Abnormal energy regulation may significantly contribute to the pathogenesis of obesity, diabetes mellitus, cardiovascular disease, and cancer. For rapid control of energy homeostasis, allosteric and posttranslational events activate or alter activity of key metabolic enzymes. For longer impact, transcriptional regulation is more effective, especially in response to nutrients such as long chain fatty acids (LCFA). Recent advances provide insights into how poorly water-soluble lipid nutrients [LCFA; retinoic acid (RA)] and their metabolites (long chain fatty acyl Coenzyme A, LCFA-CoA) reach nuclei, bind their cognate ligand-activated receptors, and regulate transcription for signaling lipid and glucose catabolism or storage: (i) while serum and cytoplasmic LCFA levels are in the 200 μM–mM range, real-time imaging recently revealed that LCFA and LCFA-CoA are also located within nuclei (nM range); (ii) sensitive fluorescence binding assays show that LCFA-activated nuclear receptors [peroxisome proliferator-activated receptor-α (PPARα) and hepatocyte nuclear factor 4α (HNF4α)] exhibit high affinity (low nM Kds) for LCFA (PPARα) and/or LCFA-CoA (PPARα, HNF4α)—in the same range as nuclear levels of these ligands; (iii) live and fixed cell immunolabeling and imaging revealed that some cytoplasmic lipid binding proteins [liver fatty acid binding protein (L-FABP), acyl CoA binding protein (ACBP), cellular retinoic acid binding protein-2 (CRABP-2)] enter nuclei, bind nuclear receptors (PPARα, HNF4α, CRABP-2), and activate transcription of genes in fatty acid and glucose metabolism; and (iv) studies with gene ablated mice provided physiological relevance of LCFA and LCFA-CoA binding proteins in nuclear signaling. This led to the hypothesis that cytoplasmic lipid binding proteins transfer and channel lipidic ligands into nuclei for initiating nuclear receptor transcriptional activity to provide new lipid nutrient signaling pathways that affect lipid and glucose catabolism and storage.

Role of Fatty Acid Binding Proteins and Long Chain Fatty Acids in Modulating Nuclear Receptors and Gene Transcription

While low levels of unesterified long chain fatty acids (LCFAs) are normal metabolic intermediates of dietary and endogenous fat, LCFAs are also potent regulators of key receptors/enzymes and at high levels become toxic detergents within the cell. Elevated levels of LCFAs are associated with diabetes, obesity and metabolic syndrome. Consequently, mammals evolved fatty acid-binding proteins (FABPs) that bind/sequester these potentially toxic free fatty acids in the cytosol and present them for rapid removal in oxidative (mitochondria, peroxisomes) or storage (endoplasmic reticulum, lipid droplets) organelles. Mammals have a large (15-member) family of FABPs with multiple members occurring within a single cell type. The first described FABP, liver-FABP (L-FABP or FABP1), is expressed in very high levels (2-5% of cytosolic protein) in liver as well as in intestine and kidney. Since L-FABP facilitates uptake and metabolism of LCFAs in vitro and in cultured cells, it was expected that abnormal function or loss of L-FABP would reduce hepatic LCFA uptake/oxidation and thereby increase LCFAs available for oxidation in muscle and/or storage in adipose. This prediction was confirmed in vitro with isolated liver slices and cultured primary hepatocytes from L-FABP gene-ablated mice. Despite unaltered food consumption when fed a control diet ad libitum, the L-FABP null mice exhibited age- and sex-dependent weight gain and increased fat tissue mass. The obese phenotype was exacerbated in L-FABP null mice pair fed a high-fat diet. Taken together with other findings, these data suggest that L-FABP could have an important role in preventing age- or diet-induced obesity.

