Transcript P - Liver Cell Biology Lab

Objective, Workpackage And Deliverables
Liver Cell Biology
VUB, Belgium
1. Objective : to characterize the effects of insulin resistance and NAFLD
in specific hepatic cell populations (O1.2)
2. Work package : to characterize the effects of insulin resistance
in specific hepatic cell populations (WP2.2)
3. Deliverables :
•
Optimisation of isolation technology applied to selected cell populations (D2.2.1)
(done)
•
Optimisation of gene expression and proteomic profiling using small amount of
biological material (D2.2.2) (ongoing)
•
Gene expression profile and comparative analysis of cell populations investigated
under normal and insulin resistant conditions (D2.2.3) (JM + AV-P + BT)
•
Proteomic profile and comparative analysis of cell populations investigated under
normal and insulin resistant conditions (D2.2.4)
Topics Of This Presentation
Liver Cell Biology
VUB, Belgium
• Isolation/purification of specific hepatic cell types
• Test of new generation Affymetrix microarray Gene1.0 ST1
• Metabolic syndrome associated factors with direct influence on sinusoidal
liver cells
– Hyperinsulinemia
– Hypoadiponectinemia
– Hyperleptinemia
– Advanced glycation end products
Isolation and Purification of Liver Cells
Liver Cell Biology
VUB, Belgium
Hepatocytes
- in situ Collagenase/DNase
- 50 x g centrifugation (2x)
- Triple Percoll cushion
- HC are in bottom layer
- Second centrifugation through
Percoll
LSEC and HSC
- in situ
Collagenase/Pronase/DNase
KC
- Blood removal
- 50 x g centrifugation
- in vitro
Collagenase/Pronase/DNase
- 500 x g centrifugations
- 50 x g centrifugation
- 18% Nycodenz density cushion
- 500 x g centrifugations
- Incubation with anti-LSEC-FITC
- 18% Nycodenz density cushion
- Sorting using 488 and 355 nm
excitation light
- Incubation with CD11b-APC
- Sorting using 633 nm
excitation light
- Selective adherence @ 37 °C
LSEC
HSC
Dot Plots of Purified Liver Cells
Liver Cell Biology
VUB, Belgium
LSEC
HSC
KC
Purified Liver Cells from Normal Mouse Liver
Liver Cell Biology
VUB, Belgium
HC
LSEC
HSC
KC
Gene1.0 ST Array family
Liver Cell Biology
VUB, Belgium
• The GeneChip®Mouse and Rat Gene 1.0ST Arrays are the latest additions to
the Gene1.0 ST Array family. The Mouse Gene 1.0 STArray interrogates 28,853
well-annotated genes with 770,317 distinct probes
• The Mouse Gene 1.0 ST Array has 100 percent coverage of NM sequences
present in the April 3, 2007, RefSeq database
• Considerably cheaper  more experiments possible
• Only small amount of DNA needed: 100 ng for the microarray (not incl.
amount for QC)
• Results not comparable with older chips – but we start with the arrays so a
possibility?
•Tested by a collaborating lab in Leuven, Belgium and found to be of excellent
quality
Sinusoidal liver cells in metabolic syndrome :
working hypothesis
Liver Cell Biology
VUB, Belgium
Normal
liver
1st hit
2nd hit
Steatosis
•Hyperglycemia
•Hyperlipidemia
•Hyperinsulinemia
•Aberant adipokine profile
•Glycation products
•....
?
Steatohepatitis
Fibrosis/
cirrhosis
Activation of HSCs
Activation of KCs ?
Activation of LSECs ?
HSC culture mimmicks in vivo activation
Liver Cell Biology
VUB, Belgium
A
B
C
Day 0
Day 2
Day 5
D
Day 8
E
Day 11
F
Day 14
100μm
Signalling Pathways Activated by Insulin
[email protected]
Liver Cell Biology
VUB, Belgium
Insulin Receptor on HSC
Liver Cell Biology
VUB, Belgium
qHSC
InsR-B
InsR-A
IRS1
IRS2
aHSC
Cellular Response to Insulin
Liver Cell Biology
VUB, Belgium
Activation of PI3K,
but weak MAPK
activation
–
+
–
45000
+
RFU
P-AKT
AKT
qHSC 0 nM
40000
qHSC 100 nM
35000
A 0 nM
30000
A 100 nM
aHSC 0 nM
25000
aHSC 100 nM
20000
P-ERK
P-
nmol
15000
10000
aHSC
Phosphorylation pattern of AKT
and ERK in qHSC (day 2) and
aHSC (day 13) after treatment
without or with 100 nM insulin for
5 minutes.
