195 hepatic synthesis and secretion of vldl
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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
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