The Hepatokine Orosomucoid 2 Mediates Beneficial Metabolic Effects of Bile Acids

Abstract

Bile acids (BAs) are pleiotropic regulators of metabolism. Elevated levels of hepatic and circulating BAs improve energy metabolism in peripheral organs, but the precise mechanisms underlying the metabolic benefits and harm still need to be fully understood. In the current study, we identified orosomucoid 2 (ORM2) as a liver-secreted hormone (i.e., hepatokine) induced by BAs and investigated its role in BA-induced metabolic improvements in mouse models of diet-induced obesity. Contrary to our expectation, under a high-fat diet (HFD), our Orm2 knockout (Orm2-KO) exhibited a lean phenotype compared with C57BL/6J control, partly due to the increased energy expenditure. However, when challenged with a HFD supplemented with cholic acid, Orm2-KO eliminated the antiobesity effect of BAs, indicating that ORM2 governs BA-induced metabolic improvements. Moreover, hepatic ORM2 overexpression partially replicated BA effects by enhancing insulin sensitivity. Mechanistically, ORM2 suppressed interferon-γ/STAT1 activities in inguinal white adipose tissue depots, forming the basis for anti-inflammatory effects of BAs and improving glucose homeostasis. In conclusion, our study provides new insights into the molecular mechanisms of BA-induced liver-adipose cross talk through ORM2 induction.

Figure 1

Identification of hepatokines in both CA-fed and FS-DKO livers. A: Gene expression profiling of secreted proteins (total of 326 genes) was analyzed in CA-fed and FS-DKO livers. B: Differentially expressed secreted proteins (P < 0.05) in CA-fed and FS-DKO livers. In both data sets, 15 genes were upregulated and 15 genes were downregulated. C: Tissue distribution of 30 candidate genes. One of the highly abundant genes in the liver was Orm2, which was commonly increased in CA-fed and FS-DKO livers. D: The mRNA expressions of representative candidates in 0.5% CA-fed (24 h after overnight fasting, left) and FS-DKO (8 weeks old, right) livers (n = 4 per group). Data are presented as mean ± SEM. Student t test, *P < 0.05, **P < 0.01, ***P < 0.005; ns, not significant.

Figure 2

ORM2 is required for metabolic benefits of BAs in obesity. A: Body weight changes of WT and Orm2-KO (KO) mice after HFD or HFDCA feeding (n = 6 per group). Body weight (B) and tissue weights (in grams and %) (C) at 16 weeks after HFD or HFDCA feeding (n = 6 per group). BAT, brown adipose tissue. D: Representative images of the liver, iWAT, and eWAT. Scale bar = 1 cm. E: Hematoxylin and eosin staining of the liver and adipose tissues. Scale bar = 100 μm. F: Hepatic triglyceride (TG) levels (n = 4 per group). Data are presented as mean ± SEM. ANOVA, followed by Tukey test, *P < 0.05, **P < 0.01, ***P < 0.005.

Figure 3

ORM2 deficiency abolishes metabolic improvements induced by BAs. A: GTT (glucose concentration of 1.5 g/kg body weight, intraperitoneal injection, n = 4 in WT and n = 3 in KO). B: The AUC and AOC. C: ITT (low dose, 0.25 units/kg body weight, n = 4 in WT and n = 3 in KO). D: AUC and the AOC for the initial 60 min (AUC₆₀ and AOC₆₀) were calculated. Data are presented as mean ± SEM. ANOVA, followed by Tukey test, *P < 0.05, ***P < 0.005.

Figure 4

Metabolic changes induced by hepatic ORM2 overexpression in obesity. A: Validation of ORM2 overexpression in the liver and serum. AAV8-GFP was used as a control. B: Body weight changes after tail vein injection of AAV8-GFP and AAV8-Orm2 (n = 5 in AAV8-GFP and n = 6 in AAV8-Orm2). C: Body weight and tissue weight at the time of euthanasia (16 weeks, n = 5 in AAV8-GFP and n = 6 in AAV8-Orm2). BAT, brown adipose tissue. D: Hematoxylin and eosin staining of the liver and adipose tissues. E: Hepatic triglyceride (TG) levels (n = 4 per group). Scale bar = 100 μm. Data are presented as mean ± SEM. Student t test, *P < 0.05, ***P < 0.005.

Figure 5

Hepatic ORM2 overexpression improves glucose tolerance and insulin sensitivity. A: GTT (glucose concentration of 1.5 g/kg body weight, intraperitoneal injection, n = 4 per group). AUC and AOC were calculated. B: ITT (0.5 units/kg body weight, n = 4 per group). AUC and AOC for 60 min (AUC₆₀ and AOC₆₀) were calculated. Data are presented as mean ± SEM. Student t test, *P < 0.05.

Figure 6

Hepatic ORM2 overexpression attenuates iWAT inflammation. A: Volcano plots of iWAT and eWAT transcriptomes (n = 4 per group). The differentially expressed genes are noted as a red circle (P < 0.05). The detailed analytic process is described in Research Design and Methods. B: KEGG pathway analysis of all differentially expressed genes in iWAT and eWAT. C: Prediction of upstream transcription factors of upregulated (left) and downregulated (right) genes in iWAT. D: Gene set enrichment analysis results of AAV8-Orm2 iWAT on IFNγ and TNF signaling cascades. ES, enrichment score; FDR, false discovery rate; NES, normalized enrichment score. Changes of IFNγ (E) and TNF (F) target gene expressions (n = 4 per group) in iWAT (top) and eWAT (bottom). Data are presented as mean ± SEM. Student t test, *P < 0.05, **P < 0.01.

Figure 7

ORM2 suppresses IFNγ/STAT1 signaling. A and B: Western blotting analysis of IFNγ/STAT1 signaling (duplicates). Differentiated adipocytes (A) or RAW264.7 monocytes (B) were pretreated with rORM2 (10 μg/mL) for 18 h and incubated with IFNγ (100 ng/mL) for 30 min. Protein levels of STAT1, JAK1, AMPK, and ERK with their phosphorylated (p) forms are visualized. C: STAT1 and JAK1 levels in AAV8-Orm2 iWAT. Protein levels of STAT1 and JAK1 were analyzed by Western blotting (n = 4 per group). D and E: IFNγ/STAT1 target gene expressions (triplicates). Adipocytes (D) and monocytes (E) were pretreated with rORM2 (5 μg/mL) for 18 h, and IFNγ (100 ng/mL) was added for an additional 6 h. F: IFNγ-STAT1 target gene expressions in WAT from HFDCA-fed WT and Orm2-KO mice (n = 4 per group). For D, E, and F, expression levels of Stat1 and its targets were quantified by qPCR. For D and E, data are presented as mean ± SD. For F, data are presented as mean ± SEM. ANOVA, followed by Tukey test, *P < 0.05, **P < 0.01, ***P < 0.005.