-5000
qHSC
6
4
aHSC
A
2
0 nM
0
qHSC
8
0
5000
ERK
DOG / mg protein
Insulin:
No effect of insulin on fatty acid and glucose
uptake
0
1000
2000
3000
1 nM
100 nM
4000
Time (s)
Uptake of LCFA in qHSC (day 1),
aHSC (day 13) and
adipocytes
(A) measured after 30 minutes without or with
100 nM insulin using the QBT TM Fatty Acid Uptake Assay Kit.
Uptake is normalized for background fluorescence.
Uptake of 2 -[³ H]deoxyglucose
(DOG) measured during 5
minutes after qHSC (day 1) and
adipocytes (A) are incubated
without or with 100 nM insulin
for 30 minutes.
Long Term Exposure to Insulin
Liver Cell Biology
VUB, Belgium
No significant effect of insulin on mHSC activation
- SMA
b.
GFAP
Relative expression
Relative expression
a.
15
10
5
0
0
2
4
Days
6
8
10
Insulin (nM):
GFAP
ß-actin
Days
Protein expression of -SMA
and GFAP after 5 days culturing
with indicated insulin
concentrations. ß-actin is used
as equal loading control.
Insulin (nM):
0
1 10 100
0.8
InsR
0.6
0.4
ß-actin
0.2
b.
InsR mRNA levels
0
0 nM
1 nM
100 nM
At day 4 mHSC are incubated with
indicated insulin concentrations for
24h followed by proliferation
measurement by the WST -1 Cell
Proliferation Assay Kit.
Relative
expression
Absorbance
Downregulation of insulin
receptor on protein steady
state level, but not mRNA level
a.
1
1 10 100
a-SMA
Relative gene expression of two markers for mHSC activation after long term
insulin exposure. Every day HSC are treated with or without insu lin and
collected on indicated time points. a) Expression of  - SMA, normally
upregulated during activation. b) Expression of GFAP, normally downregulated
during activation.
No increased mHSC
proliferation by insulin
0
2
1.5
1
0.5
0
0 nM
1 nM
10 nM 100 nM
a) Protein expression and b) relative mRNA
levels of the InsR after long term insulin
exposure. Cells are incubated with indicated
insulin concentration and harvested after 5
days.
Adipokines
[email protected]
Liver Cell Biology
VUB, Belgium
Adipo(cyto)kines = bio-active peptides which are exclusively produced and
secreted by the white adipose tissue
The viceral part of this adipose tissue drains in the portal vein which supplies
venous blood to the liver.
 It’s not unlikely that there is an effect on sinusoidal liver cells
parenchymal cells
• hepatocytes (60-65%)
sinusoidal cells
 Poor knowledge compared to hepatocytes
 Stellate cells are the main fibrogenic effector
type of the liver (fibrosis ~ end stage NAFLD)
• stellate cells (10-11%)
• endothelial cells (15%)
• Kupffer cells (7%)
• pit cells (1-2%)
Adiponectin receptors in HSC
Liver Cell Biology
VUB, Belgium
RNA
Adipo-R1
Adipo-R2
PROTEIN
Adipo-R1
+
D01 D13 +
SKM mHSC Liver
Adipo-R2
D01 D13
mHSC
+
Liver
Exposure of HSC to Adiponectin
Liver Cell Biology
VUB, Belgium
P-AMPKα
Leptin receptor expression by HSC
Relative mRNA
expression
Liver Cell Biology
VUB, Belgium
RNA
Ob-Ra
Ob-Rc
Relative mRNA
expression
Ob-Rd
Ob-Rb
Ob-Re
D01 D13
PROTEIN
Ob-R
D01
D13
mHSC
+
Lung
+
Exposure of HSC to Leptin
Liver Cell Biology
VUB, Belgium
•
α-SMA
Incubation:
time
Relative mRNA
expression
Relative mRNA
expression
GFAP
2 days
6 days
P-ERK1/2
Incubation:
time
P-AKT
2 days
6 days
Influence of Glycation Products on HSC
[email protected]
Liver Cell Biology
VUB, Belgium
Receptors that bind AGE
relative expression
Liver Cell Biology
VUB, Belgium
20
18
16
14
12
10
8
6
4
2
0
*
*
Day 1
*
Day 3
Day 8
* *
RAGE
*
SR-AI
SR-BI
RAGE (50-55kD)
CD36
Galectin-3
SR-AI (60kD)
50 kD
