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

Autor: Anna Trincone, Stephanie Breher-Esch, Jürgen Borlak, Christin Wallstab, Nishika Sahini
Rok vydání: 2018
Předmět:
0301 basic medicine
lcsh:Internal medicine
lcsh:QH426-470
Vesicle-Associated Membrane Protein 3
Palmitic Acid
Mitochondrion
Biology
Perilipin-2
Non-alcoholic fatty liver disease (NAFLD)
Transcriptome
03 medical and health sciences
Non-alcoholic Fatty Liver Disease
Lipid droplet
Gene expression
TNFα
Genetics
Humans
Oxidoreductases Acting on Sulfur Group Donors
Qc-SNARE Proteins
SOCS3
lcsh:RC31-1245
Transcriptomics
Cells
Cultured
Genetics (clinical)
Membrane Glycoproteins
Carnitine O-Palmitoyltransferase
Tumor Necrosis Factor-alpha
LD-associated proteins
Genomics
Hep G2 Cells
Lipid Droplets
Drug targets
nuclear receptor/PPARs
Transfection
Qb-SNARE Proteins
Endoplasmic Reticulum Stress
Molecular biology
Mitochondria
Gene expression profiling
lcsh:Genetics
030104 developmental biology
Gene Expression Regulation
Microscopy
Fluorescence
Hepatocytes
Perilipin
Oleic Acid
Research Article
Zdroj: BMC Medical Genomics
BMC Medical Genomics, Vol 11, Iss 1, Pp 1-20 (2018)
ISSN: 1755-8794
DOI: 10.1186/s12920-018-0438-7
Popis: 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. Electronic supplementary material The online version of this article (10.1186/s12920-018-0438-7) contains supplementary material, which is available to authorized users.
Databáze: OpenAIRE
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