Spatio-Temporal Multiscale Analysis of Western Diet-Fed Mice Reveals a Translationally Relevant Sequence of Events during NAFLD Progression
Abstract
:1. Introduction
2. Materials and Methods
2.1. Mice and Feeding Style
2.2. Induction of Acute-on-Chronic Liver Injury by Acetaminophen
2.3. Induction of Acute-on-Chronic Liver Injury by LPS
2.4. Intravital Imaging
2.5. Magnetic Resonance Imaging (MRI)
2.5.1. Tumor Detection
2.5.2. Estimation of the Fat Fraction
2.5.3. Assessment of Hepatocyte Uptake Capacity
2.6. Sample Collection
2.7. Liver Enzyme Assay
2.8. Histopathology, Immunohistochemistry, and TUNEL Staining
2.9. RNA-Seq Analysis
2.10. Bioinformatics
2.11. Western Blot Analysis and Quantification
2.12. Quantification of Plasma Metabolites
2.13. Image Analysis
2.14. Patients
2.15. Statistical Analysis
3. Results
3.1. Spatio-Temporal Accumulation of Lipid Droplets and Tumor Development after Western-Style Diet Feeding
3.2. Time-Resolved Genome-Wide Expression Analysis
3.3. Progression from Simple Steatosis to Steatohepatitis
3.4. Ductular Reaction (DR) and Fibrosis Progression
3.5. Reorganization of Zonally Expressed Enzymes and Functional Consequences
3.6. Comparison of Key Histological Features of WD-Fed Mice to NAFLD Patients
4. Discussion
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Conflicts of Interest
Abbreviations
References
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Reagent or Resource | Source | Identifier |
---|---|---|
Antibodies, Reagents, and Dyes Used for Immunohistochemistry | ||
Anti-liver arginase1 antibody, rabbit | Abcam, Cambridge, UK | ab203490 |
Anti-arginase1 antibody, goat | Novus Biologicals, Littleton, USA | NB100-59740 |
Bodipy 495/503 | Thermo Fisher Scientific, Waltham, USA | D3922 |
Anti-mouse CD45 antibody, rat | BD Bioscience, Heidelberg, Germany | 550539 |
Anti-human CD68 monoclonal antibody, mouse | DakoCytomation A/S, Glostrup, Denmark | M0876 |
Anti-K18 polyclonal antibody, rabbit | Proteintech, Manchester, UK | 10830-1-AP |
Recombinant anti-K19 antibody, rabbit | Abcam, Cambridge, UK | ab52625 |
Recombinant anti-CPS1 monoclonal antibody, rabbit | Abcam, Cambridge, UK | ab129076 |
Anti-Cyp2e1 antibody, rabbit | Sigma-Aldrich, St. Louis, USA | HPA009128 |
Anti-mouse desmin antibody, rabbit | Thermo Fisher Scientific, Waltham, USA | RB -9014-P0 |
Anti-mouse F4/80 monoclonal antibody, rat | Bio-Rad, Hercules, USA | MCA497 |
Anti-GS polyclonal antibody, rabbit | Sigma, St. Louis, USA | G2781 |
Anti-GS polyclonal antibody, rabbit | Sigma, St. Louis, USA | G2781 |
Anti-Ki67 antibody, rabbit | Cell Signaling Technology, Danvers, USA | D3B5 |
Anti- cl. Caspase 3 (rabbit) monoclonal | Cell Signaling Technology, Danvers, USA | 9661S |
Fluorescent Markers/Dyes Used for Intravital Imaging | ||
Hoechst 33258 | Thermo Fisher Scientific, Waltham, USA | H21491 |
