Our study unveils an H19/PTBP1/sterol regulatory element-binding protein 1 feedforward amplifying signaling pathway to exacerbate the development of fatty liver. (Hepatology 2018;67:1768-1783).

Based on our recent finding that disruption of bile acid (BA) homeostasis in mice results in the induction of hepatic lncRNA H19 expression, we sought to elucidate the role of H19 in cholestatic liver fibrosis. Hepatic overexpression of H19RNA augmented bile duct ligation (BDL)-induced liver fibrosis, which was accompanied by the elevation of serum ALT, AST, bilirubin, and BA levels. Multiple genes related to liver fibrosis, inflammation, and biliary hyperplasia were increased in H19-BDL vs Null-BDL mice, whereas genes in BA synthesis were decreased. Livers and spleens of H19-BDL mice showed significant enrichment of CD3+γδ+, IL-4, and IL-17 producing CD4+ and CD8+ immune cell populations. H19 downregulated hepatic zinc finger E-box-binding homeobox 1 (ZEB1) but upregulated epithelial cell adhesion molecule (EpCAM) and SRY (sex determining region Y)-box 9 (SOX9) expression. Mechanistically, ZEB1 repressed EpCAM promoter activity and gene transcription. H19RNA impeded ZEB1’s inhibitory action by interacting with ZEB1 protein to prevent its binding to the EpCAM promoter. Hepatic overexpression of ZEB1 or knockdown of EpCAM diminished H19-induced fibrosis; the latter was also prevented in H19−/− mice. H19RNA was markedly induced by bile acids in mouse small cholangiocytes (MSC) and to a lesser extent in mouse large cholangiocytes (MLC). The upregulation of H19RNA and EpCAM correlated positively with the downregulation of ZEB1 in primary sclerosing cholangitis (PSC) and primary biliary cirrhosis (PBC) liver specimens. Conclusions: The activation of hepatic H19RNA promoted cholestatic liver fibrosis in mice through the ZEB1/EpCAM signaling pathway.

Bile acid (BA) metabolism is tightly controlled by nuclear receptor signaling to coordinate regulation of BA synthetic enzymes and transporters. Here we reveal a molecular cascade consisting of the antiapoptotic protein BCL2, nuclear receptor Shp, and long non-coding RNA (lncRNA) H19 to maintain BA homeostasis. Bcl2 was overexpressed in liver of C57BL/6J mice using adenovirus mediated gene delivery for two weeks. Hepatic overexpression of Bcl2 caused drastic accumulation of serum BA and bilirubin levels and dysregulated BA synthetic enzymes and transporters. Bcl2 reactivation triggered severe liver injury, fibrosis and inflammation, which were accompanied by a significant induction of H19. Bcl2 induced rapid SHP protein degradation via the activation of caspase-8 pathway. The induction of H19 in Bcl2 overexpressed mice was contributed by a direct loss of Shp transcriptional repression. H19 knockdown or Shp re-expression largely rescued Bcl2-induced liver injury. Strikingly different than Shp, the expression of Bcl2 and H19 was hardly detectable in adult liver but was markedly increased in fibrotic/cirrhotic human and mouse liver. We demonstrated for the first time a detrimental effect of Bcl2 and H19 associated with cholestatic liver fibrosis and an indispensable role of Shp to maintain normal liver function.

Adiponectin secreted from adipose tissues plays a role in the regulation of energy homeostasis, food intake, and reproduction in the hypothalamus. We have previously demonstrated that adiponectin significantly inhibited GNRH secretion from GT1-7 hypothalamic GNRH neuron cells. In this study, we further investigated the effect of adiponectin on hypothalamic KISS1 gene transcription, which is the upstream signal of GNRH. We found that globular adiponectin (gAd) or AICAR, an artificial AMPK activator, decreased KISS1 mRNA transcription and promoter activity. Conversely, inhibition of AMPK by Compound C or AMPKa1-SiRNA augmented KISS1 mRNA transcription and promoter activity. Additionally, gAd and AICAR decreased the translocation of specificity protein-1 (SP1) from cytoplasm to nucleus; however, Compound C and AMPKa1-siRNA played an inverse role. Our experiments in vivo demonstrated that the expression of Kiss1 mRNA was stimulated twofold in the Compound C-treated rats and decreased about 60-70% in gAd-or AICAR-treated rats compared with control group. The numbers of kisspeptin immunopositive neurons in the arcuate nucleus region of Sprague Dawley rats mimicked the same trend seen in Kiss1 mRNA levels in animal groups with different treatments. In conclusion, our results provide the first evidence that adiponectin reduces Kiss1 gene transcription in GT1-7 cells through activation of AMPK and subsequently decreased translocation of SP1.

The small heterodimer partner (SHP) nuclear receptor is an important regulator of nonalcoholic fatty liver disease. However, little is known about the role of SHP in alcoholic fatty liver. In this study, we used a modified chronic ethanol-binge model to examine cyclic alterations of lipid metabolism in wild-type (WT) and Shp mice over a 24-hour period after binge. The serum and hepatic lipid profiles, as well as the expression of lipid synthesis genes and markers of endoplasmic reticulum stress, exhibited distinct variations in WT and Shp mice in response to ethanol diet plus ethanol binge (ED+E) and control diet plus maltose binge. ED+E induced steatosis in WT mice, which correlated with a marked up-regulation of activating transcription factor 4 protein (ATF4) but down-regulation of C/EBP homologous protein (CHOP) and sterol regulatory element-binding transcription factor 1c protein (SREBP-1c). On the contrary, the control diet plus maltose binge caused lipid accumulation in Shp mice, which was accompanied by a sharp elevation of CHOP, SREBP-1c, and REV-ERBα proteins but a diminished ATF4. REV-ERBα activated CHOP promoter activity and gene transcription, which were inhibited by SHP. Knockdown Rev-Erbα in Shp mice prevented steatosis induced by ED+E. Our study revealed a critical role of SHP and REV-ERBα in controlling rhythmic CHOP expression in alcoholic fatty liver.