The role of fatty acid binding proteins in metabolic syndrome and atherosclerosis

Abstract

Purpose of review

The global prevalence of obesity is increasing epidemically. Obesity causes an array of health problems, reduces life expectancy, and costs over US$100 billion annually. More than a quarter of the population suffers from an aggregation of co-morbidities, including obesity, atherosclerosis, insulin resistance, dyslipidemias, coagulopathies, hypertension, and a pro-inflammatory state known as the metabolic syndrome. Patients with metabolic syndrome have high risk of atherosclerosis as well as type 2 diabetes and other health problems. Like obesity, atherosclerosis has very limited therapeutic options.

Recent findings

Fatty acid binding proteins integrate metabolic and immune responses and link the inflammatory and lipid-mediated pathways that are critical in the metabolic syndrome. This review will highlight recent studies on fatty acid binding protein-deficient models and several fatty acid binding protein-mediated pathways specifically modified in macrophages, cells that are paramount to the initiation and persistence of cardiovascular lesions.

Summary

Adipocyte/macrophage fatty acid binding proteins, aP2 and mal1, act at the interface of metabolic and inflammatory pathways. These fatty acid binding proteins are involved in the formation of atherosclerosis predominantly through the direct modification of macrophage cholesterol trafficking and inflammatory responses. In addition to atherosclerosis, these fatty acid binding proteins also exert a dramatic impact on obesity, insulin resistance, type 2 diabetes and fatty liver disease. The creation of pharmacological agents to modify fatty acid binding protein function will provide tissue or cell-type-specific control of these lipid signaling pathways, inflammatory responses, atherosclerosis, and the other components of the metabolic syndrome, therefore offering a new class of multi-indication therapeutic agents.

Introduction

Atherosclerosis is the leading cause of death in the United States [1–4]. At the core of this syndrome is the dysregulation of lipid metabolism and aberrant inflammatory responses [5]. Although mechanistic roles for fatty acids have been put forward in the formation of obesity and diabetes by modifying glucose and lipid metabolism as well as inflammatory cascades, little is known about the mechanisms that link fatty acids or other lipid signals to inflammatory responses and the formation of atherosclerotic lesions [6–8]. This review will focus on the biology of fatty acid binding proteins (FABPs) in several mouse models with targeted mutations in adipocyte/macrophage isoforms of these proteins. Although serum fatty acid levels are not reduced in these FABP-deficient models, they are strikingly and paradoxically protected from obesity, insulin resistance, type 2 diabetes, fatty liver disease and atherosclerosis [9–12,13••,14,15••,16–18]. This phenotype emphasizes the fact that total fatty acids may not be the primary pathogenic indicator, and that individual fatty acid or metabolite action at the intracellular level and the specific responses evoked by these signals are more relevant to the pathophysiology and outcomes of atherosclerotic disease than parameters classically measured.

Fatty acids and eicosanoids in signaling

Fatty acids and cholesterol are involved in the basic maintenance of the cell structure and energy metabolism, but are also vital in cell signaling [19,20]. Many transcription factors are regulated by lipids, including the families of peroxisome proliferator-activated receptors (PPARs) and liver X receptors (LXRs), which play central roles in lipid metabolism, cell differentiation, and the inflammatory response [21]. In addition, fatty acids can transmit a stress response through the activation of multiple kinases such as the inhibitor of kappa kinase (IKK) and c-jun NH2-terminal kinase (JNK), which have been linked to insulin resistance and other aspects of the metabolic syndrome including atherosclerosis [22]. Furthermore, fatty acids can be metabolized into a diverse family of more than 100 bioactive lipid mediators called eicosanoids, which may function as pro and anti-inflammatory mediators [19,23]. In particular, the cyclopentenone prostaglandins (PGA₁, PGA₂, and PGJ₂) have potent anti-inflammatory effects through the inhibition of inflammatory kinase pathways, and are beneficial in rodent models of inflammation [24]. 15-Deoxy-Δ^12,14-prostaglandin J₂ (15d-PGJ₂) is a controversial lipid mediator, which has been shown to be anti-inflammatory, but some suggest that it is not produced in substantial quantities to be truly effective [24]. However, 15d-PGJ₂ should not be disregarded as it was recently detected in human atherosclerotic lesions and is produced in and secreted from macrophages [25]. The role of fatty acids and eicosanoids in inflammation and atherosclerosis deserves special consideration in light of the clinical profile of cyclo-oxygenase 2-specific inhibitors and the increased incidence of heart disease [26].

