Abstract
BackgroundThe carnitine acetyltransferase (CrAT) is a mitochondrial matrix protein that directly influences intramitochondrial acetyl-CoA pools. Murine CrAT is encoded by a single gene located in the opposite orientation head to head to the PPP2R4 gene, sharing a very condensed bi-directional promoter. Since decreased CrAT expression is correlated with metabolic inflexibility and subsequent pathological consequences, our aim was to reveal and define possible activators of CrAT transcription in the normal embryonic murine liver cell line BNL CL. 2 and via which nuclear factors based on key metabolites mainly regulate hepatic expression of CrAT. Here we describe a functional characterization of the CrAT promoter region under conditions of L-carnitine deficiency and supplementation as well as fenofibrate induction in cell culture cells.ResultsThe murine CrAT promoter displays some characteristics of a housekeeping gene: it lacks a TATA-box, is very GC-rich and harbors two Sp1 binding sites. Analysis of the promoter activity of CrAT by luciferase assays uncovered a L-carnitine sensitive region within −342 bp of the transcription start. Electrophoretic mobility shift and supershift assays proved the sequence element (−228/-222) to be an L-carnitine sensitive RXRα binding site, which also showed sensitivity to application of anti-PPARα and anti-PPARbp antibodies. In addition we analysed this specific RXRα/PPARα site by Southwestern Blotting technique and could pin down three protein factors binding to this promoter element. By qPCR we could quantify the nutrigenomic effect of L-carnitine itself and fenofibrate.ConclusionsOur results indicate a cooperative interplay of L-carnitine and PPARα in transcriptional regulation of murine CrAT, which is of nutrigenomical relevance. We created experimental proof that the muCrAT gene clearly is a PPARα target. Both L-carnitine and fenofibrate are inducers of CrAT transcripts, but the important hyperlipidemic drug fenofibrate being a more potent one, as a consequence of its pharmacological interaction.Electronic supplementary materialThe online version of this article (doi:10.1186/1471-2164-15-514) contains supplementary material, which is available to authorized users.
Highlights
The carnitine acetyltransferase (CrAT) is a mitochondrial matrix protein that directly influences intramitochondrial acetyl-CoA pools
Our first experiments examined the regulation of the murine CrAT gene by L-carnitine and fenofibrate
CrAT mRNA levels were increased by L-carnitine supplementation after artificially induced Lcarnitine deficiency (Figure 1A)
Summary
The carnitine acetyltransferase (CrAT) is a mitochondrial matrix protein that directly influences intramitochondrial acetyl-CoA pools. The importance of CrAT has been recently underlined by the fact that muscle-specific knock-out mice showed increased metabolic inflexibility, meaning that they failed to adjust appropriately to mitochondrial fuel selection in response to nutritional cues [14]. Another very supportive fact for our study was that NIDDM patients showed decreased levels of CrAT mRNA levels [14]. L-carnitine itself could be a pharmacological tool since supplementation after artificially induced L-carnitine deficiency induces CrAT expression in a moderate way [15]
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