Effect of dietary polyunsaturated fatty acids on the expression of peroxisomal ABC transporters

Abstract

Peroxisomal ABC transporters encoded by the ABCD genes are thought to participate in the import of specific fatty acids in the peroxisomal matrix. ABCD1 deficiency is associated with X-linked adrenoleukodystrophy (X-ALD), the most frequent peroxisomal disorder which is characterized by the accumulation of saturated very-long-chain fatty acids (VLCFA). ABCD2 (the closest homolog of ABCD1) and ABCD3 have been shown to have partial functional redundancy with ABCD1; only when overexpressed, they can compensate for VLCFA accumulation. Other lipids, for instance polyunsaturated fatty acids (PUFA), should be possible candidate substrates for the ABCD2 and ABCD3 gene products, ALDRP and PMP70 respectively. Moreover, PUFA, which are known regulators of gene expression, could therefore represent potent inducers of the ABCD genes. To test this hypothesis, littermates of n-3-deficient rats were subjected to an n-3-deficient diet or equilibrated diets containing ALA (α-linolenic acid, 18:3n-3) as unique source of n-3 fatty acids or ALA plus DHA (docosahexaenoic acid, 22:6n-3) at two different doses. We analyzed the expression of peroxisomal ABC transporters and of the peroxisomal acyl-CoA oxidase gene 1 (Acox1) in adrenals, brain and liver. Whatever the diet, we did not observe any difference in gene expression in adrenals and brain. However, the hepatic expression level of Abcd2 and Abcd3 genes was found to be significantly higher in the n-3-deficient rats than in the rats fed the ALA diet or the DHA supplemented diets. This was accompanied by important changes in hepatic fatty acid composition. In summary, the hepatic expression of Abcd2 and Abcd3 but not of Abcd1 and Abcd4 appears to be highly sensitive towards dietary PUFA. This difference could be linked to the substrate specificity of the peroxisomal ABC transporters and a specific involvement of Abcd2 and Abcd3 in PUFA metabolism.

Introduction

X-linked adrenoleukodystrophy (X-ALD, MIM 300100), the most frequent peroxisomal disorder [1], [2], is caused by mutations in the ABCD1 (ALD) gene located in Xq28 [3]. ABCD1 encodes for a peroxisomal ABC (ATP-binding cassette) half-transporter called ALDP (adrenoleukodystrophy protein). The peroxisomal membrane contains three other homologous ABC half-transporters: ALDRP, the closest homolog of ALDP, encoded by the ABCD2 gene [4], PMP70 and PMP69 (70 and 69 kDa peroxisomal membrane proteins) encoded by the ABCD3 [5] and ABCD4 [6], [7] genes respectively. X-ALD is characterized by the accumulation of saturated and monounsaturated very-long-chain fatty acids (VLCFA, C > 22) in plasma and tissues. ALDP has been hypothesized to allow the import of VLCFA (probably as acyl-CoA) into the peroxisome where they are exclusively β-oxidized. Overexpression of ALDRP or PMP70 in X-ALD fibroblasts [8], [9], [10], [11], [12], [13] and of ALDRP in the Abcd1 null mice [14] has been shown to compensate for ALDP deficiency leading to a normalization of VLCFA levels. This partial functional redundancy suggests that ALDRP and PMP70 are preferentially involved in the transport of lipids structurally related to the supposed substrates of ALDP (saturated or monounsaturated VLCFA), for instance, very-long-chain polyunsaturated fatty acids (PUFA).

Linoleic acid (LA, 18:2n-6) and α-linolenic acid (ALA, 18:3n-3), which cannot be synthesized in mammals, are the essential precursors for the n-6 and n-3 series of PUFA respectively [15]. Docosahexaenoic acid (DHA; 22:6n-3), which is a major and essential constituent of cerebral lipids, is formed from ALA through a series of desaturation and elongation steps located in the ER. The last step of the DHA synthesis is located in the peroxisomal matrix and consists of one β-oxidation cycle, which transforms tetracosahexaenoic acid (24:6n-3) in DHA [16], [17]. A similar pathway has been postulated for the synthesis of DPA (22:5n-6) from LA with a final step of peroxisomal β-oxidation of tetracosapentaenoic acid (24:5n-6). Moreover, dietary PUFA are important nutrients, not only as essential constituents of cell membranes or as precursors for eicosanoids but also for their role in gene expression in liver and brain [18], [19], [20]. They have been shown to regulate the activity of numerous nuclear receptors and transcription factors including the three PPAR isoforms, RXRα, LXRα, SREBPs, FXR and HNF-4α. PPARα, LXRα and SREBPs are known to control Abcd2 expression [21], [22]. Moreover, Abcd2, which is particularly well expressed in DHA-rich tissues [4], [23], could participate in the peroxisomal entry of 24:6n-3, the immediate precursor of DHA. Therefore, a direct regulation of the expression of Abcd2 by DHA or its precursors could be speculated.

Thus, the aim of the present study was to clarify the impact of dietary PUFA on the expression of Abcd2 and the other peroxisomal ABC transporters. To that end, by using real-time RT-PCR, we analyzed gene expression in brain, adrenals, and liver of littermates of n-3-deficient rats fed an ALA-deficient diet (n-3 PUFA deficiency was compensated by LA) or diets containing ALA as the unique source of n-3 fatty acids or ALA plus two doses of DHA. The expression of Acox1, which encodes the first enzyme of the peroxisomal β-oxidation of saturated or unsaturated straight chain fatty acids and is recognized as a target gene of PPARα was also investigated.