Tetramethylrhodamine ethyl ester (TMRE) | Thermo Fisher Scientific, Waltham, USA | T669 |
Cholyl-lysyl-fluorescein (CLF) | Corning | 451041 |
PE-F4/80 antibody | Thermo Scientific (eBioscience) , Waltham, USA | 12-4801-82 |
Rhodamine 123 | Thermo Fisher Scientific, Waltham, USA | R302 |
Bodipy 493/503 | Thermo Fisher Scientific, Waltham, USA | D3922 |
Mouse Diets | ||
ssniff R/M-H, 10 mm standard diet | Ssniff, Soest, Germany | V1534-000 |
Western-style diet | Research Diets, New Brunswick, USA | D09100301 |
Drugs/Contrast Agents/Toxins | ||
Acetaminophen | Sigma-Aldrich, St. Louis, USA | A7085-500G |
LPS | Sigma-Aldrich, St. Louis, USA | 297-473-0 |
Gadoxetic acid (Primovist) 0.25 mmol/mL | Bayer, Wuppertal, Germany | KT07561 |
Commercial Kits/Reagents | ||
DeadEnd™ Fluorometric TUNEL System | Promega, Walldorf, Germany | G3250 |
Bluing Reagent | Roche, Mannheim, Germany | 05 266 769 001 |
Discovery yellow Kit (RUO) | Roche, Mannheim, Germany | 07 698 445 001 |
Discovery Teal HRP Kit (RUO) | Roche, Mannheim, Germany | 8254338001 |
Chromo Map DAB | Roche, Mannheim, Germany | 05 266 645 001 |
Piccolo general chemistry 13 | Hitado, Möhnesee, Germany | AB-114-400-0029 |
Picrosirius Red Stain Kit | Polysciences Polysciences Inc., Warrington, USA | 24901 |
RNeasy Mini Kit | Qiagen, Hilden, Germany | 74116 |
RNase-Free DNase Set | Qiagen, Hilden, Germany | 79254 |
RNA BR Assay Kit | Thermo Fisher Scientific, Waltham, USA | Q10210 |
RNA 6000 Nano Kit | Agilent Technologies, CA, USA | 5067-1511 |
Qubit 1X dsDNA HS Assay Kit | Thermo Fisher Scientific, Waltham, USA | Q33230 |
DNA 1000 Kit | Agilent Technologies, CA, USA | 5067-1504 |
Antibodies Used for Western Blotting | ||
Anti- MLKL | Biorbyt LLC, Cambridge, UK | orb32399 |
Anti- cleaved-Caspase-3 | Cell Signaling Technology, Danvers, USA | 9661S |
Anti- GAPDH | AbD Serotec, Hercules, USA | MCA 4739 |
Software and Algorithms | ||
GraphPad Prism 9.1 Software | GraphPad, San Diego, USA | NA |
Zen | Carl-Zeiss, Jena, Germany | NA |
ImageJ 1.8.0_172 | https://imagej.nih.gov; 22 April 2021 | NA |
Instruments | ||
LSM MP7 two-photon microscope | Zeiss, Jena, Germany | NA |
Axio Scan.Z1 | Zeiss, Jena, Germany | N/A |
Confocal Laser Scanning Microscope FLUOVIEW FV1000 | Olympus, Hamburg, Germany | N/A |
DISCOVERY ULTRA Automated Slide Preparation System | Roche, Mannheim, Germany | N/A |
Piccolo Xpress® chemistry analyzer | Abaxis, Union City, USA | N/A |
PocketChem BA PA-4140 ammonia meter | Arkray.inc, Amstelveen, The Netherlands | N/A |
3Tesla MRI scanner | Prisma, Siemens Healthineers, Erlangen, Germany |
Ingredient | Grams | Kcal | % |
---|---|---|---|
Casein, 80 mesh | 200 | 800 | 22.12 |
l-cystine | 3 | 12 | 0.33 |
Maltodextrin 10 | 100 | 400 | 11.06 |
Fructose | 200 | 800 | 22.12 |
Sucrose | 96 | 384 | 10.62 |
Cellulose, BW200 | 50 | 0 | 5.53 |
Soybean oil | 25 | 225 | 2.77 |
