Angptl8 mediates food-driven resetting of hepatic circadian clock in mice

Figures

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  Fig. 1. Angptl8 responses to food signals and peripheral clocks. a Venn diagrams of high-throughput RNA sequencing results from liver samples of mice subjected to 16-h fasting, 16-h fasting followed by 20-h refeeding, or DD. b Heat map of six clustered genes derived from high-throughput RNA sequencing results. c qPCR analyses of Angptl8 mRNA expression levels in the liver of mice subjected to time-restricted feeding. d Serum Angptl8 levels. For c, d n = 5, **P < 0.01 NF12 vs. NF0, ##P < 0.01 DF12 vs. DF0, one-way ANOVA followed by Bonferroni’s post hoc test. e Serum Angptl8 levels in mice subjected to DD. n = 5, **P < 0.01, Peak vs. Nadir, one-way ANOVA followed by Bonferroni’s post hoc test. f Serum Angptl8 levels in mice subjected to 16-h fasting or 16-h fasting, followed by 20-h refeeding. n = 5, **P < 0.01 16-h fasted vs. ad libitum, ##P < 0.01 16-h fasted vs. refed, one-way ANOVA followed by Bonferroni’s post hoc test. All data are presented as the means ± SD. Source data are provided as a Source Data file

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  Fig. 2. Angptl8 resets liver clock in vivo. a Representative actograms of the wheel-running activities of mice i.p. injected recombinant Angptl8 or vehicle (PBS) for 24 consecutive days at ZT2 (or CT2 when mice switched to DD). n = 5. b Calculation of mouse circadian period in a. c Calculation of mouse circadian phase shift in a. *P < 0.05 and **P < 0.01 Angptl8 vs. PBS, one-way ANOVA followed by Bonferroni’s post hoc test. d, e qPCR analyses of clock and metabolic genes expression in the liver of mice receiving 15-day injection with recombinant Angptl8 or vehicle (PBS) at ZT2. n = 5, *P < 0.05 and **P < 0.01 Angptl8 vs. PBS at the same time point, two-way ANOVA followed by Bonferroni’s post hoc test. f Serum levels of TG, TC and glucose in d. All data are presented as the means ± SD. Source data are provided as a Source Data file

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  Fig. 3. Angptl8 resets liver clock in vitro. a Representative luminescence traces of Per2::Luc U2OS cells stimulated with either Angptl8 or 50% horse serum for 2 h. b, c The periods and amplitudes of circadian transcriptional activities of Per2 promoter in Per2::Luc U2OS cells stimulated with either 40 nM Angptl8 or 50% horse serum for 2 h. n = 3. All the data were calculated from fit curve data. d qPCR analyses of time-course expression of clock genes in mouse Hepa1c1c-7 cells similarly treated in panel a. n = 3. All data are presented as the means ± SD. Source data are provided as a Source Data file

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  Fig. 4. Angptl8 induces Per1 expression in vitro and in vivo. a qPCR analyses of the mRNA expression of core clock genes in mouse Hepa1c1c-7 hepatoma cells stimulated with either 40 nM Angptl8 or vehicle (PBS) for 2 h. n = 3, *P < 0.05 and **P < 0.01 Angptl8 vs. PBS, one-way ANOVA followed by Bonferroni’s post hoc test. b qPCR and Western blot analyses of Per1 expression in Hepa1c1c-7 cells stimulated with Angptl8 at indicated doses for 2 h. n = 3, **P < 0.01 Angptl8 vs. PBS, one-way ANOVA followed by Bonferroni’s post hoc test. c qPCR and Western blot analyses of Per1 expression in Hepa1c1c-7 cells stimulated with 40 nM Angptl8 for indicated times. n = 3, *P < 0.05 and **P < 0.01 vs.0 h, one-way ANOVA followed by Bonferroni’s posthoc test. d Reporter gene assays in Hepa1c1c-7 cells transfected with plasmids expressing Per1 promoter-luc (−2100 to +100 bp) for 36 h and then treated with 40 nM Angptl8, 50% horse serum, or indicated controls for another 2 h. n = 8, **P < 0.01 vs. No stimulus group, one-way ANOVA followed by Bonferroni’s post hoc test. e Per1 mRNA accumulation was assessed by CHX (cycloheximide) chase experiments. n = 3, **P < 0.01 Angptl8 vs. PBS, one-way ANOVA followed by…

