Hepatic TLR4 signaling in obese NAFLD.

Nonalcoholic fatty liver disease occurs frequently in the setting of metabolic syndrome, but the factors leading to nonalcoholic steatohepatitis (NASH) are not fully understood. This study investigated Toll-like receptor 4 (TLR4) signaling in human liver with the goal of delineating whether activation of this pathway segregates those with nonalcoholic fatty liver from those with NASH. Experiments were performed using liver biopsy tissue obtained from class III obese subjects undergoing bariatric surgery, and extended to an immortalized human hepatocyte HepaRG cell line and primary human hepatocytes. The bacterial endotoxin lipopolysaccharide (LPS) and total free fatty acid levels were significantly increased in plasma of NASH patients. TLR4 mRNA levels were significantly increased in subjects with NASH compared with NAFL as was interferon regulatory factor (IRF) 3 in the myeloid differentiation factor 88-independent signaling pathway. In HepaRG cells, nuclear factor-κB (NF-κB) nuclear translocation and functional activity increased following treatment with the fatty acid, palmitate, and following exposure to LPS compared with hepatocytes stimulated with a lipogenic treatment that induced de novo lipogenesis. Palmitate and LPS induction of NF-κB activity was partially attenuated by chemical- or small-interfering RNA-mediated inhibition of TLR4. Expression of TLR4 and its downstream mediators was upregulated with palmitate and LPS. Similar results were observed using primary human hepatocytes from a lean donor. Interestingly, NF-κB activity assays showed obese donor hepatocytes were resistant to chemical TLR4 inhibition. In conclusion, TLR4 expression is upregulated in a large cohort of NASH patients, compared with those with NAFL, and this occurs within the setting of increased LPS and fatty acids.

Evolutionary conservation of glucose-dependent insulinotropic polypeptide (GIP) gene regulation and the enteroinsular axis.

Glucose-dependent insulinotropic polypeptide (GIP), an important component of the enteroinsular axis, is a potent stimulator of insulin secretion, functioning to maintain nutrient efficiency. Although well-characterized in mammals, little is known regarding GIP transcriptional regulation in Danio rerio (Dr). We previously demonstrated that DrGIP is expressed in the intestine and the pancreas, and we therefore cloned the Dr promoter to compare GIP transcriptional regulation in Dr and mammals. Although no significant homology was indentified between the highly conserved mammalian promoter and the DrGIP promoter, 1072-bp of the DrGIP promoter conferred tissue-specific expression in mammalian cell lines. Deletional analysis of the DrGIP promoter identified two regions that, when deleted, reduced transcription by 75% and 95%, respectively. Mutational analysis of the upstream region suggested involvement of an Nkx binding site, although we were unable to identify the factor binding to this site. The cis element in the downstream region was found to be a GATA binding site. Lastly, overexpression and shRNA experiments identified PAX4 as a potential repressor of DrGIP expression. These findings provide evidence that despite the identification of species-specific transcriptional regulators and differences in GIP expression patterns between D. rerio and mammals, a moderate degree of regulatory conservation appears to exist.