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Orosomucoid 2 maintains hepatic lipid homeostasis through suppression of de novo lipogenesis

Nature Metabolism volume 4, pages 1185–1201 (2022)Cite this article

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Abstract

Non-alcoholic fatty liver disease (NAFLD) is caused by imbalance in lipid metabolism. In this study, we show that the hepatokine orosomucoid (ORM) 2 is a key regulator of de novo lipogenesis in the liver. Hepatic and plasma ORM2 levels are markedly decreased in obese murine models and patients with NAFLD. Through multiple loss- and gain-of function studies, we demonstrate that ORM2 is essential to maintain hepatic and systemic lipid homeostasis. At the mechanistic level, ORM2 binds to inositol 1, 4, 5-trisphosphate receptor type 2 to activate AMP-activated protein kinase signaling, thereby inhibiting sterol regulatory element binding protein 1c-mediated lipogenic gene program. Notably, intraperitoneal injections of recombinant ORM2 protein or stabilized ORM2–FC fusion protein markedly improved liver steatosis, steatohepatitis and atherosclerosis in preclinical mouse models, without adverse effects on body weight or food intake. Thus, these findings suggest that ORM2 may serve as a potential target for therapeutic intervention in NAFLD, non-alcoholic steatohepatitis and related lipid disorders.

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Fig. 1: Downregulation of ORM2 in the livers and plasma of mice and patients with NAFLD.
Fig. 2: Both ORM2 KO male and female mice exhibited hepatic TG accumulation under ND conditions.
Fig. 3: ORM2 deficiency exacerbates diet-induced steatosis and steatohepatitis in mice.
Fig. 4: ORM2 overexpression alleviates liver steatosis and hyperlipidemia in obese mice.
Fig. 5: Downregulation of lipogenic genes by ORM2.
Fig. 6: ORM2 activates AMPK signaling to suppress DNL.
Fig. 7: ITPR2 is required for the hepatic benefits of ORM2.
Fig. 8: Recombinant ORM2 protein improves diet-induced liver steatosis and steatohepatitis in mice.

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Data availability

RNA-seq data have been deposited into the Gene Expression Omnibus database (accession no. GSE186024). Proteomics data have been deposited at the ProteomeXchange Consortium (accession nos. PXD035372 and PXD035373). The data supporting the findings of this study are available in source data files. Source data are provided with this paper.

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Acknowledgements

The authors thank J. Li (Shanghai Institute of Materia Medica) for the gift of liver-specific Prkaa1 and Prkaa2 DKO mice and constitutively activated AMPK plasmids, C.-S. Zhang (Xiamen University) for the gift of CaMKK2 KO mice, C. Xie and X. Guo (Shanghai Institute of Materia Medica) for the measurement of lipogenesis rate, G. Shi (Sun Yat-sen University), C. Ruan (Fudan University), Y. Li (Shanghai Jiao Tong University School of Medicine), D. Li (East China Normal University) and J. Wang (Shanghai Jiao Tong University School of Medicine) for helpful suggestions and G. Yan (Shanghai Jiao Tong University School of Medicine) for technical support. This study was supported by grants from the National Key Research and Development Program of China (2018YFA0800402 to Y. Lu), the Shanghai Outstanding Academic Leaders Projects, the Basic Research of Science and Technology Innovation Action Plan and Shanghai Sailing Program by Shanghai Municipal Science and Technology Committee (21XD1423400 and 21JC1401300 to Y. Lu and 22YF1432800 to B.Z.), China Postdoctoral Science Foundation Funded Project (2021M702183 to B.Z.) and Youth Cultivation Project of Shanghai Jiao Tong University Affiliated Sixth People’s Hospital (ynqn202107 to B.Z.).

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Author notes

  1. These authors contributed equally: Bing Zhou, Yunchen Luo.

Authors and Affiliations

  1. Shanghai Diabetes Institute, Shanghai Key Laboratory of Diabetes Mellitus, Shanghai Clinical Centre for Diabetes, Shanghai Jiao Tong University Affiliated Sixth People’s Hospital, Shanghai, China

    Bing Zhou & Cheng Hu

  2. Key Laboratory of Metabolism and Molecular Medicine of the Ministry of Education, Department of Endocrinology and Metabolism of Zhongshan Hospital, Fudan University, Shanghai, China

    Bing Zhou & Yan Lu

  3. Department of Endocrinology and Metabolism, Shanghai General Hospital, Shanghai Jiao Tong University, Shanghai, China

    Yunchen Luo

  4. Department of Endocrinology and Metabolism, Qingpu Branch of Zhongshan Hospital, Fudan University, Shanghai, China

    Nana Ji

  5. Institute of Metabolism and Regenerative Medicine, Shanghai Jiao Tong University Affiliated Sixth People’s Hospital, Shanghai, China

    Yan Lu

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Contributions

Y. Lu and B.Z. designed and directed the study. B.Z., Y. Luo and N.J. performed all animal and cellular experiments and human sample analysis. C.H. contributed to the discussion. Y. Lu and Z.B. wrote the manuscript.