50 kD
ß-actin
ß-actin
SR-BI (70-80 kD)
CD36 (90kD)
100 kD
75 kD
ß-actin
ß-actin
Quiescent
Activated
37 kD
Quiescent
Galectin-3 (30kD)
ß-actin
Quiescent
Activated
Activated
Reactive Oxygen Species (ROS) detection
Liver Cell Biology
VUB, Belgium
2',7'- dichlorofluorescein (DCFH - DA) method
ROS Production by HSC in the presence of AGE
Liver Cell Biology
VUB, Belgium
Activated HSCs
200
150
*
*
*
*
*
*
*
100
50
0
100
µg/ml
ControlBSA
200
µg/mç
GA-BSA
400
µg/ml
100
µg/ml
200
µg/ml
CML-BSA
400
µg/ml
100
µg/ml
200
µg/ml
AGE-BSA
DCF fluorescence (% relative to control-BSA)
DCF fluorescence (% relative to control-BSA)
Quiescent HSCs
200
*
150
*
*
*
*
*
*
*
100
50
0
400
µg/ml
100
µg/ml
ControlBSA
200
µg/ml
GA-BSA
400
µg/ml
100
µg/ml
200
µg/ml
CML-BSA
400
µg/ml
100
µg/ml
200
µg/ml
400
µg/ml
AGE-BSA
ROS production by AGEs on HSCs. HSCs were incubated with DCFH -DA for 30 minutes,
followed by treatment with the indicated AGE concentrations on day 3 (quiescent HSCs) and day 10
(activated HSCs). After 10 minutes treatment, DCF fluorescence w as measured. *P <0,05 compared to
control-BSA in the respective concentrations.
ROS Production by HSC in the presence of AGE and
NADPH oxidase inhibitor
Liver Cell Biology
VUB, Belgium
Activated HSCs
*
150
*
*
100
50
relative fluorescence (%Control-BSA)
relative fluorescence (%control-BSA)
Quiescent HSCs
200
*
*
150
*
100
50
0
0
ControlBSA
CML-BSA CML-BSA GA-BSA GA-BSA AGE-BSA AGE-BSA
400 µg/ml
400
400 µg/ml
400
400 µg/ml
400
µg/ml+DPI
µg/ml+DPI
µg/ml+DPI
ControlBSA
CML-BSA
400 µg/ml
CML-BSA
400
µg/ml+DPI
GA-BSA
400 µg/ml
GA-BSA
400
µg/ml+DPI
AGE-BSA
400 µg/ml
AGE-BSA
400
µg/ml+DPI
Diphenylene iodonium (DPI), a NADPH oxidase inhibitor, decreases ROS production induced by
AGEs in HSCs. Cells were incubated with DCFH - DA and DPI for 30 minutes, followed by the indicated
AGEs concentrations on day 3 (quiescent HSCs) and day 10 (activated HSCs). After 10 minutes, DCF
fluorescence was measured. *P<0,05 compared to control - BSA and DPI treated cells in the respective
concentrations.
CONCLUSIONS I
Liver Cell Biology
VUB, Belgium
We have developed protocols for isolation and purification of murine HC, LSEC,
HSC and KC
Our initial experience with the new generation Affymetrix microarrays is positive
Despite the presence of both insulin receptor isoforms:
▪ no transcription of typical insulin responsive genes
▪ no effect on mHSC activation markers
▪ no increased glucose or fatty acid uptake
CONCLUSIONS II
Liver Cell Biology
VUB, Belgium
The main leptin receptor isoform with signal transduction capabilities
(Ob-Rb) was not detectable
▪
▪
no effect on mHSC activation markers
little phosphorylation of Akt & no phosphorylation of STAT3
Adipo-R1 (D13) & Adipo-R2 (D1) are expressed in minimal amounts
▪
no phosphorylation of AMPKα
CONCLUSIONS III
Liver Cell Biology
VUB, Belgium
AGE:
Mouse HSC express 5 potential receptors for AGEs, with 4 of them increasing
gene expression along cell activation
3 different AGE types induce ROS production in quiescent and activated HSCs
TOS inhibition by DPI pre-treatment suggests NADPH oxidase as the source of
ROS
Perspectives
Liver Cell Biology
VUB, Belgium
• Application of isolation and purification procedures on two models of
hepatic insulin resistance
• Large scale microarray experiment on these isolated and purified cells
• Further exploration of the influence of AGE on the HSC, LSEC and KC
Liver Cell Biology
VUB, Belgium