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  Fig. 5. Kinases activation mediates Angptl8-induced Per1 expression. a Western blot analyses of ERK1/2, P38, NF-κB, AKT, and GSK-3β phosphorylation in Hepa1c1c-7 cells stimulated with 40 nM Angptl8 for indicated times. b Reporter gene assays in Hepa1c1c-7 cells transfected with plasmids expressing Per1-Luc for 36 h and then treated with 40 nM Angptl8 alone or in combinations of various signaling pathway inhibitors for 2 h. n = 6, **P < 0.01 Angptl8 vs. Vehicle group, ##P < 0.01 Angptl8 + inhibitor vs. Angptl8 alone group, one-way ANOVA followed by Bonferroni’s post hoc test. c, d qPCR and Western blot analyses of Per1 expression in Hepa1c1c-7 cells treated with 40 nM Angptl8 alone or in combinations of various signaling pathway inhibitors for 2 h. n = 3, **P < 0.01 Angptl8 vs. Vehicle group, #P < 0.05 and ##P < 0.01 Angptl8 + inhibitor vs. Angptl8 alone group, one-way ANOVA followed by Bonferroni’s post hoc test. e Quantitative analysis of Per1 protein levels in d. n = 3, **P < 0.01 Angptl8 vs. Vehicle group, ##P < 0.01 Angptl8 + inhibitor vs. Angptl8 alone group, one-way ANOVA followed by Bonferroni’s posthoc test. All data are presented as the means ± SD. Concentrations: U0126, 10 μM;…

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  Fig. 6. Per1 deficiency impairs the resetting functions of Angptl8. a, b qPCR analyses of time-course expression of clock and metabolic genes in Per1 KO Hepa1c1c-7 hepatoma cell lines after Angptl8 shock. n = 3, *P < 0.05 and **P < 0.01 Per1 KO vs. wild type (WT) cells at the same time point. On the other hand, mice were transduced with adenovirus expressing either scramble shRNA or Per1 shRNA through tail-vein injection at the eighth day during the Angptl8 injection. c, d qPCR analyses of clock and metabolic genes expression in the liver of mice receiving 15-day injection with recombinant Angptl8 or vehicle (PBS) at ZT2. n = 3, *P < 0.05 and **P < 0.01 Ad-Per1 shRNA vs. Ad-scramble shRNA at the same time point, two-way ANOVA followed by Bonferroni’s post hoc test. All data are presented as the means ± SD. Source data are provided as a Source Data file

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  Fig. 7. Angptl8 binds to its receptor PirB. a qPCR analyses of PirB mRNA expression in the liver of mice subjected to DD. n = 8. b, c qPCR and western blot analyses of PirB expression. Hepa1c1c-7 cells were stimulated with either 40 nM Angptl8 or PBS for 2 h. n = 3. d Co-IP assays in the mouse liver lysates. Immunoblots of precipitated proteins were performed by using indicated antibodies. e Co-IP assays in Hepa1c1c-7 cells cotransfected with plasmids encoding mouse Angptl8 and PirB. Immunoblots of precipitated proteins were performed by using indicated antibodies. f ICC analysis of colocalization of Angptl8 and PirB on the membrane of Hepa1c1c-7 cells treated with exogenous recombinant His-Angptl8 or vehicle (PBS) for 20 min. Source data are provided as a Source Data file

