FOXO1-mediated epigenetic modifications are involved in the insulin-mediated repression of hepatocyte aquaporin 9 expression.

Aquaporin (AQP) 9 transports glycerol and water, and belongs to the aquaglyceroporin subfamily. Insulin acts as a negative regulator of AQP9, and FOXO1 has the ability to mediate the regulatory effects of insulin on target gene expression. The aim of the present study was to determine whether insulin­induced repression of AQP9 involved an epigenetic mechanism. HepG2 human hepatocyte cells were treated with 500 µM insulin for different durations. AQP9 mRNA expression levels were determined by quantitative polymerase chain reaction (qPCR), and histone H3 acetylation, phosphorylation and methylation at the insulin responsive element (IRE) of the AQP9 promoter was assessed using chromatin immunoprecipitation coupled with qPCR. The effects of lentiviral FOXO1 overexpression on AQP9 expression levels and H3 modifications at the AQP9 promoter were also determined. The insulin treatment resulted in a significant and time­dependent reduction in AQP9 mRNA expression levels in HepG2 cells, as compared with untreated cells (P<0.05). In the insulin­treated cells, the levels of H3 acetylation and phosphorylation were significantly reduced (P<0.05), but the level of H3 methylation was increased. Enforced expression of FOXO1 increased AQP9 mRNA and protein expression levels in HepG2 cells. Furthermore, FOXO1 overexpression promoted H3 acetylation and phosphorylation, and reduced H3 methylation at the IRE locus of the AQP9 promoter. These data provide, to the best of our knowledge, the first evidence that insulin­induced transcriptional suppression of AQP9 expression in hepatocytes involves FOXO1­mediated H3 modifications at the IRE locus in the promoter.