Primex shortening | 135 | 1215 | 14.93 |
Lard | 20 | 180 | 2.21 |
Mineral Mix S10026 | 10 | 0 | 1.11 |
Dicalcium phosphate | 13 | 0 | 1.44 |
Calcium carbonate | 5.5 | 0 | 0.61 |
Potassium citrate, 1 H2O | 16.5 | 0 | 1.83 |
Vitamin Mix V10001 | 10 | 40 | 1.11 |
Choline bitartrate | 2 | 0 | 0.22 |
Cholesterol | 18 | 0 | 1.99 |
FD&C Yellow dye | 0.05 | 0 | 0.006 |
Total | 904.05 | 4056 | 100 |
Total protein | 20 Kcal % | 22 | |
Total carbohydrate | 40 Kcal % | 45 | |
Total fat | 40 Kcal % | 20 |
Diet | Feeding Time (Weeks) | Number of Mice Analyzed | ||||||||
---|---|---|---|---|---|---|---|---|---|---|
Liver/Body Weight, RNA-seq, Histology, IHC | HCC | MRI | Intravital Imaging | Nitrogen Metabolism | APAP Experiment | Necroptosis Analysis | LPS | |||
+ | − | |||||||||
Western Diet | 3 | 5 | 0 | 5 | 3 | 4 | ||||
6 | 5 | 0 | 5 | 4 | ||||||
9 | 3 | - | ||||||||
12 | 5 | 0 | 5 | 4 | ||||||
18 | 5 | 0 | 5 | 4 | ||||||
24 | 5 | 0 | 5 | 6 | 4 | |||||
30 | 5 | 4 | 1 | 4 | ||||||
32 | 3 | |||||||||
36 | 5 | 1 | 4 | 4 | ||||||
42 | 4 | 5 | 4 | 3 | ||||||
48 | 8 | 6 | 2 | 4 | 3 | |||||
~50 | 7 | |||||||||
Standard Diet | 3 | 7 | 0 | 7 | 3 | 4 | ||||
6 | 5 | 0 | 5 | |||||||
30 | 5 | 0 | 5 | |||||||
36 | 7 | 0 | 7 | 4 | ||||||
42 | 3 | 0 | 3 | 4 | ||||||
48 | 5 | 0 | 5 | 4 | ||||||
~50 | 7 |
Dye/Marker | Marker for | Dose (mg/kg) | Vehicle | Two-Photon Excitation Range (nm) |
---|---|---|---|---|
Hoechst 33258 | Nuclei | 5 | PBS | 700–800 |
TMRE | Lobular zonation; mitochondrial membrane potential | 0.96 | Methanol/PBS (1:1) | 780–820 |
Rhodamine123 | 0.8 | Methanol/PBS (1:1) | 720–820 | |
Cholyl-lysyl-fluorescein | Bile acid analogue | 1 | PBS | 740–820 |
Bodipy 493/503 | Lipids | 0.004 | DMSO | 900–940 |
PE-F4/80 antibody | Macrophages | 0.06 | PBS | 720–760 |
Target | Primary Antibodies | Secondary Antibodies | ||
---|---|---|---|---|
Antibody | Dilution | Antibody | Dilution | |
Lipids | Bodipy 495/503 | 2 µg/mL | - | - |
Arginase1 | Anti-arginase1 antibody, goat | 1:100 | Cy™5-conjugated AffiniPure donkey anti-goat IgG (H + L) | 1:200 |
Anti-liver arginase1 antibody, rabbit | 1:2000 | Ultra-Map anti-rabbit HRP | Automatic Discovery Ready to use | |
Ultra-Map anti-rabbit alkaline phosphatase | ||||
Leukocyte common antigen | Anti-mouse CD45 antibody, rat | 1:400 | Ultra-Map anti-rat HRP | |
Macrophages, human | Anti-human CD68 monoclonal antibody, mouse | 1:500 | Ultra-Map anti-mouse HRP | |
Cytoskeleton | Anti-K18 polyclonal antibody, rabbit | 1:400 | Ultra-Map anti-rabbit HRP | |
Cholangiocyte, mouse | Recombinant anti-K19 antibody, rabbit | 1:500 | Ultra-Map anti-rabbit HRP | |
Cholangiocyte, human | Recombinant anti-K19 antibody, rabbit | 1:2000 | Ultra-Map anti-rabbit HRP | |
Carbamoyl-Phosphate Synthase1 | Recombinant anti-CPS1 monoclonal antibody, rabbit | 1:500 | Ultra-Map anti-rabbit HRP | |
Cyp2e1 | Anti-Cyp2e1 antibody, rabbit | 1:100 | Ultra-Map anti-rabbit HRP | |
