The Potential of the FSP1cre-Pparb/d−/− Mouse Model for Studying Juvenile NAFLD
Abstract
:1. Introduction
2. Results
2.1. Deletion of Pparb/d in FSP1+CD11b+ Cells
2.2. More and Larger Intracellular Lipid Droplets in FSP1cre-Pparb/d−/− Livers at P2 and Week 4
2.3. Higher Triglyceride Levels in the Liver of FSP1cre-Pparb/d−/− Mice at P2 and Week 4
2.4. Upregulation of Genes Involved in Fatty Acid β-Oxidation, Fatty Acid Synthesis and Triglyceride Synthesis in FSP1cre-Pparb/d−/− Liver at P2 and Week 4
2.5. Increase in Hadha, Phospho-ACLY, Phospho-ACC and GPD2 in the Liver of P2 FSP1cre-Pparb/d−/− Mice
3. Discussion
4. Materials and Methods
4.1. Chemicals and Antibodies
4.2. Animals
4.3. Genotyping Analysis
4.4. Histology
4.5. Double Immunofluorescence Staining
4.6. Isolation of Hepatic Cells
4.7. Flow Cytometry
4.8. Quantification of Total Lipids, Total Cholesterol, and Triglycerides
4.9. Total RNA Extraction and Reverse Transcription
4.10. Quantitative Real-Time PCR
4.11. Western Blot and Densitometric Analysis
4.12. Statistical Analysis
Supplementary Materials
Author Contributions
Funding
Acknowledgments
Conflicts of Interest
Abbreviations
ACAA2 | acetyl-CoA acyltransferase 2 |
ACAT1 | acetyl-CoA acetyltransferase 1 |
ACC | acetyl-CoA synthetase |
ACLY | acyl-CoA oxidase 1 |
ACS | acetyl-CoA synthetase |
ACOX1 | acyl-CoA oxidase 1 |
AGPAT3 | 1-acylglycerol-3-phosphate O-acyltransferase 3 |
AMPK | AMP-activated protein kinase |
CYP51 | cytochrome P450 family 51 subfamily A member 1 |
DGAT1 | diacylglycerol O-acyltransferase 1 |
DHCR7 | 7-dehydrocholesterol reductase |
FAS | fatty acid synthase |
FSP1 | fibroblast-specific protein 1 |
GFAP | glial fibrillary acidic protein |
GPD1 | glycerol 3-phosphate dehydrogenase 1 |
GPD2 | glycerol 3-phosphate dehydrogenase 2 |
H&E | hematoxylin and eosin |
HADHA | hydroxyacyl-CoA dehydrogenase |
HMGCS1 | hydroxymethylglutaryl-CoA synthase |
HSC | hepatic stellate cell |
IL4 | interleukin 4 |
IL13 | interleukin 13 |
IgG | immunoglobulin G |
LPIN3 | lipin 3 |
LSEC | liver sinusoidal endothelial cell |
NAFLD | non-alcoholic fatty liver disease |
NASH | non-alcoholic steatohepatitis |
PLIN5 | perilipin 5 |
PPAR | peroxisome proliferator-activated receptor |
ROS | reactive oxygen species |
SCD1 | stearoyl-CoA desaturase 1 |
SREBP1c | sterol regulatory element-binding protein 1 |
SREBP2 | sterol regulatory element-binding protein 2 |
TG | triglycerides |
TZDs | thiazolidinediones |
Appendix A
Pparb/dfl/fl Genotyping Protocol | |
PCR Reaction mixture | PCR program setting |
1.2 µL PBX10 | 95 °C 3 min |
0.6 µL AB008 | |
0.6 µL AB021 | 30 cycles: |
8.1 µL Water | 94 °C 15 s |
12.5 µL 2× KAPPA2G Fast (HotStart) genotyping mix with dye | 68 °C 10 s |
72 °C 20 s | |
72 °C 10 min | |
4 °C hold | |
FSP1cre-Pparb/d−/− Genotyping Protocol | |
PCR Reaction mixture | PCR program setting |
0.375 µL Fsp1 Ctrl Forward | 94 °C 3 min |
0.375 µL Fsp1 Ctrl Reverse | |
1.25 µL Fsp1 Forward | 30 cycles: |
1.25 µL Fsp1 Reverse | 95 °C 15 s |
8.25 µL Water | 59.2 °C 30 s |
12.5 µL 2× KAPPA2G Fast (HotStart) genotyping mix with dye | 72 °C 10 s |
1.0 µL template | |
72 °C 10 min | |
4 °C hold | |
Sry Genotyping Protocol | |
