In NAFLD models the first hit is liver fat
In NAFLD models, the “first hit” is liver fat accumulation, which causes insulin resistance, whereas the interplay of inflammatory cytokines, which causes inflammation, acts as the representative “second hit” . However, recently, the “multiple hit” hypothesis has taken into consideration that multiple insults, including insulin resistance, nutritional factors, gut microbiota, and genetic and epigenetic factors, act collectively to induce NAFLD and provides a more accurate explanation of NAFLD pathogenesis . The p300 HAT enzyme, which functions as a transcriptional coactivator, is co-recruited with nuclear factor-κB to regulate various inflammatory signaling pathways . Carbohydrate-responsive element-binding protein (ChREBP) is a transcriptional activator of lipogenic genes, which plays a major role in the development of NAFLD . p300 increases ChREBP transcriptional activity through acetylation of Lys-672, which, in turn, increases p300 occupancy on target gene promoters . Furthermore, p300 overexpression results in NAFLD, insulin resistance, and inflammation . However, little is known regarding the role of HATs in the development of NAFLD.
During an ongoing screening study using an in vitro HAT assay system to identify phytochemical compounds with HAT inhibitory activity, we identified tannic 93 3 (TA) as a novel HAT inhibitor (HATi) with specificity for major HAT enzymes. TA, a plant-derived hydrolysable tannin polyphenol, is a gallic acid polymer glucoside found in many dietary plant products such as coffee, tea, cocoa, and sorghum grain . Various studies have revealed the beneficial effects of TA , , . Recent studies have shown the anti-obesity effects of TA , ; however, no explanation has been provided regarding the exact molecular mechanism of TA activity on signaling pathways. Furthermore, no study has shown beneficial effects of TA on the development of NAFLD, even though this disease is closely associated with components of the metabolic syndrome, such as obesity, insulin resistance, and dyslipidemia .
Materials and methods
Discussion Epigenetic dysregulation is known to initiate the development of various human diseases and to contribute to their progression. Although epigenetic modulation including HDACi and DNA methyltransferase inhibition has been extensively studied, and several are currently being assessed in clinical trials , there is little information available on HAT inhibitors. HAT is a plausible target for preventive or therapeutic agents , , and there are now several known HATi derived from natural products , ,  that have favorable safety profiles . EGCG, the major polyphenol found in green tea, has potent anti-HAT activity, with global specificity for the majority of HAT enzymes . In addition, previous studies have shown that garcinol inhibits p300 and PCAF in vitro and in vivo, whereas anacardic acid inhibits TIP60 as well as p300 and PCAF, and curcumin inhibits p300 and PCAF, suggesting that dietary compounds are potent epigenetic regulators, particularly via HAT inhibition . However, to our knowledge, epigenetic regulation by TA has not previously been investigated, and thus the present study is the first to indicate that TA is a potent and specific HATi. Initially, we demonstrated the anti-HAT activity of TA. TA showed global specificity for the majority of HAT enzymes examined, but not other epigenetic enzymes, i.e., HDACs, illustrating that TA is a specific HATi. A previous acetylation study identified an astonishing 978 acetylated proteins in human liver tissue , indicating that protein acetylation is involved in a broad range of cellular activities in the liver. To date, substantial evidence has indicated the involvement of HAT activity in NAFLD , . Using qRT-PCR analysis, we observed that C-646 treatment leads to a decrease in the mRNA expression of lipogenic genes related to NAFLD, supporting the notion that p300-induced HAT activity causes NAFLD. Although TA has been shown to have an inhibitory effect on lipid accumulation in the liver , , there has been no evidence that TA suppresses the HAT-mediated acetylation of histone and non-histone proteins and the subsequent expressions of lipogenesis-related genes. In the present study, in vitro and in vivo HAT assays clearly showed the TA inhibits HAT-induced histone acetylation. The acetylation of histone H3K9 is well known as an epigenetic marker for NAFLD , , and, as previously remarked, acetylation of proteins is associated with dynamic cellular activities in the liver . In addition, we showed that TA attenuated the lipid accumulation-induced increase in acetylation levels of total proteins and histones H3K9 and H3K36 in vitro and in vivo. Thus, our study provides firm evidence to support the notion that TA suppresses HAT-mediated acetylation of both histone and non-histone proteins.