Fatty acid-binding proteins: functional understanding and diagnostic implications

Abstract

Purpose of review

Fatty acid-binding proteins (FABPs) are a family of small, abundant proteins with highly tissue-specific expression patterns whose different functions remain incompletely understood. The purpose of this review is to summarize recent findings regarding FABP functions and mechanisms of action, including their potential utilization as serum markers of tissue-specific metabolic diseases.

Recent findings

FABPs are important not only in their tissues of origin but also appear to influence the metabolism and function of tissues distal to their sites of expression. This may be secondary to metabolic changes in their primary tissues, and/or a result of FABP secretion from these tissues leading to effects on distal sites. Their levels in the circulation are increasingly explored as potential biomarkers for tissue-specific disease prognosis and progression.

Summary

The nine fatty acid-binding members of the FABP family have unique tissue-specific functions and important secondary effects on tissues in which they are not expressed. For many of the FABPs, circulating levels may be indicative of disease processes related to their primary tissues, and may influence physiological function in distal tissues.

INTRODUCTION

Fatty acid (FA)-binding proteins (FABPs) are a family of highly expressed intracellular proteins that were first identified almost 50 years ago. Initially thought to bind long-chain FAs almost exclusively, it is now appreciated that their ligand specificity extends to numerous other hydrophobic molecules including endocannabinoids and lipophilic drugs. As reviewed earlier, abundant evidence has indicated that the nine FA-binding members of the FABP family have unique, tissue-specific functions, and that they may also exert regulatory effects outside their tissues of expression [1]. Systemic effects of the FABPs may be secondary to actions within their tissues of expression, or possibly via their presence in the circulation. Indeed circulating levels of many of the FABPs are considered as diagnostic markers of the physiological status of their tissues of origin. Here, we will review recent literature that provides insight into functional aspects of each of the FABPs, in some cases including how their serum levels may be used as a diagnostic marker potentially related to their tissue-level actions.

LIVER FATTY ACID-BINDING PROTEIN OR FATTY ACID-BINDING PROTEIN 1

Liver FABP (LFABP, FABP1) is abundant in the liver and intestine. Recent evidence not only builds on its important roles in hepatic and intestinal lipid metabolism, but also sheds light on the mechanisms underlying its effects on whole-body energy homeostasis.

In hepatocytes, LFABP was recently found to be required for perilipin 5-mediated lipid droplet formation and reversal of hepatic stellate cells activation, thereby inhibiting fibrogenesis and hepatic fibrosis [2▪]; LFABP ablation was also shown to prevent fasting-induced hepatic steatosis in liver-specific microsomal triglyceride transfer protein knockout mice, but not to affect de novo lipogenesis-induced steatosis secondary to a high-fructose diet, underscoring the importance of LFABP in transporting exogenous FA into the liver [3]. In addition, with its high affinity for various hydrophobic molecules including endocannabinoids, LFABP has been found to alter the effect of a high-fat diet on endocannabinoid levels in the liver [4] as well as in the brain [5], in a sex-specific manner. In the liver, loss of LFABP prevents the diet-induced elevation in arachidonoylethanolamide (AEA) in female mice, whereas hepatic AEA levels in male wild-type mice did not respond to a high-fat diet despite their much higher AEA levels relative to females [4]. In the brain, the pattern seems to be opposite, with LFABP ablation diminishing the high-fat diet-induced increase in AEA and 2-arachidonoylglycerol (2-AG) in male but not female mice [5]. Further studies also demonstrate that loss of both LFABP and sterol carrier protein-2, another abundant endocannabinoid-binding protein in the liver, exacerbated the high-fat diet-induced elevation of hepatic endocannabinoid levels, suggesting their synergistic roles in the hepatic endocannabinoid system [6]. LFABP regulates endogenous endocannabinoid levels not only by decreasing their uptake, but also by enhancing their intracellular hydrolysis, particularly the hydrolysis of 2-AG by monoacylglycerol lipase [7▪]. Given that the endocannabinoid system is involved in promoting energy storage and fat accumulation, the effects of LFABP on whole-body energy metabolism are likely to be mediated, at least in part, by the endocannabinoid system, especially in the context of obesity.

In enterocytes, new insight into the biological role of LFABP in cell proliferation was recently reported, where significantly decreased proliferation rates were observed in an LFABP knockdown Caco-2 cell model [8]. Given the dramatic whole-body phenotypes in high-fat-fed mice lacking LFABP, as well as the alterations in brain endocannabinoid levels, it appears that the functions of LFABP extend beyond its expression sites. Indeed, we have recently observed unexpected metabolic changes in the skeletal muscle of high-fat-fed LFABP null mice [9▪], suggesting that LFABP ablation may trigger interorgan crosstalk to affect energy metabolism in other tissues.

