The human liver fatty acid binding protein (FABP1) gene is activated by FOXA1 and PPARα; and repressed by C/EBPα: Implications in FABP1 down-regulation in nonalcoholic fatty liver disease

https://doi.org/10.1016/j.bbalip.2012.12.014Get rights and content

Abstract

Liver fatty acid binding protein (FABP1) prevents lipotoxicity of free fatty acids and regulates fatty acid trafficking and partition. Our objective is to investigate the transcription factors controlling the human FABP1 gene and their regulation in nonalcoholic fatty liver disease (NAFLD). Adenovirus-mediated expression of multiple transcription factors in HepG2 cells and cultured human hepatocytes demonstrated that FOXA1 and PPARα are among the most effective activators of human FABP1, whereas C/EBPα is a major dominant repressor. Moreover, FOXA1 and PPARα induced re-distribution of FABP1 protein and increased cytoplasmic expression. Reporter assays demonstrated that the major basal activity of the human FABP1 promoter locates between − 96 and − 229 bp, where C/EBPα binds to a composite DR1-C/EBP element. Mutation of this element at − 123 bp diminished basal reporter activity, abolished repression by C/EBPα and reduced transactivation by HNF4α. Moreover, HNF4α gene silencing by shRNA in HepG2 cells caused a significant down-regulation of FABP1 mRNA expression. FOXA1 activated the FABP1 promoter through binding to a cluster of elements between − 229 and − 592 bp, whereas PPARα operated through a conserved proximal element at − 59 bp. Finally, FABP1, FOXA1 and PPARα were concomitantly repressed in animal models of NAFLD and in human nonalcoholic fatty livers, whereas C/EBPα was induced or did not change. We conclude that human FABP1 has a complex mechanism of regulation where C/EBPα displaces HNF4α and hampers activation by FOXA1 and PPARα. Alteration of expression of these transcription factors in NAFLD leads to FABP1 gen repression and could exacerbate lipotoxicity and disease progression.

Highlights

► A model for transcription regulation of human FABP1 gene in liver is proposed. ► HNF4α and FOXA1 contribute to the high constitutive FABP1 transcription. ► PPARα induces FABP1 in response to ligands such as GW7647. ► CEBPα is a repressor, competes with HNF4α and blunts activation by FOXA1 and PPARα. ► Repression of FOXA1 and PPARα is concomitant to FABP1 down-regulation in NAFLD.

Introduction

Free fatty acids (FAs) are known to be potent cytotoxic, amphipathic detergents damaging plasma, mitochondrial and lysosomal membranes and inhibiting the action of multiple receptors and enzymes [1]. To prevent FA lipotoxicity, mammals have evolved a family of small cytosolic fatty acid binding proteins to maintain the intracellular concentration of unbound free FAs and fatty acyl CoAs at a low level (i.e. nM range) [2]. The liver has a very active role in long-chain fatty acid (LCFA) uptake and metabolism and in agreement has a high level of a fatty acid binding protein, FABP1 or L-FABP, which represents 2–5% of the cytosolic protein (0.1–0.4 mM) [2]. FABP1 has a broad range/promiscuity for a variety of ligands, and high affinity for LCFAs and long-chain fatty acyl CoAs (LCFA-CoAs) [3].

Studies performed in vitro and in vivo show that FABP1 facilitates uptake of LCFA from membranes, binds LCFAs and LCFA-CoAs to minimize toxic effects, enhances intracellular transport/diffusion, functions as a donor for both peroxisomal and mitochondrial LCFA β-oxidation, and targets LCFA and LCFA-CoA to the endoplasmic reticulum for esterification into membrane lipids (phosphatidic acid, phospholipids) or into lipids for storage and secretion in very low density lipoproteins (triglycerides, cholesteryl esters) [4]. Moreover, FABP1 transfers and channels lipidic ligands into nuclei for initiating nuclear receptor transcriptional activity and activating new lipid signaling pathways that affect lipid and glucose metabolism [5].

The physiological significance of FABP1 has been extended and confirmed by numerous studies using FABP1 deficient mice. It has been found that FABP1 plays a role not only in hepatic FA metabolism, but also in hepatic cholesterol metabolism [6]. Indeed, FABP1 seems to protect against gallstone in lithogenic diet-fed mice [7]. Moreover, studies with FABP1 deficient mice [8] and with a Thr94Ala mutation in human FABP1 [9] also suggest that FABP1 is able to influence glucose homeostasis. In this regard, it has also been shown that FABP1 can bind glucose and glucose-1-phosphate resulting in altered FABP1 conformation and increased affinity, uptake and distribution of lipidic ligands [10].

FABP1 function is also relevant for body weight and, depending on the diet type and the mouse strain, weight gain [11], [12], [13], [14] or protection against obesity [8], [15], [16] has been observed in FABP1 deficient mice.

