Hedgehog signaling is a potent regulator of liver lipid metabolism and reveals a GLI-code associated with steatosis
- Leipzig University, Germany
- Leibniz Institute for Natural Product Research and Infection Biology – Hans Knöll Institute, Germany
- Regensburg University Hospital, Germany
Figures
Figure 1 with 1 supplement
Strategy for conditional and hepatocyte-specific deletion of Smo.
(A) Scheme of the tet-on system in the LC-1/rTALAP-1 mice. (B) Structure of Smo with loxP sites. (C) PCR, for Cre recombinase, yielded a 400 bp fragment in SLC-KO mice only. (D) PCR from liver …
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Figure 1—source data 1
Source data of qRT-PCR of Smo in different tissues and isolated hepatocytes of the SLC-WT and the SLC-KO mice (Figure 1F).
- https://doi.org/10.7554/eLife.13308.004
Figure 1—figure supplement 1
Influence of doxycycline on lipogenic gene expression qRT-PCR of Ppara, Pparb/d, Pparg, Elovl3, Elovl6 and Fasn in isolated hepatocytes of the male SLC-WT mice treated with doxycycline (n=3–4) for 10 days compared to male SLC-WT mice without doxycycline (n=3–4) at the same age.
https://doi.org/10.7554/eLife.13308.005
Figure 2 with 3 supplements
Liver phenotype of the SLC mice.
(A) The H&E and fat red staining of liver sections and hepatocytes showed strong steatosis in the male SLC-KO mice compared to the SLC-WT mice (bars: 200 µm, 100 µm and 50 µm) (pc: pericentral, pc: …
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Figure 2—source data 1
Source data of quantification of the fat red-stained liver sections from the male SLC-WT and SLC-KO mice (Figure 2B) and comparison of the liver/body ratio (Figure 2C).
- https://doi.org/10.7554/eLife.13308.007
Figure 2—figure supplement 1
Gene expression of insulin signaling in isolated hepatocytes from SLC mice.
Relative gene expression determined by qRT-PCR in isolated hepatocytes from the male SLC-WT (n=14) and the male SLC-KO (n=14) mice of insulin receptor (Inrs) and insulin receptor substrate 1 and 2 (I…
Figure 2—figure supplement 2
Serum lipoprotein levels of the SLC mice.
(A–D) Total cholesterol, HDL, LDL and VLDL in the serum of the male SLC-WT (n=5) and male SLC-KO mice (n=6).
Figure 2—figure supplement 3
Influence of cyclopamine on accumulation of neutral lipids in cultured mouse and human hepatocytes.
(A) Mouse and (B) human hepatocytes were cultured in the absence (left; vehicle only) or presence (right) of 10 µM cyclopamine (in 0.1% DMSO). After 72 h, cultures were fixed and stained with Nile …
Figure 3 with 2 supplements
Expression of genes and proteins related to Hh signaling in SLC mice.
(A–D) qRT-PCR data from isolated hepatocytes from the male SLC-WT (n=6–10) and the SLC-KO (n=6–10) mice illustrating the expression of (A) the ligands Ihh, Shh and Dhh (n.d.: not detectable); (B) …
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Figure 3—source data 1
Source data of the expression of genes related to Hh signaling in SLC mice (Figure 3A–D).
- https://doi.org/10.7554/eLife.13308.013
Figure 3—figure supplement 1
Western Blot of GLI3 in the SLC mice.
(A) Representative Western blot from isolated hepatocytes from male SLC-WT (n=4) and SLC-KO (n=4) mice. The blot shows the expected molecular weight from the two isoforms of GLI3, the full length …
Figure 3—figure supplement 2
The “steatotic Gli-code” extents to several mouse models and humans with steatosis.
(A) qRT-PCR data from isolated hepatocytes from the male MC4R-WT (n=4) and the MC4R-KO (n=4) mice illustrating the expression of Gli1, Gli2 and Gli3 at the age of 6 month; (B) qRT-PCR data from …
Figure 4
Gen and protein expression of hepatic TFs involved in lipid metabolism in SLC mice.
(A) Volcano blot visualizing differentially expressed genes in male SLC-KO mice detected by Affymetrix microarray analysis (n=4). All colored dots (blue and magenta) indicate an expression fold …
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Figure 4—source data 1
Gene set enrichment analysis of isolated hepatocytes from SLC-WT and SLC-KO mice.
- https://doi.org/10.7554/eLife.13308.017
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Figure 4—source data 2
Source data of gene expression of hepatic TFs involved in lipid metabolism in SLC mice (Figure 4B).
- https://doi.org/10.7554/eLife.13308.018
Figure 5
Expression of the hepatic TFs and enzymes involved in lipid metabolism in SLC mice.
(A–D) qRT-PCR data from hepatocytes from the male SLC-WT (n=6–13) and SLC-KO (n=6–13) mice. (A) Gata4, Gata6, Nfyb, Nfyg and Lxra. (B) Foxa1, Foxa2, Nr1d1 and Nr1d2. (C) Acaca, Acacb, Fasn, Gpam, …
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Figure 5—source data 1
Source data of expression of the hepatic TFs and enzymes involved in lipid metabolism in SLC mice (Figure 5A–D).
