Liver Cell Biology VUB, Belgium Objective, Workpackage And Deliverables 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)
Transcript
Liver Cell BiologyVUB, Belgium
Objective, Workpackage And Deliverables
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 resistancein 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)
Liver Cell BiologyVUB, Belgium
Topics Of This Presentation
• Isolation/purification of specific hepatic cell typesIsolation/purification of specific hepatic cell types
• Test of new generation Affymetrix microarray Gene1.0 ST1Test of new generation Affymetrix microarray Gene1.0 ST1
• Metabolic syndrome associated factors with direct influence on sinusoidal Metabolic syndrome associated factors with direct influence on sinusoidal liver cellsliver cells
– HyperinsulinemiaHyperinsulinemia
– HypoadiponectinemiaHypoadiponectinemia
– HyperleptinemiaHyperleptinemia
– Advanced glycation end productsAdvanced glycation end products
Liver Cell BiologyVUB, Belgium
Isolation and Purification of Liver Cells
LSEC and HSC
- in situ Collagenase/Pronase/DNase
- 50 x g centrifugation
- 500 x g centrifugations
- 18% Nycodenz density cushion
- Incubation with anti-LSEC-FITC
- Sorting using 488 and 355 nm excitation light
LSEC HSC
KC
- Blood removal
- in vitro Collagenase/Pronase/DNase
- 50 x g centrifugation
- 500 x g centrifugations
- 18% Nycodenz density cushion
- Incubation with CD11b-APC
- Sorting using 633 nm excitation light
- Selective adherence @ 37 °C
Hepatocytes
- in situ Collagenase/DNase
- 50 x g centrifugation (2x)
- Triple Percoll cushion
- HC are in bottom layer
- Second centrifugation through Percoll
Liver Cell BiologyVUB, Belgium
Dot Plots of Purified Liver Cells
KC
LSEC HSCLSEC HSC
Liver Cell BiologyVUB, Belgium
Purified Liver Cells from Normal Mouse Liver
HC LSEC
HSC KC
Liver Cell BiologyVUB, Belgium
Gene1.0 ST Array family
• 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
Specific gene expressionCell growth, differentiation
P
ERK1/2
General gene expressionCell growth, differentiation
IRS1-4
InsR
P
LCFA
LCFA
FATP1
Liver Cell BiologyVUB, Belgium
Insulin Receptor on HSC
InsR-BInsR-A
qHSC aHSC
IRS1
IRS2
InsR-BInsR-A
qHSC aHSC
IRS1
IRS2
Liver Cell BiologyVUB, Belgium
Cellular Response to Insulin
Activation of PI3K, but weak MAPK
activation
Phosphorylation pattern of AKT and ERK in qHSC (day 2) and aHSC(day 13) after treatment
without or with 100 nMinsulin for 5 minutes.
qHSC aHSC
Insulin: – + – +
P-AKT
AKT
P-ERK
ERK
Activation of PI3K, but weak MAPK
activation
Phosphorylation pattern of AKT and ERK in qHSC (day 2) and aHSC(day 13) after treatment
without or with 100 nMinsulin for 5 minutes.
qHSC aHSC
Insulin: – + – +
P-AKT
AKT
P-ERK
ERK
Phosphorylation pattern of AKT and ERK in qHSC (day 2) and aHSC(day 13) after treatment
without or with 100 nMinsulin for 5 minutes.
qHSC aHSC
Insulin: – + – +
P-AKT
AKT
P-ERK
ERK
qHSC aHSC
Insulin: – + – +
P-AKT
AKT
P-
ERK
nm
ol
DO
G /
mg
pro
tein
No effect of insulin on fatty acid and glucose uptake
Uptake of 2 -[³H]deoxyglucose (DOG) measured during 5 minutes after qHSC (day 1) and adipocytes (A) are incubated without or with 100 nM insulin for 30 minutes.
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 QBTTMFatty Acid Uptake Assay Kit.
Uptake is normalized for background fluorescence.
RFU
Time (s)
for 30 minutes.
Uptake of LCFA in qHSC (day 1), aHSC (day 13) and
100 nM insulin using the QBTTMFatty Acid Uptake Assay Kit. Uptake is normalized for background fluorescence.
RFU
Time (s)-5000
0
5000
10000
15000
20000
25000
30000
35000
40000
45000
0 1000 2000 3000 4000
qHSC 0 nM
qHSC 100 nM
A 0 nM
A 100 nM
aHSC 0 nM
aHSC 100 nM
0
2
4
6
8
0 nM 1 nM 100 nM
qHSC
aHSC
A
Liver Cell BiologyVUB, Belgium
Long Term Exposure to Insulin
Downregulationof insulin receptor on protein steady
state level, but not mRNA level
a) Protein expression and b) relative mRNA levels of the InsRafter long term insulin
exposure. Cells are incubated with indicated insulin concentration and harvested after 5
days.