The expression of LFABP in liver and the gastrointestinal tract suggests potential utility as a marker of tissue-level disorders. For example, LFABP was identified as a candidate biomarker for early detection of gastric cancer when highly coexpressing with FA synthase [10,11]. High serum LFABP was also found to be associated with increased risk of death in patients with acetaminophen-induced acute liver failure, which could potentially be used to improve prognostic models [12].

INTESTINAL FATTY ACID-BINDING PROTEIN OR FATTY ACID-BINDING PROTEIN 2

Intestinal FABP (IFABP, FABP2) is only highly expressed in the small intestine, where it coex-presses with LFABP (different functions of the two enterocyte FABPs previously reviewed [13]). Due to its tissue-specific localization, the potential of IFABP as a biomarker for intestinal damage has been explored in various conditions including celiac disease and necrotizing enterocolitis in preterm neonates [14–17]. Significantly, type II diabetes has been associated with increased intestinal permeability (leaky gut), as assessed by circulating IFABP, along with lipopolysaccharide (LPS) and LPS-binding protein [18]. In keeping with the notion that IFABP is an important marker for intestinal integrity, we have recently found that mice lacking IFABP display decreased intestinal villi length, alterations in goblet cell density, and increased intestinal permeability [19].

A relatively common polymorphism in human IFABP, Ala54Thr, has been linked to systemic lipid and glucose control, and was shown to be significantly associated with postprandial hypertriglyceridemia [20]. In addition, in response to a lowglycemic index diet, Thr54 allele carriers consistently exhibited a blunted decrease in serum triglycerides, with lower fasting glucose levels in women carriers [21]. The mechanisms underlying these associations are unknown.

FATTY ACID-BINDING PROTEIN 6 OR ILEAL LIPID-BINDING PROTEIN

FABP6 (ILFABP, ileal bile acid-binding protein) displays a high affinity for bile acids and is mainly expressed in the ileum, the site of bile acid uptake. As FABP6 is a downstream target of farnesoid X receptor (FXR), several recent studies have examined FABP6 in the context of FXR signaling. Under high-fat feeding, ileal Fabp6 was found increased along with serum bile acids levels in mice lacking fibroblast growth factor 15 (FGF15), a ligand for FXR signaling, indicating that the Fabp6 induction mediated by FXR signaling may be independent of FGF15 [22]. In studies examining effects of alcohol exposure, Fabp6 expression was markedly reduced in FXR null mice; minimal effects of alcohol were observed in wild-type mice [23], whereas hepatocyte-specific FXR knockout mice displayed a ~50% decrease in Fabp6 on a control diet, and elevated Fabp6 in response to alcohol feeding [24▪]. The evidence reveals the importance of intestinal FXR signaling in FABP6 expression, but it remains unclear how FABP6 and FXR signaling function in the context of high-fat or alcohol feeding. Given the complexity of FXR enterohepatic signaling and its broad implications in alcoholic/nonalcoholic fatty liver disease, further research is warranted to understand the interactions between FABP6, FXR, and environmental stimuli.

In addition to its role in lipid metabolism, Fabp6 has been identified as a differentially overexpressed gene in tumor tissues of colorectal cancer patients based on microarray analysis, and real-time polymerase chain reaction analysis supports its association with colorectal cancer metastasis [25].

HEART FATTY ACID-BINDING PROTEIN OR FATTY ACID-BINDING PROTEIN 3

Heart FABP (HFABP, FABP3) is highly expressed in cardiac and skeletal muscle cells and is thought to channel FAs toward β-oxidation, such that its deficiency leads to enhanced glucose oxidation in myocytes and increased plasma FA levels [26]. FABP3 enters the circulation soon after myocardial cell damage, and heart failure patients have elevated circulating FABP3 levels. FABP3 has long been suggested as a prognostic biomarker for ischemic heart disease and heart failure [26,27], although its diagnostic performance as a marker of acute myocardial infarction (MI) and related cardiac disorders remains controversial [28–30]. FABP3 overexpression has been shown to trigger phosphorylation of mitogen-activated protein kinase, initiating a signaling cascade implicated in cardiomyocyte apoptosis and remodeling following MI, aggravating ischemic heart injury [31▪]. Significantly, lower atrial expression of Fabp3 before cardiac surgery was shown to be strongly associated with the development of atrial fibrillation postoperatively, presumably due to impaired cardiac lipid metabolism [32]. Cardiomyocyte damage is also associated with exacerbation of chronic obstructive pulmonary disease, where increased FABP3 levels may predict adverse outcomes [33].

