(515bp) Effect Of Steatosis And Cytokines On Transcriptional Dynamics Of Hepatocyte-Derived Reporter Cells

The main aim of this research is to investigate the effect of intracellular lipid accumulation in cultures of liver cells on the regulation of inflammatory cytokine (TNF-a, interleukin (IL)-1b, IL-6) signal transduction and its impact on the cellular phenotype (e.g. release of chemokines that attract inflammatory cells to the tissue after injury) in the context of steatohepatitis. Our approach involves following key steps: Generation of H35 hepatoma reporter cell lines that monitor major events in the cytokine signaling cascade, and Induction of steatosis in H35 cells by exposure to single- or combination of free fatty acids. After 72 hours of culture in the optimized fatty medium containing albumin, insulin, glucagon, and varying levels of selected fatty acids (oleic and linoleic acid), we observed dramatic morphological changes characterized by accumulation of refractive intracellular inclusions and significant Oil Red O staining compared to controls cultured in lean medium. The amount of intracellular triglyceride correlated with the dose of fatty acids added to the culture medium. Interestingly, while the triglyceride levels appeared independent of the fatty acid species, we found that oleic and linoleic acids differentially affected proliferation, cell survival, and GFP fluorescence of some reporters. Our investigations of steatotic reporter responses also revealed that TNF-a exposure resulted in nearly complete decline of heat shock reporter responses in steatotic cells compared to lean controls. In contrast, steatosis only partially attenuated the NFkB reporter response to TNF-a. This difference is important as it might provide clues regarding the signaling mechanism linking TNF-a stimulation with heat shock responses. Not all stimuli resulted in decreased GFP responses. For example, we observed increased fluorescence in steatotic but not lean NFkB reporter cells in response to metformin, an oral hypoglycemic agent commonly taken by patients with obesity and diabetes, conditions strongly associated with steatohepatitis. This establishment of techniques for making steatotic reporter cells will provide a foundation for future studies comparing lean and steatotic responses to cytokine stress in high-throughput microfluidic devices.