Adipocyte-derived Periostin mediates glucocorticoid-induced hepatosteatosis in mice

Figures

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  Figure 1. DEX induces liver steatosis in mice but not in the hepatocytes. (A–E): C57BL/6 male mice were treated with dexamethasone (DEX, 2.5 mg/kg) or vehicle control (Con) for 14 days. Hepatic triglycerides (TG) contents (A), plasma free fatty acids levels (B), blood glucose levels (C), plasma insulin levels (D) and insulin tolerance tests (E) were determined. n = 8 per group. (F–H) Cellular TG contents were measured in mouse primary hepatocytes (MPHs) (F), HepG2 cells (G) and Hep1-6 cells (H) treated with DEX (0, 10, 50, 100 nM) or palmitate (PA, 0, 50, 100, 250 μM) or DEX plus PA (100 nM plus 250 μM). n = 4 per group. *P < 0.05,**P < 0.01, ***P < 0.001.

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  Figure 2. DEX inhibits fatty acid β-oxidation in the livers of mice but not in hepatocytes. (A–B) Relative mRNA levels of genes related to de novo lipogenesis (A) and fatty acid β-oxidation (B) in the livers of mice treated with DEX or vehicle control. n = 8 per group. (C) Representative protein levels of PPARα in the livers of mice treated with DEX or vehicle control. (D) Relative mRNA levels of genes related to fatty acid β-oxidation in MPHs treated with DEX (100 nM) or vehicle control. n = 5 per group. (E) Representative protein levels of PPARα MPHs treated with DEX or vehicle control. (F) Relative mRNA levels of genes related to fatty acid β-oxidation in HepG2 cells treated with DEX (100 nM) or vehicle control. n = 5 per group. (G) Representative protein levels of PPARα in HepG2 cells treated with DEX or vehicle control. ***P < 0.001.

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  Figure 3. Adipokines potentially mediate the hepatic steatotic roles of DEX. (A–C): 3T3-L1 preadipocytes were differentiated into mature adipocytes and then treated with DEX for 48 h. Then, the supernatants were collected to treat the mouse primary hepatocytes (MPHs) for 48 h. Cellular TG contents (A), mRNA levels of genes related to fatty acid β-oxidation (B) and protein levels of PPARα (C) were determined. n = 4 per group. (D–F): C2C12 cells were differentiated into mature skeletal muscle cells and then treated with DEX for 48 h. Then, the supernatants were collected to treat the mouse primary hepatocytes (MPHs) for 48 h. Cellular TG contents (A), mRNA levels of genes related to fatty acid β-oxidation (B) and protein levels of PPARα (C) were determined. n = 4 per group. ***P < 0.001.

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  Figure 4. DEX upregulates Periostin expression in adipocytes. (A) 3T3-L1 mature adipocytes were treated with DEX (100 nM) or vehicle control for 24 h. Then, the transcript levels of several adipokines were measured by quantitative real-time PCR, normalized to vehicle control and depicted as a fold change between DEX and controls (set as 1, dotted line). n = 5 per group. (B) Protein levels of Periostin were determined in 3T3-L1 adipocytes treated with DEX or vehicle control. (C) Relative mRNA levels of Periostin in several metabolic tissues from mice treated with DEX or vehicle control. eWAT: epididymal white adipose tissue; iWAT: inguinal white adipose tissue; SKM: skeletal muscle. n = 8 per group. (D–E) Protein levels of Periostin were analyzed in eWAT (D) and iWAT (E) from two groups of mice. (F) Plasma Periostin levels were measured by ELISA assays from two groups of mice. ***P < 0.001.

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  Figure 5. Neutralizing of Periostin attenuated the hepatosteatosis induced by DEX. (A–E): 3T3-L1 preadipocytes were differentiated into mature adipocytes and then treated with DEX for 48 h. Mouse primary hepatocytes (MPHs) were pre-incubated with Periostin neutralizing antibody for 2 h and then treated with the supernatants from 3T3-L1 cells for 48 h. Cellular TG contents (A), mRNA and protein levels of PPARα (B, C), expression of genes related to fatty acid β-oxidation (D, E) were determined. n = 4 per group. (F–N) C57BL/6 male mice were treated with DEX (2.5 mg/kg), DEX (2.5 mg/kg) plus Periostin antibody (5 mg/kg) or vehicle control. Liver TG contents (F, G), blood glucose levels (H), plasma insulin levels (I), insulin tolerance test (J), mRNA and protein levels of PPARα (K, L), expression levels of ACOX1 and MCAD (M, N) were analyzed in three groups of mice. n = 6 per group. ***P < 0.001.

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  Figure 6. Periostin knockout mice are protected from DEX-induced hepatic steatosis. (A–I) Periostin wild-type and knockout mice were treated with DEX (2.5 mg/kg) or vehicle control for 14 days. Liver TG contents (A, B), blood glucose levels (C), plasma insulin levels (D), insulin tolerance tests (E), mRNA and protein levels of PPARα (F, G), expression levels of ACOX1 and MCAD (H, I) were analyzed. n = 8 per group. **P < 0.01, ***P < 0.001.

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  Figure 7. DEX upregulates the Periostin promoter through two half GREs. (A) The transcriptional activity of the full-length mouse Periostin promoter (−2500 to +1) was upregulated by GR in the presence of DEX (100 nM). HEK293T cells were co-transfected with GR expression plasmid. (B) The transcriptional activity of truncated Periostin promoters in HEK293T cells overexpressing GR. (C) The mouse Periostin promoter contains two potential half GR response elements (GREs). Point mutations were highlighted in red. (D) The human Periostin promoter contains two potential half GR response elements (GREs). (E) The transcriptional activity of wild-type or mutant Periostin promoters in HEK293T cells overexpressing GR. (F–G) Chromatin immunoprecipitation (ChIP) assays showing the recruitment of GR onto the promoter regions of Periostin in 3T3-L1 adipocytes (F) or eWAT (G) of C57BL/6 mice treated with DEX. The proximal promoter region of β actin gene was used as a negative control for ChIP assays.