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Correspondence to Yan Lu.

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Extended data

Extended Data Fig. 1 ORM2 deficiency exacerbates TG accumulation.

Related to Figs. 2 and 3. a-i, 8-week-old ORM2 wild-type (WT), heterozygous (HE) and knockout (KO) male mice were fed a high-fat-diet for 8 weeks. (a-c) Plasma LDL-cholesterol (a), ALT (b), AST (c) levels. WT: n = 4 biologically independent mice. HE and KO: n = 6 biologically independent mice per condition. d, Relative mRNA levels of genes related to hepatic inflammation in the liver. n = 4 biologically independent mice per condition. (e-h) Body weight (e), plasma leptin levels (f), iWAT weight (g) and BAT weight (h) of three groups of mice. WT: n = 4 biologically independent mice. HE and KO: n = 6 biologically independent mice per condition. i, H&E staining of WAT and BAT. Scale bar, 50 μm. Representative result from three biologically independent mice. j-n, 8-week-old ORM2 WT, HE and KO female mice were fed a high-fat-diet for 8 weeks. Body weight (j), liver/body weight ratio (k), hepatic (l) and plasma TG (m) levels. WT and HE: n = 6 biologically independent mice per condition. KO: n = 4 biologically independent mice. n, H&E and Oil Red O staining of liver sections. Scale bar, 50 μm. Representative result from three biologically independent mice. Data are shown as mean ± SEM. (a-h, j-m). P values are determined by one-way ANOVA followed by Dunnett’s multiple comparisons test (a-d, f-h, j-m) and two-way ANOVA followed by Dunnett’s multiple comparisons test (e).

Source data

Extended Data Fig. 2 The distinct roles of ORM1 and ORM2 in the improvement of liver steatosis.

Related to Fig. 4. a-h, 8-week-old ob/ob male mice were administered with equal amount of Ad-ORM1, Ad-ORM2, or Ad-GFP through tail vein injection. a, b, Relative mRNA levels (a, n = 8 biologically independent mice per condition) and protein expression (b, n = 4 biologically independent mice per condition) of ORM1 and ORM2 in the livers from three groups of mice. c-i, Body weight (c), average daily food intake for 3 consecutive days (d), liver/body weight ratio (e), hepatic TG levels (f), plasma TG (g) and TC (i) levels. (c-i) n = 8 biologically independent mice per condition. h, Macroscopic appearance of liver, H&E and Oil Red O staining of liver sections. Scale bar, 50 μm. Representative result from three biologically independent mice. Data are shown as mean ± SEM (a, c-g, i). P values are determined by one-way ANOVA followed by Dunnett’s multiple comparisons test (a, c-g, i).

Source data

Extended Data Fig. 3 ORM2 overexpression attenuates hepatic steatosis and hyperlipidemia in db/db mice and HFD-induced obese mice.

Related to Fig. 4. a-j, 8-week-old db/db male mice were administered with adenovirus containing ORM2 (Ad-ORM2) or GFP (Ad-GFP) through tail vein injection. a, b, Protein ORM2 levels were determined by western blots using the livers (a, top) and plasma (b, bottom) from two groups of mice. n = 6 biologically independent mice per condition. c, Relative mRNA expression levels of ORM1, ORM2 and ORM3 in the liver. n = 6 biologically independent mice per condition. d-i, Body weight (d), average daily food intake for 3 consecutive days (e), liver/body weight ratio (f), hepatic TG (g), plasma TG (h) and TC (i) levels from two groups of mice. (d-i) Ad-GFP: n = 8 biologically independent mice. Ad-ORM2: n = 9 biologically independent mice. j, H&E and Oil Red O staining of liver sections. Scale bar, 50 μm. Representative result from three biologically independent mice. k-q, C57BL/6 J male mice were fed a normal diet or HFD for 12 weeks and then administered with Ad-GFP or Ad-ORM2 for 12 days. k, Protein levels of ORM2 in the livers from three groups of mice. n = 4 biologically independent mice per condition. Liver/body weight ratio (l), hepatic TG (m), plasma TG (n) and TC (p) levels and body weight (q) from three groups of mice. ND + Ad-GFP: n = 4 biologically independent mice. HFD + Ad-GFP: n = 6 biologically independent mice. HFD + Ad-ORM2: n = 7 biologically independent mice. o, Macroscopic appearance of liver, H&E and Oil Red O staining of liver sections. Scale bar, 50 μm. Representative result from three biologically independent mice. Data are shown as mean ± SEM (c-i, l-n, p, q). P values are determined by unpaired two-tailed Student’s t-test (c-i) and one-way ANOVA followed by Dunnett’s multiple comparisons test (l-n, p, q).