Ultra-Map anti-rabbit alkaline phosphatase | ||||
Hepatic stellate cells | Anti-mouse desmin antibody, rabbit | 1:400 | Ultra-Map anti-rabbit HRP | |
Macrophages, mouse | Anti-mouse F4/80 monoclonal antibody, rat | 1:50 | Ultra-Map anti-rat HRP | |
Glutamine synthetase, mouse | Anti-GS polyclonal antibody, rabbit | 1:15,000 | Ultra-Map anti-rabbit HRP | |
Apoptosis | Anti- cl. Caspase 3 monoclonal antibody, rabbit | 1:500 | Ultra-Map anti-rabbit HRP | |
Glutamine synthetase, human | Anti-GS polyclonal antibody, rabbit | 1:5000 | Ultra-Map anti-rabbit HRP | |
Cell proliferation antigen | Anti-Ki-67 antibody, rabbit | 1:100 | Ultra-Map anti-rabbit HRP |
Similarities | Differences |
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Ghallab, A.; Myllys, M.; Friebel, A.; Duda, J.; Edlund, K.; Halilbasic, E.; Vucur, M.; Hobloss, Z.; Brackhagen, L.; Begher-Tibbe, B.; Hassan, R.; Burke, M.; Genc, E.; Frohwein, L.J.; Hofmann, U.; Holland, C.H.; González, D.; Keller, M.; Seddek, A.-l.; Abbas, T.; Mohammed, E.S.I.; Teufel, A.; Itzel, T.; Metzler, S.; Marchan, R.; Cadenas, C.; Watzl, C.; Nitsche, M.A.; Kappenberg, F.; Luedde, T.; Longerich, T.; Rahnenführer, J.; Hoehme, S.; Trauner, M.; Hengstler, J.G. Spatio-Temporal Multiscale Analysis of Western Diet-Fed Mice Reveals a Translationally Relevant Sequence of Events during NAFLD Progression. Cells 2021, 10, 2516. https://doi.org/10.3390/cells10102516
Ghallab A, Myllys M, Friebel A, Duda J, Edlund K, Halilbasic E, Vucur M, Hobloss Z, Brackhagen L, Begher-Tibbe B, Hassan R, Burke M, Genc E, Frohwein LJ, Hofmann U, Holland CH, González D, Keller M, Seddek A-l, Abbas T, Mohammed ESI, Teufel A, Itzel T, Metzler S, Marchan R, Cadenas C, Watzl C, Nitsche MA, Kappenberg F, Luedde T, Longerich T, Rahnenführer J, Hoehme S, Trauner M, Hengstler JG. Spatio-Temporal Multiscale Analysis of Western Diet-Fed Mice Reveals a Translationally Relevant Sequence of Events during NAFLD Progression. Cells. 2021; 10(10):2516. https://doi.org/10.3390/cells10102516
Chicago/Turabian StyleGhallab, Ahmed, Maiju Myllys, Adrian Friebel, Julia Duda, Karolina Edlund, Emina Halilbasic, Mihael Vucur, Zaynab Hobloss, Lisa Brackhagen, Brigitte Begher-Tibbe, Reham Hassan, Michael Burke, Erhan Genc, Lynn Johann Frohwein, Ute Hofmann, Christian H. Holland, Daniela González, Magdalena Keller, Abdel-latif Seddek, Tahany Abbas, Elsayed S. I. Mohammed, Andreas Teufel, Timo Itzel, Sarah Metzler, Rosemarie Marchan, Cristina Cadenas, Carsten Watzl, Michael A. Nitsche, Franziska Kappenberg, Tom Luedde, Thomas Longerich, Jörg Rahnenführer, Stefan Hoehme, Michael Trauner, and Jan G. Hengstler. 2021. "Spatio-Temporal Multiscale Analysis of Western Diet-Fed Mice Reveals a Translationally Relevant Sequence of Events during NAFLD Progression" Cells 10, no. 10: 2516. https://doi.org/10.3390/cells10102516