Glucocorticoid receptor-PPARα axis in fetal mouse liver prepares neonates for milk lipid catabolism
- University of Lausanne, Switzerland
- Nanyang Technological University, Singapore
- INRA, Université de Toulouse, France
- IFR 150 Plateforme Metatoul, Institut Fédératif de Recherche Bio-Médicale de Toulouse INSERM U563, France
Figures
Figure 1
GR directly controls fetal PPARα expression.
(A, B) Ontogenic expression of Nr3c1 (A) and Ppara (B) mRNA in the developing mouse liver. *p<0.05, **p<0.01 vs. E13 samples; #p<0.05, ##p<0.01 vs. WT counterparts. (C) Enrichment of the DNA …
Figure 2 with 1 supplement
PPARα is a functional transcription factor in the term fetus.
(A) Heat map of genes significantly altered at E19.5 (left panel) or P2 (right panel) in Ppara-/- livers (red: up-regulation; blue: down-regulation). Genes were grouped by fetal (E19.5) or postnatal …
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Figure 2—source data 1
A list of PPARα-regulated genes and pathways in prenatal and neonatal livers.
The dataset provides a list of PPARα-regulated genes (A) and pathways (B) in E19.5 and P2 mouse livers. Significant genes based on a false discovery rate < 0.05 were classified as regulated at both E19.5 and P2, in E19.5 only, or P2 only. The logarithmic fold change (logFC) cut-off value was set at 1.3. For each group, the significant enrichment of underlying KEGG, GO, and Reactome curated pathways was determined from the hypergeometric distribution and corrected for multiple comparisons.
- https://doi.org/10.7554/eLife.11853.005
Figure 2—figure supplement 1
PPARα deficiency leads to compensatory up-regulation of genes.
(A) Venn diagram depicting the number of up-regulated genes in the Ppara-/- liver with overlapping PPARα regulation (false discovery rate <0.05) before (E19.5) and after (P2) birth. The absolute …
Figure 3
Defective mitochondrial function, fatty acid oxidation and energy production in Ppara-/- hepatocytes.
(A,B) Flow cytometric analyses of intracellular peroxisomes and mitochondria in primary hepatocytes isolated from Ppara-/- and Ppara+/+ livers at E19.5 (A) and P2 (B) using Alexa Fluor 488-labeled …
Figure 4 with 2 supplements
PPARα deficiency leads to congenital hepatic steatosis after birth.
(A) Photographs showing dissected Ppara-/- and Ppara+/+ pups (left) and livers (right). The pallid liver of a Ppara-/- pup is indicated by an arrow. The white content of the stomach indicates milk …
Figure 4—figure supplement 1
Postnatal lipid catabolic derangements in Ppara-/- pups.
(A,B) Boxplots showing the ω-3 (A) and ω-6 (B) indexes of the P2 livers calculated from the ratios of docosahexaenoic acid to α-linolenic acid and arachidonic acid to linoleic acid, respectively. …
Figure 4—figure supplement 2
Anaplerotic compensation for defective oxidative metabolism and ketogenesis in suckling Ppara-/- pups.
(A,B) Body weight (A) and plasma glucose levels (B) of Ppara+/+ and Ppara-/- pups at the indicated postnatal days. (C) Alanine aminotransferase (ALT) activity measured in liver lysates at P2. (D) …
Figure 5
A temporal dichotomy in the regulation of PPARα target genes before and after birth.
(A) Immunoblots showing the ontogenic expression of cytoplasmic and nuclear GR, HDAC3, PPARα, and its target genes, including ACOX1, Cyp4A14, FGF21, and full-length (FL)-ANGPTL4, in Ppara-/- and Ppar…
Figure 6
PPARα-mediated regulation of Fgf21 expression is dictated by the occupancy of HDAC3 on its promoter.
(A–C) Enrichment of the DNA fragment containing the ACOX1 (A), Cyp4A14 (B), or FGF21 (C) TSS in primary hepatocytes isolated from Ppara+/+ pups using antibodies against acetyl-histone four (AcH4), …
Figure 7
β-hydroxybutyrate acts as an endogenous inhibitor of HDAC3 to activate Fgf21 expression upon milk suckling.
