Development of an in vitro cell culture model of hepatic steatosis using hepatocyte-derived reporter cells

Abstract

Fatty liver disease is a problem of growing clinical importance due to its association with the increasingly prevalent conditions of obesity and diabetes. While steatosis represents a reversible state of excess intrahepatic lipid, it is also associated with increased susceptibility to oxidative and cytokine stresses and progression to irreversible hepatic injury characterized by steatohepatitis, cirrhosis, and malignancy. Currently, the molecular mechanisms underlying progression of this dynamic disease remain poorly understood, particularly at the level of transcriptional regulation. We recently constructed a library of stable monoclonal green fluorescent protein (GFP) reporter cells that enable transcriptional regulation to be studied dynamically in living cells. Here, we adapt the reporter cells to create a model of steatosis that will allow investigation of transcriptional dynamics associated with the development of steatosis and the response to subsequent "second hit" stresses. The reporter model recapitulates many cellular features of the human disease, including fatty acid uptake, intracellular triglyceride accumulation, increased reactive oxygen species accumulation, decreased mitochondrial membrane potential, increased susceptibility to apoptotic cytokine stresses, and decreased proliferation. Finally, to demonstrate the utility of the reporter cells for studying transcriptional regulation, we compared the transcriptional dynamics of nuclear factor kappaB (NFkappaB), heat shock response element (HSE), and glucocorticoid response element (GRE) in response to their classical inducers under lean and fatty conditions and found that intracellular lipid accumulation was associated with dose-dependent impairment of NFkappaB and HSE but not GRE activation. Thus, steatotic reporter cells represent an efficient model for studying transcriptional responses and have the potential to provide important insights into the progression of fatty liver disease.

Figure 1

Intracellular TG accumulation in H35 cells as a function of fatty acid dose (a) measured by TG assay, (b) optical microscopy, and (c) Oil-Red-O staining. All measurements were performed after the H35 cells were exposed to different FFA doses for 72 h. Error bars represent 95% confidence intervals. * p ≤ 0.05 compared to values for 0.25 mmol/l fatty acid dose. • p ≤ 0.05 compared to values for same OA dose. # cells treated with 4 mmol/l LA did not survive over the 72 h culture period.

Figure 2

Effect of steatosis on (a) generation of intracellular reactive oxygen species and (b) mitochondrial membrane potential. All measurements were performed after the H35 cells were exposed to different FFA doses for 72 h. Error bars represent 95% confidence intervals. * p ≤ 0.05 compared to values for 0 mmol/l FFA dose. • p ≤ 0.05 compared to values for same OA dose. # Cells treated with 4 mmol/l LA did not survive over the 72 h culture period.

Figure 3

Effect of steatosis on (a) proliferation and (b) viability of H35 cells. All measurements were performed after the H35 cells were exposed to different FFA doses for 72 h. Error bars represent 95% confidence intervals. * p ≤ 0.05 compared to values for 0 mmol/l FFA dose. • p ≤ 0.05 compared to values for same OA dose. # Cells treated with 4 mmol/l LA did not survive over the 72 h culture period.

Figure 4

Effect of steatosis on (a) dexamethasone mediated activation of GRE and TNF-α mediated activation of (b) NFκB and (c) HSE in H35 cells. All reporter cells were exposed to different FFA doses for 72 h. GRE reporter cells were exposed to 4 mmol/l dexamethasone for 18 h before the flow cytometry. NFκB and HSE reporter cells were exposed to 10 ng/ml TNF-α for 18 h before the flow cytometry. Error bars represent 95% confidence intervals. * p ≤ 0.05 compared to values for 0 mmol/l FFA dose. • p ≤ 0.05 compared to values for same OA dose.