Source data

Extended Data Fig. 4 ORM2 has little effects on the expression levels of genes related to fatty acid esterification and oxidation and lipid transport.

Related to Fig. 5. a, b, Relative mRNA level (a, n = 8 biologically independent mice per condition) and protein expression of lipogenic genes (b, n = 4 biologically independent mice per condition) in ob/ob male mice administered with Ad-ORM1, Ad-ORM2 or Ad-GFP. c-e, Relative mRNA levels of genes related to fatty acid esterification (c), fatty acid oxidation (d) and lipid transport (e) in the livers from db/db male mice administered with Ad-ORM2 or Ad-GFP. n = 6 biologically independent mice per condition. f-h, Relative mRNA levels of genes related to fatty acid esterification (f), fatty acid oxidation (g) and lipid transport (h) in the livers from adenovirus-treated HFD male mice as indicated. n = 4 biologically independent mice per condition. i-k, Relative mRNA levels of genes related to fatty acid esterification (i), fatty acid oxidation (j), and lipid transport (k) in the livers from AAV-treated HFD male mice as indicated. n = 4 biologically independent mice per condition. l-n, Relative mRNA levels of genes related to fatty acid esterification (l), fatty acid oxidation (m), and lipid transport (n) in the livers from ORM2 WT, HE and KO male mice fed a normal diet. WT and HE: n = 6 biologically independent mice per condition. KO: n = 4 biologically independent mice. o-q, Relative mRNA levels of genes related to fatty acid esterification (o), fatty acid oxidation (p), and lipid transport (q) in the livers from ORM2 WT, HE and KO male mice fed a high fat diet for 8 weeks. WT: n = 4 biologically independent mice. HE and KO: n = 6 biologically independent mice per condition. Data are shown as mean ± SEM (a, c-q). P values are determined by unpaired two-tailed Student’s t-test (c-e) and one-way ANOVA followed by Dunnett’s multiple comparisons test (a, f-q).

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Extended Data Fig. 5 ORM2 activates CaMKK2-AMPK signaling pathway.