(A–C) Ex vivo liver explants isolated from Ppara+/+ fetuses at E19.5 were used to study the effects of butyrate, β-hydroxybutyrate, and trichostatin in the presence or absence of WY-14643 (WY) on …
Figure 8
Schematic illustrations of GR-mediated regulation of PPARα expression, HDAC3-dependent and -independent regulation of PPARα target genes before and after birth, and the affected liver metabolic processes with or without PPARα.
(A) In fetal liver, hormone-activated GR binds to the GR response element (GRE) spanning -1007 to -993 within the PPARα promoter region to directly activate the transcription of Ppara. NTD: …
Author response image 1
Tables
Table 1
Standard chow diet formulation.
| Manufacturer: SPECIALITY FEEDS Product ID: AIN93M | |||
|---|---|---|---|
Standard AIN93M rodent diet | |||
A semi-pure diet formulation for laboratory rats and mice based on AIN-93M. This formulation satisfies the maintenance nutritional requirements of rats and mice. Some modifications have been made to the original formulation to suit locally available raw materials.
| |||
| Calculated nutritional parameters | Ingredients | ||
| Protein | 13.6% | Casein (acid) | 140 g/kg |
| Total fat | 4.0% | Sucrose | 100 g/kg |
| Total digestible carbohydrate as defined by FSANZ Standard 1.2.8 | 64.8% | Canola oil | 40 g/kg |
| Cellulose | 50 g/kg | ||
| Crude fiber | 4.7% | Wheat starch | 472 g/kg |
| AD fiber | 4.7% | Dextrinised starch | 155 g/kg |
| Digestible energy | 15.1 MJ/kg | DL methionine | 1.8 g/kg |
| % Total calculated digestible energy from lipids | 9.0% | Calcium carbonate | 13.1 g/kg |
| % Total calculated digestible energy from protein | 15.0% | Sodium chloride | 2.6 g/kg |
| AIN93 trace minerals | 1.4 g/kg | ||
| Diet form and features | Potassium citrate | 1.0 g/kg | |
| Potassium dihydrogen phosphate | 8.8 g/kg | |
| Potassium sulphate | 1.6 g/kg | ||
| Choline chloride (75%) | 2.5 g/kg | ||
| AIN93 vitamins | 10 g/kg | ||
| Calculated amino acids | Calculated total vitamins | ||
| Valine | 0.90% | Vitamin A (retinol) | 4000 IU/kg |
| Leucine | 1.30% | Vitamin D (cholecalciferol) | 1000 IU/kg |
| Isoleucine | 0.60% | Vitamin E (α-tocopherol acetate) | 75 mg/kg |
| Threonine | 0.60% | Vitamin K (menadione) | 1 mg/kg |
| Methionine | 0.60% | Vitamin C (ascorbic acid) | None added |
| Cystine | 0.05% | Vitamin B1 (thiamine) | 6.1 mg/kg |
| Lysine | 1.00% | Vitamin B2 (riboflavin) | 6.3 mg/kg |
| Phenylanine | 0.70% | Niacin (nicotinic acid) | 30 mg/kg |
| Tyrosine | 0.70% | Vitamin B6 (pryridoxine) | 7 mg/kg |
| Tryptophan | 0.20% | Pantothenic acid | 16.5 mg/kg |
| Histidine | 0.42% | Biotin | 200 μg/kg |
| Calculated total minerals | Folic acid | 2 mg/kg | |
| Calcium | 0.47% | Inositol | None added |
| Phosphorus | 0.35% | Vitamin B12 (cyancobalamin) | 103 μg/kg |
| Magnesium | 0.09% | Choline | 1670 mg/kg |
| Sodium | 0.15% | Calculated fatty acid composition | |
| Chloride | 0.16% | Myristic acid 14:0 | No data |
| Potassium | 0.40% | Palmitic acid 16:0 | 0.20% |
| Sulphur | 0.17% | Stearic acid 18:0 | 0.10% |
| Iron | 75 mg/kg | Palmitoleic acid 16:1 | No data |
| Copper | 6.9 mg/kg | Oleic acid 18:1 | 2.40% |
| Iodine | 0.2 mg/kg | Gadoleic acid 20:1 | trace |
| Manganese | 19.5 mg/kg | Linoleic acid 18:2 n6 | 0.80% |
| Cobalt | No data | α-Linolenic acid 18:3 n3 | 0.40% |
| Zinc | 47 mg/kg | Arachidonic acid 20:4 n6 | No data |
| Molybdenum | 0.15 mg/kg | EPA 20:5 n3 | No data |
| Selenium | 0.3 mg/kg | DHA 22:6 n3 | No data |
| Cadmium | No data | Total n3 | 0.45% |
| Chromium | 1.0 mg/kg | Toal n6 | 0.76% |
| Fluoride | 1.0 mg/kg | Total mono-unsaturated fats | 2.46% |
| Lithium | 0.1 mg/kg | Total polyunsaturated fats | 1.21% |
| Boron | 3.1 mg/kg | Total saturated fats | 0.28% |
| Nickel | 0.5 mg/kg | ||
| Vanadium | 0.1 mg/kg | ||
-
Calculated data uses information from typical raw material composition. It could be expected that individual batches of diet will vary from this figure. Diet post treatment by irradiation or autoclave could change these parameters. We are happy to provide full calculated nutritional information for all of our products, however we would like to emphasise that these diets have been specifically designed for manufacture by Specialty Feeds.