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Liver fatty acid-binding protein and obesity

Peroxisome proliferator-activated receptors (PPAR) are ligand-activated members of the steroid/thyroid nuclear hormone receptor superfamily. PPAR have been identified in several species, with at least three isotypes recognized (α, β/δ, γ). Each isotype, encoded by a different gene, is differentially expressed in select tissues and expression levels depend on cellular processes (rev. in (1). Although there is some overlap in ligand specificity, unique isotype binding occurs by a variety of xenobiotics depends upon specific amino acid substitutions within the PPAR ligand-binding pocket (1,2). Independent of ligand binding, a basal level of transcription occurs by PPAR forming a heterodimer with the retinoid X receptor (RXR) which binds to a specific DNA response element (PPRE) within the target genes (3,4). Ligand binding to one and, even more so, both receptors greatly potentiates transcription (rev. in (4). PPRE exist within many of the genes involved in fatty acid oxidation and cell differentiation, suggesting that PPAR are responsible for regulating transcription of these genes (5).

An especially important role of PPARα is inducing transcription of multiple peroxisomal enzymes required for β-oxidation of very-long-chain fatty acids (VLCFA) and branched-chain fatty acids (BCFA). VLCFA and BCFA accumulation are associated with human neurological disorders including Zellweger syndrome, X-linked adrenoleukodystrophy, Refsum disease, and MFP-2 deficiency (6-8). These genetic disorders arise from defects in enzymes of the peroxisomal β-oxidation pathway which result in accumulation of VLCFA, BCFA, and their thioesters (6-8). Both VLCFA and isoprenoid-derived BCFA preferentially undergo peroxisomal β-oxidation following acylation of the respective fatty acids to CoA thioesters. The classical peroxisomal β-oxidation pathway catalyzed by palmitoyl-CoA oxidase (AOX), L-bifunctional protein (L-PBE), and 3-ketoacyl-CoA thiolase (PTL) is responsible for the oxidation of straight-chain fatty acids (9). All three enzymes of the classical peroxisomal β-oxidation system are transcriptionally activated by PPARα ligands (rev. in (10). In contrast, although the branched-chain phytanic acid is considered a β-oxidation pathway precursor, a different set of peroxisomal enzymes are utilized for oxidation of BCFA. For example, phytanic acid must first undergo α-oxidation to produce pristanoyl-CoA before entering the β-oxidation cycle (11). BCFA-CoAs are then utilized by the branched-chain acyl-CoA oxidase, D-bifunctional protein (D-PBE), and sterol carrier protein-x (SCP-x). Both D-PBE (9) and SCP-x (12,13) are inducible upon administration of branched-chain PPARα ligands.

While there is evidence indicating that VLCFA and BCFA are peroxisome proliferators and may be PPARα ligands, it is not completely clear whether the unesterified VLCFA and BCFA directly and/or metabolites thereof activate PPARα. For example, early transactivation assays show: (i) PPARα is activated by polyunsaturated (20:4, 20:5, 22:6), but not monounsaturated (24:1, 22:1) VLCFA (3), (ii) n-3 polyunsaturated VLCFA elicited similar PPARα activation levels as n-6 polyunsaturated VLCFA (3). These findings of VLCFA being potential PPARα ligands are supported by gel-shift assays (14) and co-activator-dependent receptor ligand assays (15). Likewise, transactivation studies with branched-chain fatty acids also show PPARα activation (11). It must be noted that most of these assays utilize very high concentrations of VLCFA (i.e. 30-100 μM) and BCFA (20 μM)--several orders of magnitude higher than concentrations of unesterified fatty acids detected in nuclei of living cells (16,17). These data suggested that affinities of PPARα for these ligands are very weak, i.e. Kds in the μM range, and the free fatty acids may not represent the endogenous ligands. The finding that, since unesterified fatty acids are potent detergents, they are rapidly activated/metabolized to their CoA thioesters in cells and tissues (rev. (18-20), suggests that metabolites such as the CoA thioesters may represent the endogenous PPARα ligands. This possibility is supported by recent findings in mice. For instance, peroxisome proliferators increase the level of CoA thioesters of the peroxisome proliferators in mice (21,22). Inhibitors of fatty acid thioesterification such as 2-bromopalmitic acid inhibit the effects of bezafibrate on peroxisomal proliferation (23). Mice with a disruption of the AOX gene exhibit peroxisomal proliferation, elevated serum levels of VLCFA, as well as elevated VLCFA-CoAs, hyperactivation of PPARα, and up-regulation of down-stream genes which contain PPRE (23). On the other hand, ablation of the adrenoleukodystrophy gene inhibits VLCFA transport into peroxisomes and results in elevated serum VLCFA, unaltered liver VLCFA-CoA, and normal PPARα activity (10). Taken together with other studies (24,25) these data in both normal and gene-targeted mice suggest that intermediates of peroxisomal β-oxidation are PPARα ligands.

