Genomics of lipid-laden human hepatocyte cultures enables drug target screening for the treatment of non-alcoholic fatty liver disease.

BACKGROUND: Non-alcoholic fatty liver disease (NAFLD) is a major health burden in need for new medication. To identify potential drug targets a genomic study was performed in lipid-laden primary human hepatocyte (PHH) and human hepatoma cell cultures. METHODS: PHH, HuH7 and HepG2 hepatoma cell cultures were treated with lipids and/or TNFα. Intracellular lipid load was quantified with the ORO assay. The Affymetrix HG-U133+ array system was employed to perform transcriptome analysis. The lipid droplet (LD) growth and fusion was determined by fluorescence microscopy. LD associated proteins were imaged by confocal immunofluorescence microscopy and confirmed by Western immunoblotting. Bioinformatics defined perturbed metabolic pathways. RESULTS: Whole genome expression profiling identified 227, 1031 and 571 significant regulated genes. Likewise, the combined lipid and TNFα treatment of PHH, HuH7 and HepG2 cell cultures revealed 154, 1238 and 278 differentially expressed genes. Although genomic responses differed among in-vitro systems, commonalities were ascertained by filtering the data for LD associated gene regulations. Among others the LD-growth and fusion associated cell death inducing DFFA like effector C (CIDEC), perilipins (PLIN2, PLIN3), the synaptosome-associated-protein 23 and the vesicle associated membrane protein 3 were strongly up-regulated. Likewise, the PPAR targets pyruvate-dehydrogenase-kinase-4 and angiopoietin-like-4 were up-regulated as was hypoxia-inducible lipid droplet-associated (HILPDA), flotilin and FGF21. Their inhibition ameliorates triglyceride and cholesterol accumulation. TNFα treatment elicited strong induction of the chemokine CXCL8, the kinases MAP3K8, MAP4K4 and negative regulators of cytokine signaling, i.e. SOCS2&SOCS3. Live cell imaging of DsRED calreticulin plasmid transfected HuH7 cells permitted an assessment of LD growth and fusion and confocal immunofluorescence microscopy evidenced induced LD-associated PLIN2, CIDEC, HIF1α, HILPDA, JAK1, PDK4 and ROCK2 expression. Notwithstanding, CPT1A protein was repressed to protect mitochondria from lipid overload. Pharmacological inhibition of the GTPase-dynamin and the fatty acid transporter-2 reduced lipid uptake by 28.5 and 35%, respectively. Finally, a comparisons of in-vitro/NAFLD patient biopsy findings confirmed common gene regulations thus demonstrating clinical relevance. CONCLUSION: The genomics of fat-laden hepatocytes revealed LD-associated gene regulations and perturbed metabolic pathways. Immunofluorescence microscopy confirmed expression of coded proteins to provide a rationale for therapeutic intervention strategies. Collectively, the in-vitro system permits testing of drug candidates.

A unifying mathematical model of lipid droplet metabolism reveals key molecular players in the development of hepatic steatosis.

The liver responds to elevated plasma concentrations of free fatty acids (FFAs) with an enhanced uptake of FFAs and their esterification to triacylglycerol (TAG). On the long term, this may result in massive hepatic TAG accumulation called steatosis hepatitis. In hepatocytes, the poor water-soluble TAG is packed in specialized organelles: Lipid droplets (LDs) serving as transient cellular deposit and lipoproteins (LPs) transporting TAG and cholesterol esters to extra-hepatic tissues. The dynamics of these organelles is controlled by a variety of regulatory surface proteins (RSPs). Assembly and export of VLDLs are mainly regulated by the microsomal transfer protein (MTP) and apoprotein B100. Formation and lipolysis of LDs are regulated by several RSPs. The best studied regulators belong to the PAT (Perilipin/Adipophilin/TIP47) and CIDE families. Knockdown or overexpression of SRPs may significantly affect the total number and size distribution of LDs. Intriguingly, a large cell-to-cell heterogeneity with respect to the number and size of LDs has been found in various cell types including hepatocytes. These findings suggest that the extent of cellular lipid accumulation is determined not only by the imbalance between lipid supply and utilization but also by variations in the expression of RSPs and metabolic enzymes. To better understand the relative regulatory impact of individual processes involved in the cellular TAG turnover, we developed a comprehensive kinetic model encompassing the pathways of the fatty acid and triglyceride metabolism and the main molecular processes governing the dynamics of LDs. The model was parametrized such that a large number of experimental in vitro and in vivo findings are correctly recapitulated. A control analysis of the model revealed that variations in the activity of FFA uptake, diacylglycerol acyltransferase (DGAT) 2, and adipose triglyceride lipase (ATGL) have the strongest influence on the cellular TAG level. We used the model to simulate LD size distributions in human hepatoma cells and hepatocytes exposed to a challenge with FFAs. A random fold change by a factor of about two in the activity of RSPs was sufficient to reproduce the large diversity of droplet size distributions observed in individual cells. Under the premise that the same extent of variability of RSPs holds for the intact organ, our model predicts variations in the TAG content of individual hepatocytes by a factor of about 3-6 depending on the nutritional regime. Taken together, our modeling approach integrates numerous experimental findings on individual processes in the cellular TAG metabolism and LD dynamics metabolism to a consistent state-of-the-art dynamic network model that can be used to study how changes in the external conditions or systemic parameters will affect the TAG content of hepatocytes.