Table 2
Primer sequences for mouse genes used in quantitative real-time PCR.
| Gene | Forward primer (5’-3’) | Reverse primer (5’-3’) |
|---|---|---|
| Acaa2 | ATGTGCGCTTCGGAACCAAA | CAAGGCGTATCTGTCACAGTC |
| Acetyl-coenzyme A acyltransferase 2/short chain-specific 3-ketoacyl-CoA thiolase (mitochondrial) | ||
| Acadl | TGCCCTATATTGCGAATTACGG | CTATGGCACCGATACACTTGC |
| Acyl-coenzyme A dehydrogenase, long chain | ||
| Acadvl | TGACCTTGGTGTTAGCGTTAC | CTGGGCCTTTGTGCCATAGAG |
| Acyl-coenzyme A dehydrogenase, very long chain | ||
| Acox1 | TCGAAGCCAGCGTTACGAG | ATCTCCGTCTGGGCGTAGG |
| Acyl-CoA oxidase 1, palmitoyl | ||
| Acsl1 | ACCAGCCCTATGAGTGGATTT | CAAGGCTTGAACCCCTTCTG |
| Acyl-CoA synthetase, long chain family member 1 | ||
| Angptl4 | TCCAACGCCACCCACTTAC | TGAAGTCATCTCACAGTTGACCA |
| Angiopoietin-like 4 | ||
| Cpt1a | CTATGCGCTACTCGCTGAAGG | GGCTTTCGACCCGAGAAGA |
| Carnitine palmitoyltransferase 1a (liver) | ||
| Cpt2 | CAAAAGACTCATCCGCTTTGTTC | CATCACGACTGGGTTTGGGTA |
| Carnitine palmitoyltransferase 2 | ||
| Cyp4a14 | TCATGGCGGACTCTGTCAATA | GCAGGCGAAAGAAAGTCAGG |
| Cytochrome P450, family 4, subfamily a, polypeptide 14 | ||
| Ehhadh | ACAGCGATACCAGAAGCCAG | TGGCAATCCGATAGTGACAGC |
| Enoyl-coenzyme A, hydratase/3-hydroxyacyl coenzyme A dehydrogenase | ||
| Fabp1 | AAGGCAGTCGTCAAGCTGG | CATTGAGTTCAGTCACGGACTT |
| Fatty acid bind protein 1 (liver) | ||
| Fgf21 | CTGCTGGGGGTCTACCAAG | CTGCGCCTACCACTGTTCC |
| Fibroblast growth factor 21 | ||
| Gck | AGACGAAACACCAGATGTATTCC | GAAGCCCTTGGTCCAGTTGAG |
| Glucokinase | ||
| Nr3c1 | CCGGGTCCCCAGGTAAAGA | TGTCCGGTAAAATAAGAGGCTTG |
| Glucocorticoid receptor | ||
| Hadha | AGCAACACGAATATCACAGGAAG | AGGCACACCCACCATTTTGG |
| Hydroxyacyl-coenzyme A dehydrogenase, alpha subunit | ||