In the liver, FABP1 could influence the level of triglycerides. A lower concentration of triglycerides have been found in FABP1 deficient mice under different diets [6], [8], [15], [16] and in response to 48-h fasting [17]. Lipid analysis of FABP1 gene-ablated hepatocytes has also revealed lower triglyceride levels [18]. Moreover, the Thr94Ala mutation in the FABP1 gen, which supposedly contributes negatively to FA binding, caused decreased triglycerides compared to the wild-type cells, when incubated with extracellular FAs [19].

A different question is whether FABP1 levels are altered or not by metabolic conditions such as NAFLD. Very few studies have investigated this point. A clinical study has shown that FABP1 is overexpressed in simple steatosis, but paradoxically underexpressed in nonalcoholic steatohepatitis [20]. FABP1 levels were also decreased in a steatotic rat model established by administration of 17α-ethynylestradiol [21]. Lower FABP1 level/function could lead to a lower capacity to attenuate the cytotoxic detergent effect of free FAs and potentiate lipotoxicity in NAFLD [20]. The underlying mechanism for FABP1 deregulation in NAFLD has not yet been investigated.

Given the key role of FABP1 in lipid metabolism and its potential influence in metabolic disorders [22], the study of FABP1 gene regulation is of particular relevance. FABP1 is induced by both fibrate hypolipidemic drugs and LCFA, and a functional peroxisome proliferator activated receptor (PPAR)-responsive element has been identified in the proximal 5′-flanking region of the murine FABP1 [23], [24]. FABP1 is also induced by statins in rodents through a mechanism involving PPARα upregulation and FABP1 activation via its PPAR-responsive element [25]. Other major transcription activator of FABP1 in the liver is HNF1α, and mice with HNF1α gene null mutation exhibit complete loss of FABP1 expression [26], [27]. In addition, several liver enriched transcription factors including C/EBPs [28], FOXAs [27], and HNF4α [29] have been found to be capable of transactivating rodent FABP1. Thus, the regulation of FABP1 gene has been investigated in rodents but, despite substantial differences in the regulation of tissue-specific genes between rodents and humans [30], very few studies have extrapolated rodent data and investigated the regulation of the FABP1 gene in the human liver.

Here we present a comprehensive study of the regulation of human FABP1 and demonstrate that FOXA1 and HNF4α contribute to the high expression of FABP1 in the liver through newly identified cognate elements. We have also confirmed the regulation of human FABP1 by PPARα and uncovered C/EBPα as a dominant transcriptional repressor of FABP1 in human hepatocytes. We also show that FABP1 is repressed in NAFLD and that this repression correlates with an altered expression of its transcription factors, thus suggesting that the mechanisms of FABP1 regulation could have potential clinical implications in NAFLD.

Section snippets

Animals

To induce NAFLD, male C57BL/6J mice (8–10 weeks old) were fed a MCD diet for 5 weeks (cat. no. 960439, ICN, Aurora, OH, USA). Control mice received the same MCD diet supplemented with DL-methionine (3 g/kg) and choline chloride (2 g/kg) (cat. no. 960441, ICN). Mice (five per group) were allowed food and water ad libitum up to 5 weeks. At the end of the feeding experiment, mice were euthanized and livers were excised. A part of the right posterior lobe was fixed in 10% formalin. The presence of

The human FABP1 gene is induced by PPARα and FOXA1, and repressed by C/EBPα

Human hepatoma HepG2 cells were transduced with adenoviral vectors encoding 10 transcription factors. The selection of these factors was based on two criteria: 1) a proven role in transcription regulation of energy/metabolism genes (particularly fatty acid metabolism genes) and 2) an important expression level in the liver. Based on these criteria, we selected four lipid-sensor nuclear receptors: PPARα, LXRα, PXR and CAR; their heterodimeric partner: RXRα; one liver-enriched nuclear receptor:

Discussion

We have accomplished a comprehensive study of FABP1 gene regulation in human liver cells. Most of the previous work on FABP1 regulation has focused on the rat or the mouse FABP1 gene [26], [27], [29]. However, marked differences between rodents and humans in tissue-specific gene regulation [30], [57] point to the need to investigate the regulation of human genes. Indeed, our results have revealed substantial differences between the regulation of rodent and human FABP1 that are discussed below.

Acknowledgments

This work has been supported by grants PI 10/00194 from Fondo de Investigación Sanitaria (FIS, Instituto de Salud Carlos III, Ministerio de Economía y Competitividad), BFU2010-15784 from Ministerio de Educación y Ciencia and GRS 482/A/10 from Junta de Castilla y León. C.G. was a recipient of a contract (CA 09/00122) from the Instituto de Salud Carlos III. M.B. (EHD‐10‐DOC2) and M.V. G.‐M (EHD-24-DOC) were recipients of CIBERehd contracts. The authors also wish to thank to Dr. David Hervás

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