Article Figures & Data
Figures
- FIG. 1.
Hepatic genes activated by Wy14643 and fasting show a variable dependence on PPARα. Livers from wild-type and PPARα−/− mice treated with the PPARα agonist Wy14643 for 6 h or fasted for 24 h were used for gene expression profiling (n = 4 to 5 mice per group). (A) Overlap in top-regulated pathways between Wy14643 treatment and fasting according to gene set enrichment analysis. Gene sets with a false discovery rate P value of <0.1 were considered significant. (B) Overlap of upregulated genes between Wy14643 treatment and fasting (criteria for inclusion: P < 0.01 and a change of >1.5-fold). (C) Scatter plot showing the effects of fasting in genes significantly upregulated by Wy14643. The y axis and x axis show the effects of fasting in wild-type and PPARα−/− mice, respectively. Red dots represent classical PPARα target genes, while blue dots are Wy14643-responsive genes that are more significantly upregulated by fasting in the PPARα−/− mouse compared to wild type. (D) Heatmap showing the changes (n-fold) of genes compared to the wild-type control/fed state. (Upper panel) Classical PPARα target genes, showing a PPARα-dependent increase in gene expression upon Wy14643 treatment as well as fasting. Genes in the lower panel exhibit a PPARα-dependent induction upon Wy14643 treatment but are induced independently of PPARα upon fasting.
- FIG. 2.
Lpin2 and St3gal5 are induced during fasting independently of PPARα. Livers from wild-type and PPARα−/− mice fasted for 24 h or treated with tridocosahexaenoin (DHA) for 6 h were used for gene expression profiling (n = 4 to 5 mice per group). (A and B) Gene expression of classical PPARα targets Aldh3a2 and Cpt2 (A) and Lpin2 and St3gal5 (B) in fed and fasted wild-type and PPARα−/− mice. (C and D) Gene expression of Aldh3a2 and Cpt2 (C) and Lpin2 and St3gal5 (D) after treatment with the dietary fatty acid tridocosahexaenoin. Error bars represent standard errors of the means. *, significantly different according to Student's t test (P < 0.05).
- FIG. 3.
PPARβ/δ as alternative transcription factor to PPARα in mouse liver. (A) Lpin2 and St3gal5 expression in livers of wild-type mice (n = 5) treated with the PPARβ/δ agonist GW501516 for 6 h or PPARα−/− mice (n = 5) treated with the PPARβ/δ agonist L165041 for 5 days. Error bars represent standard errors of the means. (B) PPREs conserved between mouse and human were identified 1,291 bp and 23,333 bp downstream of the TSS of the Lpin2 and St3gal5 genes. (C) HepG2 cells were transfected with a PPARβ/δ expression vector and a simian virus 40 reporter vector containing 201-nucleotide and 183-nucleotide fragments with the putative PPREs within the Lpin2 and St3gal5 genes, respectively. The reporter vector (PPRE)3-TK-luciferase served as a positive control. Luciferase and β-galactosidase activities were determined 24 h after exposure of the cells to 1 μM GW501516. Error bars represent standard errors of the means. (D) Chromatin was extracted from livers of fed or 24-h-fasted wild-type and PPARα−/− mice (n = 3 per group). ChIP was performed with antibodies against PPARα and PPARβ/δ on the TSS of Lpin2, St3gal5, Aldh3a2, Cpt2, and Rplp0. Rabbit IgG was used as a specificity control. Gray bars, fed state; black bars, 24-h fasted state. Error bars represent standard deviations. (E) Expression of Lpin2 and St3gal5 in livers of fed and 24-h-fasted wild-type and PPARβ/δ−/− mice (n = 4 to 5 per group). Relative induction by fasting is indicated. Error bars represent standard errors of the means. (F and G) Plasma FFA levels in wild-type and PPARα−/− mice (F) or wild-type and PPARβ/δ−/− mice (G) sacrificed in a fed or 24-h-fasted state. Error bars represent standard errors of the means. *, significantly different according to Student's t test (P < 0.05).
- FIG. 4.