| Hadhb | TGAATATGCACTGCGTTCTCAT | CCTTTCCTGGTACTTTGAAGGG |
| Hydroxyacyl-coenzyme A dehydrogenase, beta subunit | ||
| Hk1 | CGGAATGGGGAGCCTTTGG | GCCTTCCTTATCCGTTTCAATGG |
| Hexokinase 1 | ||
| Fkbp51 | TTTGAAGATTCAGGCGTTATCCG | GGTGGACTTTTACCGTTGCTC |
| FK506 binding protein 51 | ||
| Pex19 | GACAGCGAGGCTACTCAGAG | GCCCGACAGATTGAGAGCA |
| Peroxisomal biogenesis factor 19 | ||
| Ppara | TCGGCGAACTATTCGGCTG | GCACTTGTGAAAACGGCAGT |
| Peroxisome proliferator activated receptor alpha | ||
| Slc25a20 | GCGCCCATCATTGGAGTCA | CACACCAGATAACATCCCAGC |
| Solute carrier family 25 (mitochondrial carnitine/acylcarnitine translocase), member 20 | ||
| 36B4/RplP0 | CGAGGACCGCCTGGTTCTC | GTCACTGGGGAGAGAGAGG |
| Ribosomal protein P0 | ||
Table 3
Primer sequences for mouse genes used in chromatin immunoprecipitation.
| Gene | Forward primer (5’-3’) | Reverse primer (5’-3’) |
|---|---|---|
| ACOX1_TSS | TCCCGGAAAGATCACGTGAACC | TCCCCGAGCGGCTCCTCGCCA |
| ACOX1_PPRE | TAGCCAACGACAATGAACC | CGGAAACCAGAAGGGAATG |
| ANGPTL4_TSS | CCAGCAAGTTCATCTCGTCC | TCCCTCCCACTCCCACACC |
| CYP4A14_TSS | ATTCCCCCTCCCACAAGTAG | CCCATGGTTAGTAGTTTCTGGA |
| CYP4A14_PPRE | AAGGAAAAGGCCACCGTCTA | TCCATCTCACTGAACTTTACCC |
| FGF21_TSS | ATATCACGCGTCAGGAGTGG | TCCCCAGCTGAGAAGACACT |
| FGF21_PPRE | AGGGCCCGAATGCTAAGC | AGCCAAGCAGGTGGAAGTCT |
| PPARα_TSS | GTTGTCATCACAGCTTAGCG | CAGATAAGGGACTTTCCAGGTC |
| PPARα_GRE (−1007 to −993) | GGGACTCGGGGAACAAGCTGTGCGATCTAG | GGAAGGGTGCGCCTTGGCGCGCACTCC |
| PPARα_GRE (−2080 to −2066) | CTTTCCTCTCAATACAGTCTGTCAAACAAAA | GTTTTGTTTGTTTTGACTCTCTGTCCAG |
| PPARα_GRE (−2953 to −2939) | AAGGGTGAACACACTTTGTTTCCTGGATG | TCCACCAGGGCAGGGGAAGTAGGTATT |
Table 4
Control diet formulation.