Related to Fig. 6. a, b, Immunoblots of AMPK signaling pathway in MPHs (a) and HepG2 cells (b). Cells were treated with ORM2 recombinant protein (100 ng/ml) or vehicle control (PBS) for 6 hr. n = 3 independent samples per condition. c, d, MPHs were pre-incubated with palmitic acid (200 μM) for 12 hr, and then treated with ORM2 protein (100 ng/ml) or vehicle control (PBS) for 6 hr, in the presence or absence of Compound C (20 μM). c, Cellular TG contents. n = 3 independent samples per condition. d, Relative mRNA levels of genes related to de novo lipogenesis. n = 3 independent samples per condition. e, f, HepG2 Cells were pre-incubated with palmitic acid (200 μM) for 12 hr, and then treated with ORM2 protein (100 ng/ml) or vehicle control (PBS) for 6 hr, in the presence or absence of Compound C (20 μM). e, Cellular TG contents. n = 3 independent samples per condition. f, Relative mRNA levels of genes related to de novo lipogenesis. n = 3 independent samples per condition. g-i, MPHs from ORM2 WT or KO mice were administered with Ad-GFP, Ad-CA-AMPK α1 (T172D TC312) or Ad-CA-AMPK α2 (T172D TC312). g, Immunoblots of AMPK signaling. h, Cellular TG contents. n = 3 independent samples per condition. i, Relative mRNA levels of genes related to de novo lipogenesis. n = 3 independent samples per condition. j, k, MPHs were pre-incubated with palmitic acid (200 μM) for 12 hr, and then treated with ORM2 protein (100 ng/ml) or vehicle control (PBS) for 6 hr, in the presence or absence of CaMKK2 inhibitor (STO-609, 5 μM) or LKB1 inhibitor (PIM1, 10 μM). j, Cellular TG contents in MPHs. n = 3 independent samples per condition. k, Relative mRNA levels of genes related to lipogenesis in MPHs. n = 3 independent samples per condition. l, Immunoblots of CaMKK2 and AMPK signaling in MPHs. Cells were transfected with adenoviral shRNA targeting CaMKK2 or negative control for 24 hr, then treated with ORM2 protein (100 ng/ml) or vehicle control (PBS) for 6 hr. m, Immunoblots of LKB1 and AMPK signaling in MPHs. Cells were transfected with adenoviral shRNA targeting LKB1 or negative control for 24 hr, then treated with ORM2 protein (100 ng/ml) or vehicle control (PBS) for 6 hr. n, Immunoblots of AMPK in the MPHs from ORM2 WT and KO mice. Cells were treated with ORM2 recombinant protein, Ca2+ ionophore (Ionomycin, 0.5 μM) or vehicle control (DMSO) for 3 hr. o, Genomic sequencing of WT or ACC1 S80A knockin mutation (KI) HepG2 cells. p, Immunoblots of AMPK signaling in WT and KI cells treated with metformin (2 mM) or vehicle control (PBS) for 6 hr. q, r, Cells were incubated for 6 hr with 13C-acetate (1 mM) containing vehicle control (PBS), ORM2 (100 ng/ml), AMPK allosteric activators (A-769662, 10μM and AICAR, 100μM). The rate of de novo lipogenesis in WT cells (q, n = 4 independent samples per condition) and KI cells (r, n = 3 independent samples per condition) were measured. s, t, Relative mRNA levels of genes related to de novo lipogenesis in WT (s) and KI cells (t) were analyzed. n = 3 independent samples per condition. u, v, Mouse primary hepatocytes were transfected with adenoviral shRNA targeting ACC1 or negative control. Cells were then treated with ORM2 (100 ng/ml) or vehicle control (PBS) for 6 hr. ACC1 protein expression (u, n = 3 independent samples per condition) and relative mRNA levels of genes related to de novo lipogenesis (v, n = 3 independent samples per condition) were measured. Data are shown as mean ± SEM (c-f, h-k, q-t, v). P values are determined by one-way ANOVA followed by Dunnett’s multiple comparisons test (q-t) and Bonferroni’s multiple comparisons test (c-f, h-k, v).

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Extended Data Fig. 6 ITPR2 is required for the anti-lipogenic role of ORM2 in hepatocytes.

Related to Fig. 7. a, b, Immunoblots of AMPK signaling in MPHs. a, MPHs were transfected with adenoviral shRNA targeting ITPR2 or negative control for 24 hr, and then treated with ORM2 protein (100 ng/ml) or vehicle control (PBS) for 6 hr. b, MPHs were transfected with adenoviral shRNA targeting ITPR2 or negative control for 24 hr, then treated with Ca2+ ionophore (Ionomycin, 0.5 μM) or vehicle control (DMSO) for 3 hr. c, Cellular TG contents in MPHs. n = 3 independent samples per condition. d, Relative mRNA levels of genes related to de novo lipogenesis in MPHs. n = 3 independent samples per condition. e, f, Cells were exposed to palmitic acid (200 μM) for 12 hr, and then treated with ORM2 protein (100 ng/ml) or vehicle control (PBS) for 6 hr, in the presence or absence of an ITPR2 inhibitor (2-APB, 50 μM). e, Cellular TG contents. n = 3 independent samples per condition. f, Relative mRNA levels of genes related to de novo lipogenesis. n = 3 independent samples per condition. Data are shown as mean ± SEM (c-f). P values are determined by one-way ANOVA followed by Bonferroni’s multiple comparisons test (c-f).

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Extended Data Fig. 7 ORM2 recombinant protein treatment improves liver steatosis in ob/ob mice.

Related to Fig. 8. a-j, ob/ob male mice were intraperitoneally injected with recombinant ORM2 protein (0.1 mg/kg, 1.0 mg/kg) or vehicle control (PBS, 0) for 10 days. a-d, Body weight (a), average daily food intake for 3 consecutive days (b), iWAT (c) and BAT weight (d). (a-d) n = 8 biologically independent mice per condition. e, H&E staining of WAT and BAT from three groups of mice. Scale bar, 50 μm. Representative result from three biologically independent mice. f-i, Liver TC (f), plasma TC (g), ALT (h) and AST (i) levels from three groups of mice. n = 8 biologically independent mice per condition. j, Relative mRNA levels of genes related to hepatic inflammation in the livers from three groups of mice. n = 8 biologically independent mice per condition. Data are shown as mean ± SEM. (a-d, f-j). P values are determined by one-way ANOVA followed by Dunnett’s multiple comparisons test (a-d, f-j).

