Fig 2: PZP promotes UCP1 expression via a P38 MAPK-ATF2 pathway. A) Immunoblots of cell lysate from differentiated BA which were cultured in refeeding medium supplemented with PZP protein (10, 100, 500 ng mL-1) using indicated antibodies which showed PZP significantly increased the phosphorylation of p38 MAPK in condition of refeeding. B) Immunoblots of cell lysate from differentiated BA which were cultured supplemented with or without thapsgargin (600 µm) and PZP protein (100 ng mL-1) using indicated antibodies. C) Immunoblots of BAT lysates from control mice and PZP KO mice when mice were fed upon HFD and IF after 2 weeks without difference in body weight between groups using indicated antibodies which showed phosphorylation of p38 MAPK and ATF2 were decreased in PZP KO mice. The samples were collected when mice were refed for 6 h after 24-h fast. D) Immunoblots of cell lysate from siGRP78-transfected BA and controls which were cultured in refeeding medium supplemented with or without PZP protein (100 ng mL-1) using indicated antibodies, which showed UCP1 expression and phosphorylation of p38 MAPK and ATF2 upon PZP treatment were significantly inhibited by GRP78 siRNA. E) Immunoblots of cell lysate from differentiated BA which were cultured in refeeding medium supplemented with or without SB203580 (10 µm) and PZP protein (100 ng mL-1) using indicated antibodies, which showed PZP-induced UCP1 expression under refeeding condition was significantly inhibited by SB203580 treatment. F) Immunoblots of cell lysate from differentiated BA which were cultured supplemented with or without SB203580 (10 µm) and PZP protein (100 ng mL-1) after thapsigargin (600 µm) stimulation using indicated antibodies, which showed PZP induced UCP1 expression under thapsigargin treatment was significantly inhibited by SB203580 treatment. Data are shown as mean ± SEM. One-way (A–C) or two-way ANOVA (D–F) with multiple comparisons and Tukey's post-test were performed; *** p <0.001, ** p < 0.01, and * p < 0.05 were considered to be significant.

Fig 3: PZP promotes UCP1 expression through binding to cell surface GRP78 during refeeding. A) Immunoblots of cell lysate from differentiated BA which were cultured in refeeding medium supplemented with PZP protein (10, 100, 500 ng mL-1). B) Oxygen consumption of differentiated BA which were cultured in refeeding medium supplemented with PZP protein (100 ng mL-1) showed higher basal cellular respiration, maximal respiration, and proton leak than control group. C) SDS-PAGE gel of anti-Flag immunoprecipitation from 293FT cell lysate which co-overexpressed BAT cDNA library and PZP-Flag or Control-Flag. Red square indicates the chopped gel which we sent for orbitrap analysis. D) 293FT was overexpressed with GRP78-Flag and PZP. PZP-GRP78 complex was immunoprecipitated by anti-Flag antibody and blotted with indicated antibodies. E) Whole mount tissue staining images of iBAT from fasted and refed mice using anti-GRP78 which showed GRP78 in BAT translocated from cytoplasm to cell membrane under IF. F) Immunoblots of cell membrane and cytoplasmic fractions from BAT using indicated antibodies which showed GRP78 dramatically increased in cell membrane fraction rather than in cytoplasm upon refeeding. G) Quantification of GRP78 protein level in cell membrane and cytoplasmic fractions of BAT, iWAT, muscle and liver from fasted and refed mice. H) Representative fluorescence images of BA which expressed GRP78-cherry fusion protein after refeeding and thapsigargin treatment which showed refeeding and thapsigargin treatment induced translocation of GRP78 to cell surface of BA. I) Cell membrane extraction from BAT incubated with PZP-His protein. PZP-GRP78 complex was immunoprecipitated by anti-His beads and blotted with indicated antibodies which showed refeeding increased GRP78-PZP complex formation in the plasma membrane fraction of BAT. F, fasting; R, refeeding. Right panel shows result of quantitative analysis. J) Immunoblots of cell surface protein from differentiated BA which were cultured in fasting or refeeding medium supplemented with PZP protein (100 ng mL-1). Cell surface proteins were biotinylated and isolated by EZLabel Protein Biotin Labeling Kit and Streptavidin beads. K) Immunoblots of cell lysate from differentiated BA which were cultured supplemented with thapsigargin (600 µm) and PZP protein (100 ng mL-1) using indicated antibodies. Data are shown as mean ± SEM. One-way (A) or two-way ANOVA (B,G) with multiple comparisons and Tukey's post-test were performed; *** p <0.001, ** p < 0.01, and * p < 0.05 were considered to be significant.