| Manufacturer: KLIBA NAFAG, SWITZERLAND Product ID: 2222 | |
| Mouse and rat | Experimental diet, purified diet |
AIN-93G | |
| Major nutrients | |
| Dry matter | 90.0% |
| Crude protein | 18.0% |
| Crude fat | 7.0% |
| Crude fiber | 3.5% |
| Crude ash | 3.0% |
| Nitrogen-free extract (NFE) | 58.5% |
| Gross energy | 17.5 MJ/kg |
| Metabolic energy | 15.9 MJ/kg |
| Starch | 35.0% |
| Amino acids |
|
| Arginine | 0.65% |
| Lysine | 1.40% |
| Methionine | 0.50% |
| Methionine + cystine | 0.85% |
| Tryptophan | 0.22% |
| Threonine | 0.70% |
| Major mineral elements |
|
| Calcium | 0.52% |
| Phosphorus | 0.32% |
| Magnesium | 0.08% |
| Sodium | 0.22% |
| Potassium | 0.36% |
| Chlorine | 0.15% |
| Trace elements |
|
| Iron | 65 mg/kg |
| Zinc | 45 mg/kg |
| Copper | 6 mg/kg |
| Iodine | 0.6 mg/kg |
| Manganese | 12 mg/kg |
| Selenium | 0.2 mg/kg |
| Vitamins |
|
| Vitamin A | 4000 IU/kg |
| Vitamin D3 | 1000 IU/kg |
| Vitamin E | 100 mg/kg |
| Vitamin K3 | 4 mg/kg |
| Vitamin B1 | 6 mg/kg |
| Vitamin B2 | 6 mg/kg |
| Vitamin B6 | 7 mg/kg |
| Vitamin B12 | 0.05 mg/kg |
| Nicotinic acid | 30 mg/kg |
| Pantothenic acid | 16 mg/kg |
| Folic acid | 2 mg/kg |
| Biotin | 0.2 mg/kg |
| Choline | 1200 mg/kg |
| Ingredients |
|
| Corn starch, casein, dextrose, sucrose, refined soybean oil, cellulose, minerals, vitamins, amino acids |
|
| Remarks |
|
| - Experimental diet for mice and rats |
|
| - Given values are calculated averages in air-dry feed |
|
| - Production on demand |
|
| Delivery form |
|
| Pellets 10 mm round |
|
| 2222.PH.A05: |
|
| 5 kg in welded aluminium bag 2222.MA.A05: 5 kg in welded aluminium bag |
|
| KLIBA NAFAG | PROVIMI KLIBA AG | CH-4303 Kaiseraugst | Tel. +41 61 816 16 16 | Fax. +41 61 816 18 00 | kliba-nafag@provimi-kliba.ch | www.kliba-nafag.ch | |
Table 5
High-fat diet formulation.
| Manufacturer: KLIBA NAFAG, SWITZERLAND Product ID: 2201 | |
|---|---|
| Mouse and rat | Experimental diet, purified diet |
Ketogenic diet XL75:XP10 | |
| Major nutrients | |
| Dry matter | 99.1% |
| Crude protein | 9.9% |
| Crude fat | 74.4% |
| Crude fiber | 5.5% |
| Crude ash | 6.3% |
| Nitrogen-free extract (NFE) | 3.0% |
| Metabolic energy | 7208 kcal/kg |
| Starch | 0.7% |
| Amino acids | |
| Arginine | 0.35% |
| Lysine | 0.79% |
| Methionine | 0.28% |
| Methionine + cystine | 0.90% |
| Tryptophan | 0.13% |
| Threonine | 0.38% |
| Major mineral elements | |
| Calcium | 0.98% |
| Phosphorus | 0.61% |
| Magnesium | 0.15% |
| Sodium | 0.40% |
| Potassium | 0.69% |
| Chlorine | 0.57% |
| Trace elements | |
| Iron | 151 mg/kg |
| Zinc | 97 mg/kg |
| Copper | 16 mg/kg |
| Iodine | 1.4 mg/kg |
| Manganese | 31 mg/kg |
| Selenium | 0.6 mg/kg |
| Vitamins | |
| Vitamin A | 8000 IU/kg |
| Vitamin D3 | 2000 IU/kg |
| Vitamin E | 200 mg/kg |
| Vitamin K3 | 9 mg/kg |
| Vitamin B1 | 12 mg/kg |
| Vitamin B2 | 13 mg/kg |
| Vitamin B6 | 14 mg/kg |
| Vitamin B12 | 0.1 mg/kg |
| Nicotinic acid | 66 mg/kg |
| Pantothenic acid | 32 mg/kg |
| Folic acid | 5 mg/kg |
| Biotin | 0.4 mg/kg |
| Choline | 1975 mg/kg |
| Ingredients | |
| Beef fat, casein, cellulose, minerals, vitamins, amino acids | |
| Remarks | |
| - Experimental diet for mice and rats | |
| - Given values are calculated averages in air-dry feed | |
| - Production on demand | |
| Delivery form | |
| Paste | |
| 2201.MA.A05: | |
| 5 kg in welded aluminium bag | |
| KLIBA NAFAG | PROVIMI KLIBA AG | CH-4303 Kaiseraugst | Tel. +41 61 816 16 16 | Fax. +41 61 816 18 00 | kliba-nafag@provimi-kliba.ch | www.kliba-nafag.ch | |