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Extended Data Fig. 8 ORM2 recombinant protein treatment improves liver steatosis in db/db mice.

Related to Fig. 8. a-j, db/db male mice were intraperitoneally injected with recombinant ORM2 protein (0.1 mg/kg, 1.0 mg/kg) or vehicle control (PBS, 0) for 10 days. a-f, Body weight (a), average daily food intake for 3 consecutive days (b), liver/body weight ratio (c), hepatic TG levels (d), plasma TG (e) and TC levels (f) from three groups of db/db mice. (a-f) n = 8 biologically independent mice per condition. g, Macroscopic appearance of liver, H&E and Oil Red O staining of liver sections. Scale bar, 50 μm. Representative result from three biologically independent mice. h, Relative mRNA levels of genes related to de novo lipogenesis in the livers. n = 8 biologically independent mice per condition. i, Protein levels of lipogenic genes in the livers. n = 4 biologically independent mice per condition. j. Immunoblots of AMPK and mTOR signaling pathways in the livers. n = 4 biologically independent mice per condition. Data are shown as mean ± SEM (a-f, h). P values are determined by one-way ANOVA followed by Dunnett’s multiple comparisons test (a-f, h).

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Extended Data Fig. 9 ORM2 recombinant protein treatment improves liver steatosis in HFD-induced obese mice.

Related to Fig. 8. a-i, C57BL/6 J male mice were fed a normal diet or high-fat-diet for 12 weeks, and then treated with ORM2 protein (1.0 mg/kg) or vehicle control (PBS) for 14 days. a-f, Body weight (a), average daily food intake for 3 consecutive days (b), liver/body weight ratio (c), hepatic TG (d), plasma TG (e) and leptin (f) levels from three groups of mice. (a-f) ND + PBS and HFD + PBS: n = 6 biologically independent mice per condition. (a-f) HFD + ORM2: n = 5 biologically independent mice. g, Macroscopic appearance of liver, H&E and Oil Red O staining of liver sections. Scale bar, 50 μm. Representative result from three biologically independent mice. h, Relative mRNA levels of genes related to de novo lipogenesis in the livers. n = 4 biologically independent mice per condition. i, Protein levels of lipogenic genes in the livers. n = 4 biologically independent mice per condition. Data are shown as mean ± SEM (a-f, h). P values are determined by one-way ANOVA followed by Dunnett’s multiple comparisons test (a-f, h).

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Extended Data Fig. 10 ORM2 protein protects against western diet-induced atherosclerotic lesion formation in ApoE null mice.

Related to Fig. 8. a-h, 10-week-old ApoE null male mice were fed a western diet for 8 weeks, and then were intraperitoneally injected with recombinant ORM2 protein (1.0 mg/kg) or vehicle control (PBS) for 14 days. a, b, Liver/body weight ratio (a) and liver TG levels (b). PBS: n = 7 biologically independent mice. OMR2: n = 6 biologically independent mice. c, H&E and Oil Red O staining of liver sections. Scale bar, 50 μm. Representative result from three biologically independent mice. d, Relative mRNA levels of genes related to hepatic TG metabolism in the livers. n = 6 biologically independent mice per condition. e, f, Plasma (e) and hepatic TC (f) levels. PBS: n = 7 biologically independent mice. OMR2: n = 6 biologically independent mice. g, Relative mRNA levels of genes related to hepatic cholesterol metabolism in the livers. n = 6 biologically independent mice per condition. h, Oil Red O staining showing the lipid accumulation in the aorta from two groups of mice. i, Relative mRNA levels of genes related to inflammation in the aorta. n = 6 biologically independent mice per condition. Data are shown as mean ± SEM (a, b, d-g, i). P values are determined by one-way ANOVA followed by unpaired two-tailed Student’s t-test (a, b, d-g, i).

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Zhou, B., Luo, Y., Ji, N. et al. Orosomucoid 2 maintains hepatic lipid homeostasis through suppression of de novo lipogenesis. Nat Metab 4, 1185–1201 (2022). https://doi.org/10.1038/s42255-022-00627-4

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