Fig 4: Identification of PZP as a hepatokine which is upregulated during refeeding. A) Scatter diagram of transcriptomic data-mining strategy showing the three cohort of genes. Yellow dots indicate a cluster of genes which meet our determinative criteria. B) Venn diagram showing the overlap of three screened list. Gene name in red letter indicates a secreted protein. C) Relative mRNA expression of candidates in liver from WT or Ob/Ob mice (n = 7–10). 7 of total 17 detected genes were decreased in Ob/Ob mice compared with WT mice. D) Relative mRNA expression of candidates in liver from Fasted (24 h) or refed (6 h) mice (n = 9–10). 10 of total 17 detected genes were increased under refed condition. E) qPCR analysis of PZP expression in mouse tissues (n = 6–8). PZP was specific high expressed in liver than other tissue. F) Western blot analysis of PZP protein level in liver and serum from HFD mice or Ob/Ob mice which showed PZP protein levels were significantly down-regulated in obese mice. G) Western blot analysis of PZP protein level in liver and serum after feeding or fasting (24 h) or refeeding (6 h) are shown. H) Serum concentration of PZP and FGF21 in mice after fasting (24 h) or different refeeding food intake (0.4, 0.8, 1.2 gram) which showed PZP expression were regulated by food intake in a dose-dependent manner. I) Spearman correlation between circulating PZP concentration and log2 BMI which showed circulating PZP level inversely correlated with log2 (BMI). ? 2 tests were used for statistical analysis (n = 44). J) Serum concentration of PZP was significantly increased in human participants during OGTT. Paired sample T-test was used for statistical analysis (n = 24). Data are shown as mean ± SEM. Two-tailed Student's t-test (C,D) or one-way ANOVA (H) with multiple comparisons and Tukey's post-test were performed; *** p <0.001, ** p < 0.01, and * p < 0.05 were considered to be significant.

Fig 5: Increased circulating PZP augments UCP1-dependent energy expenditure and protects DIO during IF treatment WT and UCP1-/- mice chronically subjected to HFD for 7 weeks. A) Schematic shows the procedure of in vivo therapeutic experiments. Mice were fed in IF regimen, comprising 1 day of fasting followed by 3 days of feeding per cycle for 8 weeks. We performed i.h. of PZP protein at the beginning of refeeding. Blue color indicating fasting; red color indicating refeeding. B) Body weight and C) GTT of WT and UCP1-/- mice treated with or without PZP protein (1 mg/kg) are shown (n = 8–10). D) Oxygen consumption rate (left panel) and energy expenditure (right panel) of WT and UCP1-/- mice treated with or without PZP protein (n = 8–9). These experiments were performed when mice were upon HFD and IF after 2 weeks without difference in body weight between groups, which showed PZP protein injection dramatically increased the oxygen consumption and energy expenditure of WT mice but not UCP1-/- mice. E) Representative H&E staining images of iBAT from WT and UCP1-/- mice treated with or without PZP protein (scale bar = 100 µm). F) Relative mRNA expression of adipogenic, thermogenic, and inflammatory genes in BAT from WT mice treated with or without PZP protein (n = 7–10). G) Immunoblots of total BAT lysates from WT mice treated with or without PZP protein using indicated antibodies which showed PZP protein treatment activated p38 MAPK-ATF2-UCP1 signaling in WT mice (n = 3). H) Schematic overview of main findings. Refeeding signals induced liver to produce and release PZP into circulation and followed by the translocation of GRP78 to cell surface in BAT. Subsequently, circulating PZP could bind to cell surface GRP78 to promote UCP1 expression via a p38 MAPK-ATF2 signaling pathway in BAT. Data are shown as mean ± SEM. Two-tailed Student's t-test (F,G) or two-way repeated measures ANOVA (B–D) with multiple comparisons and Tukey's post-test were performed; ***p<0.001, ** p < 0.01, and * p < 0.05 were considered to be significant.