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  Fig. 8. PirB mediates Angptl8-induced Per1 activation. a, b qPCR and Western blot analyses of PirB and Per1 expression in Hepa1c1c-7 cells transduced with adenoviruses expressing either PirB shRNA or scramble shRNA for 36 h followed by a 2-h Angptl8 shock. n = 3. c Quantitative analysis of PirB and Per1 protein levels. n = 6. For a–c **P < 0.01 Ad-PirB shRNA vs. Ad-scramble shRNA, one-way ANOVA followed by Bonferroni’s post hoc test. d qPCR analyses of time-course expression of clock genes. n = 3, *P < 0.05 and **P < 0.01 Ad-PirB shRNA vs. Ad-scramble shRNA at the same time point, two-way ANOVA followed by Bonferroni’s post hoc test. e Western blot analyses of P38, NF-κB, and AKT protein phosphorylation. Hepa1c1c-7 cells were transduced with adenoviruses expressing either PirB shRNA or scramble shRNA for 36 h followed by a 15-min Angptl8 stimulation. f Assessment of tyrosine phosphorylation of PirB protein and interactions between PIR-B and SHP-1/2 by using Co-IP assay. Hepa1c1c-7 cells were stimulated with either 40 nM Angptl8 for indicated times. Immunoblots of precipitated proteins were performed by using indicated antibodies. All data are presented as the means ± SD. Source data are…

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  Fig. 9. Angptl8 knockdown dampens the liver clock reset by food. Mice were transduced with AAVs carrying either Angptl8 shRNA or scramble shRNA for 45 days. a Serum Angptl8 levels in mice subjected to 12-h food deprivation (ZT13-1) or 12-h food deprivation followed by 20 min refeeding. n = 5, **P < 0.01 Refed vs. Fasted mice, ##P < 0.01 AAV-Angpptl8 shRNA vs. AAV-scramble shRNA, one-way ANOVA followed by Bonferroni’s post hoc test. b, c qPCR analyses of Per1 and Per2 mRNA expression in the liver of mice from a. n = 5, **P < 0.01 Refed vs. Fasted mice. ##P < 0.01 AAV-Angpptl8 shRNA vs. AAV-scramble shRNA, one-way ANOVA followed by Bonferroni’s post hoc test. d A schematic diagram illustrating the time design of DD and a fasting/refeeding cycle for liver-specific Angptl8 knockdown mice. e, f Time-course mRNA and serum levels of Angptl8 in mice subjected to 7-day DD and fasted for 12 h at CT13 or12-h food deprivation followed by refeeding for 21 h. g qPCR analyses of clock genes. h Serum levels of certain metabolites. For e–h n = 3, *P < 0.05 and **P < 0.01 refed vs. fasted mice. #P < 0.05 and ##P < 0.01 AAV-Angpptl8 shRNA vs. AAV-scramble shRNA, two-way ANOVA followed by Bonferroni’s post…

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  Fig. 10. Angptl8 neutralization dampens the liver clock reset by food. a, b qPCR analyses of Per1 and Per2 mRNA expression in the liver of mice subjected to 12-h food deprivation (ZT13-1) or 12-h food deprivation followed by 20 min refeeding. One hour before refeeding (at ZT0), an acute injection of Angptl8-neutralized antibody (50 μg/25 g) or control IgG was administered to mice. n = 5, **P < 0.01 Refed vs. Fasted mice. #P < 0.05 and ##P < 0.01 Anti-Angpptl8 vs. control IgG, one-way ANOVA followed by Bonferroni’s post hoc test. c A schematic diagram illustrating the time design of DD, a fasting/refeeding cycle and injection of Angptl8-neutralized antibody. d qPCR analyses of time course expression of clock genes in the liver of mice subjected to an acute injection of Angptl8-neutralized antibody (50 μg/25 g) or control IgG. e Serum levels of certain metabolites. For d, e n = 3, *P < 0.05 and **P < 0.01 Refed vs. Fasted mice, #P < 0.05 and ##P < 0.01 Anti-Angpptl8 vs. control IgG at the same time point, two-way ANOVA followed by Bonferroni’s posthoc test. All data are presented as the means ± SD. f A model illustrating the mediating role of Angptl8 in the food-driven resetting of the…