Angptl4 stimulates adipose tissue lipolysis. (A) Glycerol concentration in medium of 3T3-L1 cells treated for 30 min with isoproterenol or with concentrated conditioned medium of HEK293 cells transfected with mAngptl4. Control cells were treated with conditioned medium of nontransfected HEK293 cells. Error bars represent standard errors of the means. *, significantly different according to Student's t test (P < 0.05). (B) Increase in fatty acid and glycerol concentrations in medium of adipose tissue explants from transgenic mice overexpressing Angptl4 (Tg), wild-type (+/+), and homozygous knockout (−/−) mice. Values are corrected for weight of explants. (C and D) Plasma FFAs (C) and triglycerides (D) in transgenic mice overexpressing Angptl4 (Angptl4-Tg) and wild-type (Angptl4+/+), heterozygous (Angptl4+/−), and homozygous (Angptl4−/−) mice fed or in a 24-h-fasted state (n = 5). Gray bars, fed state; black bars, 24-h-fasted state. Error bars represent standard errors of the means. Different letters indicate statistically significant differences (Student's t test; P < 0.05). (E) Eosin and hematoxylin staining of epididymal adipose tissue.
- FIG. 5.
Plasma FFAs do not activate hepatic PPARα. Transgenic mice overexpressing Angptl4 (Angptl4-Tg) or wild-type (Angptl4+/+), heterozygous (Angptl4+/−), and homozygous knockout (Angptl4−/−) mice were sacrificed in the fed state or after a 24-h fast (n = 5). Results show hepatic gene expression of classical PPARα targets Aldh3a2 and Cpt2 (A), Pparα (B), and Pparβ/δ (F). Gray bars, fed state; black bars, 24-h-fasted state. Error bars represent standard errors of the means. *, P < 0.05. (C) Fatty acids activate PPARβ/δ in a transactivation assay using a GAL4-LBDPPARβ/δ fusion. Fatty acids were used at 125 μM or 250 μM (normal or bold plus sign). (D) A nuclear receptor PamChipH assay was used to measure the interaction between PPARβ/δ and immobilized peptides corresponding to specific coregulator-nuclear receptor binding regions in the presence and absence of fatty acids (125 μM). (E) Fatty acids (100 μM) upregulated expression of PPARβ/δ target Adfp in rat FaO hepatoma cells. (G) Chromatin was extracted from livers of 24-h-fasted transgenic mice overexpressing Angptl4 (Tg) and wild-type (+/+) and homozygous Angptl4 knockout (−/−) mice (n = 3 per group). ChIP was performed with antibodies against PPARα and PPARβ/δ on the TSS of Lpin2, St3gal5, and Rplp0. Rabbit IgG was used as a specificity control. Error bars represent standard deviations. Different lowercase letters indicate statistically significant differences (Student's t test; P < 0.05). (H) Hepatic gene expression of Lpin2 and St3gal5. Gray bars, fed state; black bars, 24-h-fasted state. Error bars represent standard errors of the means. Different lowercase letters indicate statistically significant differences (Student's t test; P < 0.05).
- FIG. 6.
PPARα activation during fasting may be mediated by PGC1α upregulation. (A) Expression of Pgc1a in livers of fed or fasted wild-type mice (n = 5). Fasting statistically significantly induced gene expression of Pgc1α (P < 0.05). Error bars represent standard errors of the means. (B) Chromatin was extracted from livers of fed and 24-h-fasted wild-type and PPARα−/− mice (n = 3 per group). ChIP was performed with antibodies against PGC1α on the TSS of Aldh3a2 and Cpt2 and the negative control gene Rplp0. Rabbit IgG was used as a specificity control. Gray bars, fed state; black bars, 24-h-fasted state. Error bars represent standard deviations. Fasting significantly induced binding of PGC1α in wild-type but not PPARα−/− mice (P < 0.05).
Additional Files
Supplemental material
Files in this Data Supplement:
- Supplemental file 1
-
Fig. S1 (Classical PPARα target genes do not follow plasma FFAs).
JPEG Image file, 627K.
- Supplemental file 1
-
Fig. S1 (Classical PPARα target genes do not follow plasma FFAs).
