamp Studies)Hyperinsulinemic Clamp Studies)

Adeli K. et al. (2000) J. Biol. Chem. 275: 8416-8425.

Evidence for Development of Hepatic VLDL Overproduction:Evidence for Development of Hepatic VLDL Overproduction: • Enhanced hepatic VLDL secretion In Vivo (Triton method)Enhanced hepatic VLDL secretion In Vivo (Triton method)• Enhanced VLDL secretion by primary hamster hepatocytes Enhanced VLDL secretion by primary hamster hepatocytes ex vivoex vivo• Increased intracellular apoB stabilityIncreased intracellular apoB stability• Enhanced MTP expression (mRNA, protein, activity)Enhanced MTP expression (mRNA, protein, activity)

Insulin Resistance ModelFructose-Fed Syrian Golden Hamster

Hypothesis I:Hypothesis I:Insulin Resistance Induces Hepatic VLDL Overproduction

Published Data:

0

1

2

ControlFructose-Fed

Free

Fat

ty A

cids

(mm

ol/L

)p=0.0045

0

100

200

300 p=0.0110

Plas

ma

Insu

lin(m

mol

/L)

0.0

2.5

5.0

7.5

p=0.9452

Plas

ma

Glu

cose

(mm

ol/L

)

0

1

2

3

4

5 p=0.0309

Plas

ma

Tri

glyc

erid

e(m

mol

/L)

p= 0.0550

0.0

2.5

5.0

7.5

Plas

ma

Cho

lest

erol

(mm

ol/L

)

A B

C D E

Increased Plasma Triglyceride, FFA, Increased Plasma Triglyceride, FFA, & Insulin in Fructose-Fed Hamsters& Insulin in Fructose-Fed Hamsters

Glu

cose

(mm

ol/l)

0

1

2

3

4

5

6

Control (n=10)Fructose fed (n=9)

Insu

lin (p

mol

/l)

0

500

1000

1500

2000

2500

3000

Gin

f (m

ol.k

g-1 .min-1

)

0

10

20

30

40

50

60

S I (1

06 l2 .kg-1

.min-1

)

0

1

2

3

4

5

6

p < 0.01

p < 0.01

p = ns

p = 0.03

A B

C D

In Vivo Evidence of Insulin ResistanceIn Vivo Evidence of Insulin Resistance(Euglycemic-hyperinsulinemic Clamp)(Euglycemic-hyperinsulinemic Clamp)

Reduced Insulin Sensitivity in Fructose-Fed HamstersReduced Insulin Sensitivity in Fructose-Fed Hamsters

Enhanced Hepatic VLDL-apoB100 Secretion Enhanced Hepatic VLDL-apoB100 Secretion in Fructose-Fed Hamsters in Fructose-Fed Hamsters

(In Vivo Triton WR 1339 Studies)

Time (min)0 20 40 60 80 100

VLD

L-ap

oB (

g/m

l)

100

150

200

250

300

350

400

VLD

L-ap

oB s

ecre

tion

(g/

min

)

0

2

4

6

8

10

12

*

Control

Fructose fed

0

100

200

300

400

500

Fructose-FedControl

VL

DL

apo

B S

ecre

ted

(% o

f con

trol

)ApoB100

*

Overproduction of VLDL-apoB by Overproduction of VLDL-apoB by Hepatocytes from Fructose-Fed HamstersHepatocytes from Fructose-Fed Hamsters

050

100150200250

Control Fructose-Fed

MTP

Act

ivity

(Per

cent

of C

ontro

l)P=0.042

0

5

10

15

20

Control

MTP

RN

A

P<0.02

050

100

200250

MTP

Pro

tein

Mas

s(p

erce

nt o

f con

trol)

150

Fructose-Fed

P=0.011

Control

Fructose-Fed

tota

l RN

A/

gpg

)(

Protein Mass

mRNA Lipid Transfer Activity

Evidence for Enhanced Hepatic Microsomal Triglyceride Transfer Protein

(MTP) in Fructose-Fed Hamsters

Insulin Signaling Status in Hepatocytes: Insulin Signaling Status in Hepatocytes: • Ex vivoEx vivo Analysis of Insulin Receptor, IRS-1, PI3-kinase, Analysis of Insulin Receptor, IRS-1, PI3-kinase,

PTP-1B in Control and Fructose-Fed Hamster LiversPTP-1B in Control and Fructose-Fed Hamster Livers• In VitroIn Vitro Analysis of Insulin Receptor, IRS-1, PI3-kinase, Analysis of Insulin Receptor, IRS-1, PI3-kinase,

PTP-1B in Primary Hepatocytes Exposed to High InsulinPTP-1B in Primary Hepatocytes Exposed to High Insulin

Link between Insulin Signaling & VLDL-apoB Secretion:Link between Insulin Signaling & VLDL-apoB Secretion: • In VitroIn Vitro Analysis of ApoB Secretion in Primary Hepatocytes Analysis of ApoB Secretion in Primary Hepatocytes

Exposed to High InsulinExposed to High Insulin• Inhibition of Protein Phosphatases by Inhibition of Protein Phosphatases by VanadateVanadate and its Impact on and its Impact on

VLDL-apoB SecretionVLDL-apoB Secretion(J. Biol. Chem. (2002) 277, 793-803)

Hypothesis II:Hypothesis II:VLDL-apoB Overproduction is Linked to Hepatic Insulin ResistanceVLDL-apoB Overproduction is Linked to Hepatic Insulin Resistance

Insulin Resistance Model(Fructose-Fed Hamster)

Recent Data:

Y

Insulin Signaling PathwayInsulin

Insu

lin R

ecep

tor

Insu

lin R

ecep

tor

Y PP

IRSIRSProteinsProteinsSHC

Grb2

mSoS

Grb2

mSoS

RaS

p85p85

p110p110PI 3-KinasePI 3-Kinase

PDK1 (PDK2)

AktAkt

PTP-1BPTP-1B

PP PP

PTEN

aPKCs PDE BAD

Anti- apoptosis

Anti-lipolysis

Glucosetransport

Gsk3ToRp70rak

Glycogensynthesis

Proteinsynthesis

RAF

MEK

MAPK

Gene Expression/ mitogenesis

90rak

Plasma membrane

Protein kinase CK2

Ser/Ther-p

CAP

CblcrkII

Caveolae

Glucose & Lipid metabolism

Gab 1Shp-2

VanadateVanadate