Analysis of gene regulatory functions of the human acyl-CoA-binding-protein in lipid metabolism

Vock, Christina

The human acyl-CoA binding protein (ACBP) is an ubiquitary expressed multifunctional protein. ACBP is localized in the cytosol, nucleus, endoplasmatic reticulum and Golgi apparatus. As an intracellular pool former and carrier of acyl-CoAs, ACBP profoundly influences fatty acid metabolism. ACBP transports acyl-CoAs to the nucleus and interacts with the transcription factor hepatocyte nuclear factor 4 alpha (HNF-4α). The gene encoding ACBP is up-regulated by glucose and lipogenic factors and is down-regulated under fasting conditions. Here, the up- and down-regulation of the ACBP gene was simulated in liver HepG2 cells by an overexpression and RNAi approach to study the gene regulatory function of ACBP. In order to identify ACBP target genes, genome-wide transcript profiling under siRNA-mediated ACBP knockdown in human liver HepG2 cells was performed. Based on a single sided permutation T-test (p<0.05) 256 down-regulated and 198 up-regulated transcripts with a minimal fold change of 1.32 (log 0.5) were identified. TaqMan-based qRT-PCR of 10 selected genes confirmed high accuracy of the array data. Gene annotation enrichment analysis revealed ACBP-mediated down-regulation of 18 genes encoding key enzymes in glycerolipid (i.e. mitochondrial glycerol-3-phosphate acyltransferase), cholesterol (i.e. HMG-CoA synthase 1 and HMG-CoA reductase) and fatty acid (i.e. fatty acid synthase) metabolism. Integration of these genes in common pathways suggested a decrease of lipid biosynthesis under ACBP knockdown. Accordingly, saturated (16:0) and monosaturated (16:1, 18:1) fatty acids were significantly reduced to 75% in ACBP-depleted cells. In order to assess the gene regulatory function of ACBP in more detail, reporter gene analysis was performed. Using this approach, ACBP repressed the HNF-4α-induced activity of a 617bp HMGCS1 promoter fragment by about 75% in HepG2 as well as in non-endodermal HeLa cells devoid of HNF-4α. Interestingly, reporter assays without co-transfection of HNF-4α revealed an ACBP-induced reduction of HMGCS1 promoter activity by about 60-80% in both cell lines. Activities of 417bp and 317bp HMGCS1 promoter fragments were 2-4-fold decreased by ACBP. Concordantly, HMGCS1-mRNA and -protein levels were diminished to about 40% and 30% in ACBP-expressing HeLa cells, respectively. Additionally, ACBP diminished promoter activity and transcript levels of the cholesterogenic HMGCR. Taken together, by using the RNAi approach in combination with genome-wide expression profiling in HepG2 cells, evidence for the function of human ACBP in lipid metabolism at the level of gene expression was obtained. This effect seems to be translated to the cellular level of certain fatty acids. An overexpression approach suggested an essential function for human ACBP as a transcriptional regulator for HMG-CoA synthase 1 and HMG-CoA reductase, encoding the rate-limiting enzymes of cholesterol synthesis. The 454 identified ACBP-regulated genes represent a first reference of the human ACBP-regulon.

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Vock, Christina: Analysis of gene regulatory functions of the human acyl-CoA-binding-protein in lipid metabolism.

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