Regularly, down-regulation of PPAR and its target genes, was also blocked by Periostin antibody (Figure?5BCE)
Regularly, down-regulation of PPAR and its target genes, was also blocked by Periostin antibody (Figure?5BCE). levels in two groups of subjects. S5: (A-E): 3T3-L1 preadipocytes were differentiated into mature adipocytes and then treated with DEX for 48 h. HepG2 were pre-incubated with Periostin neutralizing antibody FAE for 2 hr and then treated with the supernatants from 3T3-L1 cells. Cellular TG contents (A), mRNA and protein levels of PPAR (B, C), expression of genes related to fatty acid -oxidation (D, E) were decided. (F-G) 3T3-L1 preadipocytes were differentiated into mature adipocytes. Then, cell were treated with DEX and Periostin antibody for 48 hr and 2 hr, respectively. Mouse main hepatocytes (MPHs) were treated with the supernatants from 3T3-L1 cells for 48 hr. Cellular TG contents (F) and mRNA levels of PPAR (G) were decided. n=4 per group. S6: (A-B) Body weight and food intake of wild-type and Periostin knockout mice treated with DEX or vehicle control. n=8 per group. mmc1.pptx (2.2M) GUID:?1B35FCD0-8281-4B9C-B46B-1831E067EB5D Abstract Objective Long-term glucocorticoids (GCs) therapy usually causes many metabolic side effects, including fatty liver. However, the molecular mechanisms remain poorly comprehended. Herein, we explored the molecular basis of GCs in the development of fatty liver. Methods C57BL/6 male mice were injected with Dexamethasone (DEX) while mouse main hepatocytes (MPHs), HepG2 and Hep1-6 cells were cultured in the presence of DEX. Genes expression in liver tissues and hepatocytes were assessed by quantitative real-time PCR and western blotting, respectively. To explore whether Periostin is usually involved in the development of GCs-induced fatty liver, wild-type and Periostin knockout mice were treated with DEX or vehicle control. Luciferase reporter and chromatin immunoprecipitation assays were used to determine the regulatory functions of GCs on Periostin expression. Results We show that treatment of dexamethasone (DEX), a synthetic analog of GCs, led to the accumulation of triglycerides in the livers of mice, but not in cultured hepatocytes, suggesting that GCs may promote liver steatosis through integrative organ crosstalk mediated by systemic BI-9627 factors. We further found that DEX upregulated the expression levels of Periostin in white adipose tissues, which in turn promoted liver steatosis. Administration of a Periostin-neutralizing antibody or genetic ablation of Periostin largely attenuated DEX-induced hepatic steatosis in mice. Conclusions Our findings provided a novel insight that GCs could promote liver steatosis through integrative organ crosstalk mediated by white fat-secreted Periostin. These results establish Periostin as an endocrine factor with therapeutic potential for the treatment of GCs-associated fatty liver. gene were amplified from your mouse genomic DNA template and inserted into pGL4.15 empty vectors (Promega). Mutant promoters were generated using a PCR mutagenesis kit BI-9627 (Toyobo). All of the BI-9627 transient transfections were conducted using Lipofectamine 2000 (Invitrogen) according to the manufacturer’s instructions. For luciferase reporter assays, cells were seeded in 24-well plates and transfected with the indicated plasmids. Renilla luciferase pRL-SV40 (Promega) was used to normalize the luciferase activity, which was further measured using the dual-luciferase reporter assay system (Promega). 2.7. Chromatin immunoprecipitation A chromatin immunoprecipitation (ChIP) assay kit was used (Upstate Biotechnology). In brief, lysates from 3T3-L1 cells were fixed with formaldehyde. DNA was sheared to fragments at 200C1,000 bp using sonication. The chromatins were incubated and precipitated with antibodies against GR (Santa Cruz Biotechnology), or IgG (Santa Cruz Biotechnology). 2.8. Statistical analysis All values are offered as.