Expression analysis of a human hepatic cell line in response to palmitate

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Abstract

Saturated fat plays a role in common debilitating diseases such as obesity, type 2 diabetes, and coronary heart disease. It is also clear that certain fatty acids act as regulators of metabolism via both direct and indirect signalling of target tissues. As the molecular mechanisms of saturated fatty acid signalling in the liver are poorly defined, hepatic gene expression analysis was undertaken in a human hepatocyte cell line after incubation with palmitate. Profiling of mRNA expression using cDNA microarray analysis revealed that 162 of approximately 18,000 genes tested were differentially expressed after incubation with palmitate for 48 h. Altered transcription profiles were observed in a wide variety of genes, including genes involved in lipid and cholesterol transport, cholesterol catabolism, cell growth and proliferation, cell signalling, β-oxidation, and oxidative stress response. While palmitate signalling has been examined in pancreatic β-cells, this is the first report showing that palmitate regulates expression of numerous genes via direct molecular signalling mechanisms in liver cells.

Section snippets

Methods

Cell culture and fatty acid preparation. The human hepatic cell line, Huh-7, was cultured in Dulbecco’s modified Eagle’s medium (DMEM) (Invitrogen, California, USA), supplemented with 10% foetal calf serum (Invitrogen, California, USA), and 1× antibiotic and antimycotic (Invitrogen, California, USA). Fatty acid was prepared by binding palmitate (16:0) to bovine serum albumin (Invitrogen, California, USA).

Cell counting. Cell number was calculated by hemocytometer counting and cell viability was

Microarray analysis revealed altered mRNA expression

In order to identify genes that are transcriptionally regulated by palmitate in the human hepatocyte cell line, Huh-7, mRNA expression was analysed using cDNA microarray analysis. Huh-7 cells were chosen as the model system to study the direct molecular mechanisms of saturated fatty acid signalling and to avoid the complexities inherent in whole animal experiments. Fluorescently labelled mRNA from untreated Huh-7 control cells and cells incubated with 150 μM palmitate for 48 h was hybridised to

Discussion

This study used cDNA microarray and q-PCR analysis to investigate the effect of saturated fatty acid signalling on hepatic gene regulation. These experiments have shown that palmitate is a potent regulator of gene expression for a wide variety of genes. These changes not only affect the expression of genes related to nutrient transport and metabolism, they also extend to other cellular functions such as cytoskeletal architecture, cell growth, protein synthesis, and oxidative stress response.

Acknowledgments

We thank Alistair Forrest, Chris Johns, and Sean Grimmond from the Microarray Facility, Institute of Molecular Biosciences, The University of Queensland, for their technical advice and use of facilities. This work was supported by the Australian Cooperative Research Centres Program. C.D.S. was supported by a Cooperative Research Centre for Diagnostics Postgraduate Research Award.

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