- https://doi.org/10.7554/eLife.13308.020
Figure 6 with 1 supplement
Expression of genes and proteins involved in lipid and mitochondrial energy metabolism in SLC mice.
(A) Immunohistochemistry of FASN in liver sections of male SLC-WT and SLC-KO mice (pp: periportal, pc: pericentral). (B–H) Measurements in freshly isolated hepatocytes from male SLC-WT and SLC-KO …
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Figure 6—source data 1
Source data of expression of genes and proteins involved in lipid and mitochondrial energy metabolism in SLC mice (Figure 6B–J).
- https://doi.org/10.7554/eLife.13308.022
Figure 6—figure supplement 1
Expression of respiratory chain complexes in SLC mice.
Enzyme activity of citrate synthase (CS) and respiratory chain complexes I-IV in liver tissue of the male SLC-WT (n=6) and the male SLC-KO (n=6) mice.
Figure 7 with 2 supplements
Influence of siRNA-mediated knockdown of Gli1, Gli2 and Gli3 on expression of genes of lipid metabolism.
(A–C) Isolated hepatocytes from male C57BL6/N mice (n=4–9) treated with the control, Gli1, Gli2 and Gli3 siRNAs. (A) Relative expression of Ppara, Pparg, Srebf1 and Srebf2, determined by qRT-PCR. …
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Figure 7—source data 1
Source data of the influence of siRNA-mediated knockdown of Gli1, Gli2 and Gli3 on expression of genes of lipid metabolism(Figure 7A,C).
- https://doi.org/10.7554/eLife.13308.025
Figure 7—figure supplement 1
Influence of siRNA-mediated knockdown of Gli1, Gli2 and Gli3 on gene expression of lipogenic enzymes.
(A–D) Isolated hepatocytes from male C57BL6/N mice (n=4–7) treated with the control, Gli1, Gli2 and Gli3 siRNAs. Relative expression of (A) Chrebp, (B) Fasn, (C) Elovl6 and (D) Elovl3 was determined …
Figure 7—figure supplement 2
Chromatin immunoprecipitation experiments for GLI3 binding sites.
( A) Consensus sequence of the GLI3 binding site at -1767 bp of the Fasn promoter, at -2674 bp of the Ppara promoter and at -1156 bp of the Srebf1 promoter according to MotivMap. (B) qRT-PCR …
Figure 8
Influence of siRNA-mediated knockdown of Sufu on lipid metabolism in vitro.
(A-C) Isolated hepatocytes from male C57BL6/N mice (n=8) treated with the control and Sufu siRNA. Relative expression of the following genes was determined by qRT-PCR 48 hr post-transfection (A) Sufu…
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Figure 8—source data 1
Data source of the influence of siRNA-mediated knockdown of Sufu on lipid metabolism in vitro(Figure 8 A-C, D).
- https://doi.org/10.7554/eLife.13308.029
Figure 9
Influence of GLI1, GLI2 and GLI3 overexpression on lipid content in hepatocytes of ob/ob mice.
Isolated hepatocytes from male ob/ob mice (n=3) transfected with the control plasmid (MOCK) and human overexpression plasmids of GLI1, GLI2, and GLI3 or the combination of GLI1 and GLI3, 72 hr …
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Figure 9—source data 1
Source data of the influence of GLI1, GLI2 and GLI3 overexpression on lipid content in hepatocytes of ob/ob mice (Figure A-D).
- https://doi.org/10.7554/eLife.13308.031
Figure 10
Regulation of hepatic lipid metabolism by Hh signaling.
(A) Intact Hh signaling allows a self-supporting network of the GLI TFs (Schmidt-Heck et al., 2015). Gli1 and Gli3 exert attenuating effects on lipogenic TFs resulting in balanced liver lipid …
Tables
Table 1
Serum concentrations of glucose, insulin and enzyme activities of ALAT, ASAT and GLDH in SLC mice.
| parameter | SLC-WT | n | SLC-KO | n |
|---|---|---|---|---|
| insulin (pmol/l) | 80.06 ± 9.98 | 13 | 110.88 ± 31.31 | 7 |
| glucose (mmol/dl) | 10.16 ± 1.80 | 5 | 7.68 ± 1.02 | 6 |
| ALAT (µkat/l) | 1.02 ± 0.32 | 5 | 0.81 ± 0.03 | 5 |
| ASAT (µkat/l) | 2.95 ± 0.90 | 5 | 2.50 ± 0.41 | 5 |
| GLDH (µkat/l) | 0.42 ± 0.13 | 5 | 0.28 ± 0.05 | 5 |
Additional files
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Supplementary file 1. (A)
Primers for genotyping the SLC mice.
(B) Primers for qRT-PCR on murine mRNA. (C) Primers for siRNA mediated knockdown of Sufu. (D) Primers for ChIP (E) Primers for human Gli factors
- https://doi.org/10.7554/eLife.13308.033