In summary, while many of the above experiments are suggestive that the CoA thioesters of VLCFA and BCFA represent functional ligands for PPARα, it remains to be shown whether the VLCFA, BCFA, or their CoA thioesters exhibit characteristic hallmarks of endogenous ligands that bind to PPARα: (i) high (Kds in nM range) affinity, (ii) physically alter the structure/conformation of the PPARα protein, and (iii) affect co-factor recruitment PPARα (26). The objective of the present study was to begin to resolve these issues through use of direct ligand binding assays (i.e. non-fluorescent ligand binding assays based on quenching of tyrosine emission), circular dichroism to characterize potential ligand-induced changes in PPARα secondary structure, and co-immunoprecipitation to study co-factor recruitment.

Very-Long-Chain and Branched-Chain Fatty Acyl-CoAs are High Affinity Ligands for the Peroxisome Proliferator-Activated Receptor α (PPARα)

Phytanic acid alpha-oxidation, new insights into an old problem: a review.

RATIONALE

Both traditional electron ionization and electrospray ionization tandem mass spectrometry have demonstrated limitations in the unambiguous identification of fatty acids. In the former case, high electron energies lead to extensive dissociation of the radical cations from which little specific structural information can be obtained. In the latter, conventional collision-induced dissociation (CID) of even-electron ions provides little intra-chain fragmentation and thus few structural diagnostics. New approaches that harness the desirable features of both methods, namely radical-driven dissociation with discrete energy deposition, are thus required.



METHODS

Herein we describe the derivatization of a structurally diverse suite of fatty acids as 4-iodobenzyl esters (FAIBE). Electrospray ionization of these derivatives in the presence of sodium acetate yields abundant [M + Na]+ ions that can be mass-selected and subjected to laser irradiation (λ = 266 nm) on a modified linear ion-trap mass spectrometer.



RESULTS

Photodissociation (PD) of the FAIBE derivatives yields abundant radical cations by loss of atomic iodine and in several cases selective dissociation of activated carbon–carbon bonds (e.g., at allylic positions) are also observed. Subsequent CID of the [M + Na – I]•+ radical cations yields radical-directed dissociation (RDD) mass spectra that reveal extensive carbon–carbon bond dissociation without scrambling of molecular information.



CONCLUSIONS

Both PD and RDD spectra obtained from derivatized fatty acids provide a wealth of structural information including the position(s) of unsaturation, chain-branching and hydroxylation. The structural information obtained by this approach, in particular the ability to rapidly differentiate isomeric lipids, represents a useful addition to the lipidomics tool box. Copyright © 2013 John Wiley & Sons, Ltd.

https://onlinelibrary.wiley.com/doi/pdf/10.1002/rcm.6503

Rapid differentiation of isomeric lipids by photodissociation mass spectrometry of fatty acid derivatives

Branched-chain fatty acids are potent regulators of gene expression. Among them are the vitamin A-derived retinoic acids, which are involved in cell growth and differentiation, and the chlorophyll-derived phytol metabolites such as phytanic and pristanic acids, which affect catabolic lipid metabolism. Gene expression regulated by these signaling molecules is mediated by two protein families. These are, on the one hand, the intracellular lipid binding proteins, i.e. cellular retinoic acid binding protein and liver-type fatty acid binding protein, which are responsible for ligand-transport to the nucleus. On the other hand are the ligand-activated nuclear receptors, i.e. the retinoic acid receptors for retinoic acids and the peroxisome proliferator-activated receptors for the phytol metabolites. In this review, we discuss the cross-talk between the two protein families and how this cross-talk contributes to targeted signaling with branched-chain fatty acids.

A. Adida

Papers

Intracellular lipid binding proteins and nuclear receptors involved in branched-chain fatty acid signaling.