| Fat accumulation in the liver is a natural process in goose, which prepares goose for long-distance migration. In contrast to mammalian fatty liver that usually progresses into an irreversible status, steatohepatitis, goose fatty liver can return to normal without obvious pathological damage, suggesting a protective system exists in goose liver. Adipor1/2 (Adiponectin receptor 1 and 2) have ceramidase activity, and can cleave ceramide, a group of proinflammatory signaling lipid species by binding Adiponectin. Previous studies have shown that the expression of Adipor1/2 in mammalian fatty liver is reduced compared to that in normal liver, and this reduction contributes to inflammation. So, this study was to reveal whether Adipor1/2 play an important role in protective system of goose fatty liver by gene cloning, real-time PCR, western bolt, cell culture and dual luciferase reporter system. The main results are as follows:1. Tnfα mRNA was down-regulated significantly in goose fatty liver after 19 days of overfeeding (P<0.01). This inhibition of Tnfα was accompanied with the expression level of Adiponectin in the adipose tissues and the expression level of Adipor1/2 in the livers of the overfed geese. We get complete CDS sequence of Adipor1/2. Data indicated that Adiponectin was reduced in the adipose tissues and Adipor1/2 was increased in liver (P<0.05). These findings suggested that Adipor1/2 but not Adiponectin contributed to the suppression of Tnfα.2. To investigate the regulation of goose Adipor2 in the context of fatty liver, we treated goose primary hepatocytes with fatty liver-associated factors. Data indicated that Adipor2 was up-regulated by glucose and oleate but not palmitate. Its expression was even suppressed by high level of insulin. The regulation of Adipor1 by these factors was quite similar to that of Adipor2 except that glucose did not induce Adipor1.3. To investigate the regulatory mechanism of Adipor2 and to lay a foundation for its functional study, we amplified and sequenced about its lkb upstream sequence. Based on the upstream sequence, we predicted Barbie box and v-maf as regulatory elements of Adipor2 expression. We treated goose primary hepatocytes with 100uM TBHQ for 12h and determined the expression of Adipor2 and date indicated that Adipor2 mRNA level was significantly reduced (P<0.05), suggested v-Maf was involved in Adipor2 expression as an upstream transcription factor. In addition, we predicted that miR-19a was the target miRNA of Adipor2 using TargetScan and miRDB online tools. Data showed that miR-19a was suppressed by overfeeding (P<0.05). To verify the relationship of miR-19a with Adipor2, a dual luciferase reporter system was employed. Data indicated that miR-19a could reduce the expression of Adipor2, but this reduction was not statistically significant levels (P>0.05).Taken together, the findings from this study suggest the upregulation of Adiporl/2 contributes to the inhibition of inflammation, and the genes are the protective component in goose fatty liver. Moreover, the expression of Adiporl/2 can be regulated by fatty liver-associated factors, and v-maf is a transcription factor of Adipor2. Finally, Adipor2 may be regulated by miR-19a with its reduced expression in goose fatty liver, however, it is unclear whether miR-19a is responsible for the difference in the occurrence of inflammation between mammalian and goose fatty livers, which needs to be validated by further study. |
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