FABP3 is also expressed in brain, and in dopaminergic neurons FABP3 appears to regulate the activity of D2 receptors, with deletion of Fabp3 leading to behavioral traits resembling posttraumatic stress disorder [34]. Significantly, binding of n − 6 polyunsaturated FAs (PUFAs) by FABP3 may decrease the PUFA-induced neuronal oligomerization of alpha-synuclein, thus suppressing Parkinson’s disease-associated cell death [35].

ADIPOCYTE FATTY ACID-BINDING PROTEIN OR FATTY ACID-BINDING PROTEIN 4

Adipocyte FABP (AFABP, FABP4) is primarily expressed in white and brown adipocytes, macrophages, monocytes, and endothelial cells, and is widely used as a cellular marker of adipocyte maturation. In recent years FABP4 has been suggested to be an adipose-derived cytokine, with circulating FABP4 serving to integrate adipose energy storage and distant organs. It is worth noting that FABP4 has no identifiable secretory signal sequence. Nevertheless, its lipolysis-associated secretion has been proposed to impact hepatic gluconeogenesis, pancreatic insulin secretion, and cardiomyocyte contraction [36,37▪,38], with implications for immunometabolic diseases such as diabetes and atherosclerosis [37▪]. FABP4 is thought to be secreted through endosomal enclosure and lysosomal exocytosis [38], and is transferred into the bloodstream through adipocyte-derived extracellular vesicles, a transport mode particularly active in obese individuals undergoing bariatric surgery, presumably due to increased lipolysis [39]. Its secretion may also involve sirtuin-1 regulation and early stages of autophagy [40▪]. Although the specificity of FABP4 secretion via endolysosomal, exososomal, and autophagy pathways is unclear, its role as a circulating modulator of downstream tissues is of great potential interest. Renal glomerular filtration can remove FABP4 from the circulation followed by megalin-mediated reabsorption in the proximal tubule epithelial cells [41], and it has thus been suggested as a new biomarker for renal injury [42] and diabetic nephropathy [43], with increased urine levels of FABP4 possibly revealing impaired renal glomerular filtration [42].

Increased circulating FABP4 in acute MI patients may result from adrenergic stimulation of lipolysis and FABP4 release from myocardial and epicardial adipose tissue [44], and epidemiological evidence suggests that baseline FABP4 levels are positively associated with 5-year risk of cardiovascular outcomes [45] and as an independent predictor of 3-year and annual risk of carotid intima–media thickness change [46]. The mechanisms underlying these potential associations are unknown. FABP4 expression is also reported induced in airway inflammation and appears to play a proinflammatory role in the development of allergic asthma by promoting eosinophil migration and adhesion [47].

Several adipokines may be involved in both pathogenesis and progression of cancer, mainly in obesity-associated tumors [48]. FABP4 is highly expressed in several cancer types, particularly in the adipose tissue surrounding the tumor, with a concomitant increase in serum [48]. Decreased Fabp4 expression and protein levels were proposed to be predictive of hepatocellular carcinoma (HCC) cell proliferation and invasiveness in vitro, as well as increased recurrence and poor overall survival in vivo [49]. In invasive bladder cancers, FABP4 deficiency was predictive of poor overall and recurrence-free survival, as well as tumor progression [50]. These findings are in contrast with evidence showing that FABP4 exhibits increased expression in animals and cell models of obesity-associated hepatic diseases including HCC, and that human HCC cells show increased proliferation and migration activities when challenged with exogenously administered FABP4 [51]. FABP4 is also proposed as a functional marker for breast cancer as well as a potential therapeutic target [52]. Thus, the role of FABP4 expression and its circulating levels in the pathogenesis, progression and invasiveness of cancers, and its utility as a diagnostic marker, warrant further investigation.

EPIDERMAL FATTY ACID-BINDING PROTEIN OR FATTY ACID-BINDING PROTEIN 5

Epidermal FABP (EFABP, FABP5) is abundantly expressed in skin epidermis but is also present in other cell types including adipocytes, macrophages, dendritic cells, and airway epithelia. In skin its expression is upregulated more than two-fold in fibrotic lesions induced by radiation exposure [53].

In adipocytes and macrophages, FABP5 expression is robustly associated with carcinogenesis and metastasis. Knocking down Fabp5 results in down-regulation of genes involved in both lipolysis and de novo FA synthesis in prostate and breast cancer cell lines, leading to impaired lipid metabolism and reduced cancer development and aggressiveness [54▪]. Under conditions of oxidative stress in such cancer cells, FABP5 appears to trigger the nuclear factor-kappa B-mediated production of cytokines and inflammatory biomarkers, exacerbating cancer progression and invasiveness [54▪]. In prostate cancer, Fabp5 deletion leads to downregulation of estrogen-related receptor α-dependent genes resulting in cell cycle interruption, apoptosis, and reduced cell proliferation and migration [55].