PCR Reaction mixture | PCR program setting |
1 µL Sry Forward | 94 °C 5 min |
1 µL Sry Reverse | |
3.5 µL Water | 33 cycles: |
7.5 µL 2× KAPPA2G Fast (HotStart) genotyping mix with dye | 94 °C 30 s |
1.0 µL template | 60 °C 30 s |
72 °C 45 s | |
72 °C 10 min | |
4 °C hold |
Appendix B
Gene | Primer Sequence |
Acaa2 | Forward (5′-3′) TCTTGACCCCAGCAAAACCA |
Reverse (5′-3′) CCACCTCGACGCCTTAACTC | |
Acc | Forward (5′-3′) GACAACACCTGTGTGGTGGA |
Reverse (5′-3′) GAGGTTGGAGGCAAAGGACA | |
Acat1 | Forward (5′-3′) AATGCTGGAGATTGACCCCC |
Reverse (5′-3′) CGGGCTCCAGACATCCCAAT | |
Acly | Forward (5′-3′) GTCCCAAGTCCAAGATCCCTG |
Reverse (5′-3′) TGTGATCCCCAGTGAAAGGG | |
Acox1 | Forward (5′-3′) GAGCAGCAGGAGCGTTTCTT |
Reverse (5′-3′) CAGGACTATCGCATGATTGGAAG | |
Agpat3 | Forward (5′-3′) TACTGCGAAGGAACACGCTT |
Reverse (5′-3′) TCTTGCAGGGCATCCTTCTC | |
Cd36 | Forward (5′-3′) TGATACTATGCCCGCCTCTCC |
Reverse (5′-3′) TTTCCCACACTCCTTTCTCCTCTA | |
Cyp51 | Forward (5′-3′) CGTCTACCTGTTCCGTCTCG |
Reverse (5′-3′) ATGTGGTGGACTTTTCGCTC | |
Dgat1 | Forward (5′-3′) TAGAAGAGGACGAGGTGCGAG |
Reverse (5′-3′) GTAGAGACAGCTTTGGCCTTGA | |
Dhcr7 | Forward (5′-3′) ATGGCTTCGAAATCCCAGCA |
Reverse (5′-3′) GAACCAGTCCACTTCCCAGG | |
Fas | Forward (5′-3′) AGTGTCCACCAACAAGCG |
Reverse (5′-3′) GATGCCGTCAGGTTTCAG | |
Fsp1 | Forward (5′-3′) CTCTCTTGGTCTGGTCTCAAC |
Reverse (5′-3′) AACTTGTCACCCTCTTTGCCT | |
Gpd1 | Forward (5′-3′) CATCACGACCTGCTATGGGG |
Reverse (5′-3′) GCTGCTCAATGGACTTTCCAG | |
Gpd2 | Forward (5′-3′) ACTCCGTTCTGGCTGGAGGAT |
Reverse (5′-3′) CATATGCCAGGCTCACTTGCTTC | |
Hadha | Forward (5′-3′) TGCTCAAGATGGTGGCGTCC |
Reverse (5′-3′) AATGCAGCCTCTGGAGCGTAG | |
Hmgcs1 | Forward (5′-3′) TCCCCTTTGGCTCTTTCACC |
Reverse (5′-3′) GGGCAACGATTCCCACATCT | |
Lpin3 | Forward (5′-3′) ACAGCTCATTGTACGTGGCA |
Reverse (5′-3′) ATCTCCACATGGCTTTCCCG | |
Plin5 | Forward (5′-3′) GCCATCAGACATGGTGGTGA |
Reverse (5′-3′) CCTCAGTCATGGGCAGGAAG | |
Ppara | Forward (5′-3′) TACTGCCGTTTTCACAAGTGC |
Reverse (5′-3′) AGGTCGTGTTCACAGGTAAGA | |
Pparb/d | Forward (5′-3′) CGGCAGCCTCAACATGG |
Reverse (5′-3′) AGATCCGATCGCACTTCTCATAC | |
Pparg1 | Forward (5′-3′) TGTGAGACCAACAGCCTGAC |
Reverse (5′-3′) CCGCTTCTTTCAAATCTTGTCTGT | |
Scd1 | Forward (5′-3′) GCCCACATGCTCCAAGAGAT |
Reverse (5′-3′) GGGCACTGTCTTCACCTTCT | |
Srebp1c | Forward (5′-3′) AAGCGCTACCGGTCTTCTATC |
Reverse (5′-3′) TGTGCACTTCGTAGGGTCAG | |
Srebp2 | Forward (5′-3′) TGGGCGATGGATGAGAGCAG |
Reverse (5′-3′) AACTGTAGCATCTCGTCGATGTC |
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Chen, J.; Zhuang, Y.; Sng, M.K.; Tan, N.S.; Wahli, W. The Potential of the FSP1cre-Pparb/d−/− Mouse Model for Studying Juvenile NAFLD. Int. J. Mol. Sci. 2019, 20, 5115. https://doi.org/10.3390/ijms20205115
Chen J, Zhuang Y, Sng MK, Tan NS, Wahli W. The Potential of the FSP1cre-Pparb/d−/− Mouse Model for Studying Juvenile NAFLD. International Journal of Molecular Sciences. 2019; 20(20):5115. https://doi.org/10.3390/ijms20205115
Chicago/Turabian StyleChen, Jiapeng, Yan Zhuang, Ming Keat Sng, Nguan Soon Tan, and Walter Wahli. 2019. "The Potential of the FSP1cre-Pparb/d−/− Mouse Model for Studying Juvenile NAFLD" International Journal of Molecular Sciences 20, no. 20: 5115. https://doi.org/10.3390/ijms20205115