ß-actin
InsR
Insulin (nM): 0 1 10 100
Rela
tive
exp
ress
ion
InsRmRNA levels
00.5
11.5
2
0 nM 1 nM 10 nM 100 nM
a.
b.
No significant effect of insulin on mHSCactivation
Relative gene expression of two markers for mHSCactivation after long term insulin exposure. Every day HSC are treated with or without insulin and collected on indicated time points. a) Expression of -SMA, normally
upregulatedduring activation. b) Expression of GFAP, normally downregulatedduring activation.
Protein expression of -SMA and GFAP after 5 days culturing with indicated insulin concentrations. ß-actin is usedas equalloadingcontrol.
-SMA GFAPa. b.
Rela
tive e
xpre
ssio
n
Rela
tive e
xpre
ssio
n
Insulin (nM): 0 1 10 100
a-SMA
GFAPß-actin
0
5
10
15
0 2 4 6 8 10Days Days
No increased mHSCproliferation by insulin
0
0.2
0.4
0.6
0.8
1
0 nM 1 nM 100 nM
At day 4 mHSCare incubated with indicated insulin concentrations for 24h followed by proliferation measurement by the WST-1 Cell Proliferation Assay Kit.
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 was measured. *P <0,05 compared to control-BSA in the respective concentrations.
Liver Cell BiologyVUB, Belgium
ROS Production by HSC in the presence of AGE and NADPH oxidase inhibitor
Activated HSCsQuiescent HSCs
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.
*
**
0
50
100
150
Control-BSA
CML-BSA400 µg/ml
CML-BSA400
µg/ml+DPI
GA-BSA400 µg/ml
GA-BSA400
µg/ml+DPI
AGE-BSA400 µg/ml
AGE-BSA400
µg/ml+DPI
rela
tive
flu
ore
scen
ce (
%co
ntr
ol-
BS
A)
*
*
*
0
50
100
150
200
Control-BSA
CML-BSA400 µg/ml
CML-BSA400
µg/ml+DPI
GA-BSA400 µg/ml
GA-BSA400
µg/ml+DPI
AGE-BSA400 µg/ml
AGE-BSA400
µg/ml+DPI
rela
tive
flu
ore
scen
ce (
%C
on
tro
l-B
SA
)
Liver Cell BiologyVUB, Belgium
CONCLUSIONS I
We have developed protocols for isolation and purification of murine HC, LSEC, We have developed protocols for isolation and purification of murine HC, LSEC, HSC and KCHSC and KC
Our initial experience with the new generation Affymetrix microarrays is positiveOur initial experience with the new generation Affymetrix microarrays is positive
Despite the presence of both insulin receptor isoforms:Despite the presence of both insulin receptor isoforms:
▪▪ no transcription of typical insulin responsive genesno transcription of typical insulin responsive genes▪▪ no effect on mHSC activation markersno effect on mHSC activation markers▪▪ no increased glucose or fatty acid uptakeno increased glucose or fatty acid uptake
Liver Cell BiologyVUB, Belgium
CONCLUSIONS II
The main leptin receptor isoform with signal transduction capabilitiesThe main leptin receptor isoform with signal transduction capabilities
(Ob-Rb) was not detectable(Ob-Rb) was not detectable
▪▪ no effect on mHSC activation markersno effect on mHSC activation markers
▪▪ little phosphorylation of Akt & no phosphorylation of STAT3little phosphorylation of Akt & no phosphorylation of STAT3
Adipo-R1 (D13) & Adipo-R2 (D1) are expressed in minimal amountsAdipo-R1 (D13) & Adipo-R2 (D1) are expressed in minimal amounts
▪▪ no phosphorylation of AMPKno phosphorylation of AMPKαα
Liver Cell BiologyVUB, Belgium
CONCLUSIONS III
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
Liver Cell BiologyVUB, Belgium
Perspectives
• Application of isolation and purification procedures on two models of Application of isolation and purification procedures on two models of hepatic insulin resistancehepatic insulin resistance
• Large scale microarray experiment on these isolated and purified cellsLarge scale microarray experiment on these isolated and purified cells
• Further exploration of the influence of AGE on the HSC, LSEC and KCFurther exploration of the influence of AGE on the HSC, LSEC and KC
