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Liver Cell Biology VUB, Belgium Objective, Workpackage And Deliverables 1. Objective : to characterize the effects of insulin resistance and NAFLD in specific.

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Presentation on theme: "Liver Cell Biology VUB, Belgium Objective, Workpackage And Deliverables 1. Objective : to characterize the effects of insulin resistance and NAFLD in specific."— Presentation transcript:

1 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)

2 Liver Cell Biology VUB, 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 liver cellsMetabolic syndrome associated factors with direct influence on sinusoidal liver cells –Hyperinsulinemia –Hypoadiponectinemia –Hyperleptinemia –Advanced glycation end products

3 Liver Cell Biology VUB, Belgium Isolation and Purification of Liver Cells LSEC and HSC - in situ Collagenase/Pronase/DNase - 50 x g centrifugation 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 x g centrifugations - 18% Nycodenz density cushion - Incubation with CD11b-APC - Sorting using 633 nm excitation light - Selective 37 °C Hepatocytes - in situ Collagenase/DNase - 50 x g centrifugation (2x) - Triple Percoll cushion - HC are in bottom layer - Second centrifugation through Percoll

4 Liver Cell Biology VUB, Belgium Dot Plots of Purified Liver Cells KC LSECHSCLSECHSC

5 Liver Cell Biology VUB, Belgium Purified Liver Cells from Normal Mouse Liver HC LSEC HSC KC

6 Liver Cell Biology VUB, 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

7 Liver Cell Biology VUB, Belgium 1 st hit 2 nd hit SteatosisSteatohepatitis Fibrosis/ cirrhosis Normal liver Hyperglycemia Hyperlipidemia Hyperinsulinemia Aberant adipokine profile Glycation products.... Sinusoidal liver cells in metabolic syndrome : working hypothesis ? Activation of HSCs Activation of KCs ? Activation of LSECs ?

8 Liver Cell Biology VUB, Belgium HSC culture mimmicks in vivo activation

9 Liver Cell Biology VUB, Belgium Signalling Pathways Activated by Insulin

10 Liver Cell Biology VUB, Belgium Insulin Receptor on HSC

11 Liver Cell Biology VUB, 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 100nMinsulin for 5 minutes. qHSCaHSC Insulin:–+–+ P-AKT P-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 100nMinsulin for 5 minutes. qHSCaHSC Insulin:–+–+ P-AKT P-ERK Phosphorylation pattern of AKT and ERK in qHSC (day 2) and aHSC(day 13) after treatment without or with 100nMinsulin for 5 minutes. qHSCaHSC Insulin:–+–+ P-AKT P-ERK qHSCaHSC Insulin:–+–+ P-AKT P- ERK nmol DOG / mg protein 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 100nMinsulin for 30 minutes. Uptake of LCFA in qHSC (day 1),aHSC(day 13) and adipocytes(A) measured after 30 minutes without or with 100nMinsulin using the QBT TM Fatty 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 100nMinsulin using the QBT TM Fatty Acid Uptake Assay Kit. Uptake is normalized for background fluorescence. RFU Time (s) qHSC 0 nM qHSC 100 nM A 0 nM A 100 nM aHSC 0 nM aHSC 100 nM nM1 nM100 nM qHSC aHSC A

12 Liver Cell Biology VUB, 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 theInsRafter long term insulin exposure. Cells are incubated with indicated insulin concentration and harvested after 5 days. ß-actin InsR Insulin (nM): Relative expression InsRmRNA levels nM1 nM10 nM100 nM a. b. No significant effect of insulin onmHSCactivation Relative gene expression of two markers formHSCactivation 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, normallydownregulated during activation. Protein expression of  -SMA and GFAP after 5 days culturing with indicated insulin concentrations.ß-actinisused asequalloadingcontrol.  -SMA GFAPa.b. Relative expression Insulin (nM): a -SMA GFAP ß-actin Days No increasedmHSC proliferation by insulin nM1 nM100 nM At day 4 mHSCare incubated with indicated insulin concentrations for 24h followed by proliferation measurement by the WST-1 Cell Proliferation Assay Kit. Absorbance

13 Liver Cell Biology VUB, Belgium Adipokines 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  Poor knowledge compared to hepatocytes  Stellate cells are the main fibrogenic effector type of the liver (fibrosis ~ end stage NAFLD) parenchymal cells hepatocytes (60-65%) sinusoidal cells stellate cells (10-11%) endothelial cells (15%) Kupffer cells (7%) pit cells (1-2%)

14 Liver Cell Biology VUB, Belgium Adiponectin receptors in HSC RNAPROTEIN Adipo-R1Adipo-R2 D01 D13 + mHSC Liver Adipo-R2 + D01 D13 + SKM mHSC Liver Adipo-R1

15 Liver Cell Biology VUB, Belgium Exposure of HSC to Adiponectin P-AMPKα

16 Liver Cell Biology VUB, Belgium RNAPROTEIN Leptin receptor expression by HSC D01 D13 + Ob-Rc Ob-Rd Ob-Re Relative mRNA expression Ob-Ra Ob-Rb Relative mRNA expression D01 D13 + mHSC Lung Ob-R

17 Liver Cell Biology VUB, Belgium P-ERK1/2 P-AKT α-SMAGFAP Relative mRNA expression 2 days 6 days 2 days 6 days Incubation: time Exposure of HSC to Leptin

18 Liver Cell Biology VUB, Belgium Influence of Glycation Products on HSC

19 Liver Cell Biology VUB, Belgium Receptors that bind AGE * * * * * * RAGESR-AISR-BICD36Galectin-3 relative expression Day 1 Day 3 Day 8 50 kD ß-actin RAGE (50-55kD) SR-BI (70-80 kD) ß-actin 75 kD SR-AI (60kD) 50 kD CD36 (90kD) 100 kD ß-actin ß- Galectin-3 (30kD) ß-actin 37 kD QuiescentActivatedQuiescentActivated QuiescentActivated

20 Liver Cell Biology VUB, Belgium Reactive Oxygen Species (ROS) detection 2',7'-dichlorofluorescein (DCFH-DA) method

21 Liver Cell Biology VUB, Belgium ROS Production by HSC in the presence of AGE Activated HSCsQuiescent HSCs 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.

22 Liver Cell Biology VUB, 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. * * * Control- BSA 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 relative fluorescence (%control-BSA) * * * Control- BSA 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 relative fluorescence (%Control-BSA)

23 Liver Cell Biology VUB, Belgium CONCLUSIONS I 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

24 Liver Cell Biology VUB, Belgium CONCLUSIONS II 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

25 Liver Cell Biology VUB, 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

26 Liver Cell Biology VUB, Belgium Perspectives Application of isolation and purification procedures on two models of hepatic insulin resistanceApplication of isolation and purification procedures on two models of hepatic 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

27 Liver Cell Biology VUB, Belgium


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