FABP 5 was reported to mediate the differentiation of proinflammatory macrophages by saturated FAs [56▪]. Further, FABP5 levels have recently been proposed as a novel diagnostic biomarker for Sjögren syndrome, an inflammatory autoimmune disease affecting the lacrimal and salivary glands, as these patients have significantly decreased FABP5 levels in their tears, associated with other features of dry eye and opthalmological damage [57]. In the lung, by contrast, FABP5 has been proposed as an anti-inflammatory protein, possibly functioning via activation of peroxisome proliferator-activated receptor gamma (PPARγ) [58].

BRAIN FATTY ACID-BINDING PROTEIN OR FATTY ACID-BINDING PROTEIN 7

Brain FABP (BFABP, FABP7) is abundantly expressed in astrocytes and radial glial cells, and is thought to be critical during the early stages of brain and central nervous system (CNS) development, with levels gradually decreasing postnatally [59]. The FABP7 ligand docosahexaenoic acid (DHA) induces astrocyte proliferation, which is arrested under conditions of FABP7 deficiency, highlighting the direct involvement of FABP7 in astrocyte proliferation [60].

Fabp7 ablation in mice led to decreased food intake and weight gain after challenge with high-fat feeding or a single leptin injection, implicating FABP7 in hypothalamic-regulated food intake and energy homeostasis via leptin sensing [61▪].

FABP7 has also been associated with several cancers, including glioma, breast cancer and renal cell carcinoma. A number of possible mechanisms have been suggested, including regulation of lipid rafts and the transcription of proliferation-associated genes, interaction with PPARγ and subsequent altered transcriptional programing, and altered FA uptake and lipid storage mediated by hypoxiainducible factor 1-α [62▪]. Fabp7 deficiency was reported to interrupt DNA replication and cell growth in a glioma cell line, possibly mediated by reduced extracellular-signal-regulated kinase pathway activity and increased p53 [63], whereas cell migration in glioma is aggravated by increases in the ratio of the FABP7 ligands arachidonic acid/DHA, possibly via alterations in protein kinase C activities which are implicated in cell proliferation, differentiation and migration [64]. Although Fabp7 deficiency has also been implicated in phenotypes associated with psychiatric disorders such as schizophrenia, this has recently been challenged [65].

MYELIN FATTY ACID-BINDING PROTEIN/FATTY ACID-BINDING PROTEIN 8 AND TESTIS FATTY ACID-BINDING PROTEIN/FATTY ACID-BINDING PROTEIN 9

Myelin FABP (MFABP, FABP8) is a constituent protein of myelin in the CNS and Scwann cells in the peripheral nervous system, and is thought to play a role in the regulation of myelin structural and functional integrity, contributing to the assembly of the multiple lipid bilayers in the myelin structure. Certain mutations in Fabp8 result in conformational changes that may lead to protein structural instability, resulting in defective myelin structure [66]. Such protein instability may compromise remyelination after peripheral neural injury, possibly due to shorter internodes and enlarged nodes after de/remyelination, and hence associated with decreased neural conduction velocity [67].

FABP9 is expressed in testis, salivary gland, and mammary gland. A recent study explores the potential of FABP9 as a prognostic marker for prostate cancer [68]: FABP9 was found upregulated in malignant prostate cell lines and human prostate tissues with carcinoma compared with cells or tissues in benign condition, whereas FABP9 knockdown blunted the invasion of a highly malignant prostate cell line [68].

CONCLUDING REMARKS

Recent research has continued to demonstrate the unique functions of different members of the FABP family. All the FABPs indeed bind long-chain FA, but their varying functional mechanisms appear related to the protein and membrane partners that they interact with, to the variety of hydrophobic ligands with which they interact, and to their potential regulatory roles in interorgan crosstalk. The mechanisms by which tissue-specific FABPs exert actions on distal organs remain an important area of investigation, with implications not only for understanding their cellular functions but also for their use as diagnostic markers.

KEY POINTS.

• Accumulating evidence indicates that FABPs not only regulate lipid metabolism in their tissues of expression, but may also exert regulatory effects in downstream tissues where they are not expressed.

• Some FABPs may be released into the circulation via atypical secretory mechanisms that remain to be fully elucidated.

• Circulating levels of several of the FABPs, related to their tissue-specific functions, may serve as biomarkers for metabolic and inflammatory diseases as well as specific cancers.