Transcriptional pathways and cholesterol trafficking in the macrophage

Several key steps of cholesterol uptake and efflux in the macrophage are regulated by the nuclear hormone receptors PPAR-γ and LXR-α [21]. When modified LDL is taken up and degraded, fatty acids and eicosanoid ligands for PPAR-γ are generated, driving PPAR-γ transcription, which plays a role in macrophage biology as it relates to atherosclerosis by regulating cholesterol trafficking and the inflammatory response [21,27,28,29•]. PPAR-γ-driven scavenger receptor CD36 upregulation results in enhanced lipoprotein entry, which triggers a protective mechanism via the PPAR-γ–LXR-α–ATP binding cassette (ABC) A1 cholesterol efflux pathway [27,30,31]. Briefly, the transcription factor LXR-α is positively regulated upon the activation of PPAR-γ and by oxysterol ligands [21]. LXR-α activation leads to an increase in the cholesterol transporters ABCA1 and apolipoprotein E, which drive free cholesterol efflux from macrophages to acceptors such as apolipoprotein A1 or HDL [30–32]. The activation of PPAR-γ and LXR-α, or the overexpression of ABCA1 and apolipoprotein E result in decreased atherosclerosis, whereas blocking or deleting these genes resulted in greater atherosclerosis in mice [21,33]. Early clinical results also suggest a protective effect of PPAR-γ ligands in humans [34]. In addition, PPAR-γ and LXR-α have been linked to anti-inflammatory action [21,35–38]. However, the impact of the PPAR-γ pathway on inflammatory responses has not yet been fully established.

A very poorly understood part of lipid-activated transcription factors has been the upstream mechanisms controlling their access to ligands. Recent discoveries have indicated a critical role for FABPs in this important regulation [13••] (also see below).

Lipids and pro-inflammatory kinase pathways in the macrophage

The IKK–nuclearfactor kappa B (NF-κB) and JNK-AP-1 pathways are two important intermediaries in the control of macrophage pro-inflammatory activity as well as insulin receptor signaling and insulin action [22,39,40]. For example, the IKK–NF-κB pathway has been shown to be highly sensitive to inactivation by certain lipids; α-lipoic acid, 15d-PGJ₂, PGA₁, and PGA₂ block NF-κB activity through inhibition of IKK and directly hinder transcription by blocking the ability of NF-κB to bind to DNA [24,41]. Many of the pro-inflammatory components downstream of NF-κB have been shown to be essential to the formation of the atherosclerotic lesion. For example, when monocyte chemoattractant protein 1 or its receptor are deleted from a mouse model prone to develop atherosclerotic lesions, the size of the lesions is reduced [42,43]. Similarly, JNK is regulated by lipids and plays a critical role in both type 2 diabetes and atherosclerosis [22]. The targeted disruption of JNK-1 provides strong protection against insulin resistance associated with obesity, whereas JNK-2 deficiency results in a significant reduction in vascular lesions associated with an apolipoprotein E-deficient model of atherosclerosis [44,45]. These show that manipulations in the pro-inflammatory processes controlled by these and other lipid-responsive pathways may lead to substantial benefits for the prevention of atherosclerosis. Interestingly, both of these pathways are also regulated by FABPs [13••].

Fatty acid binding proteins

The recent expansion of mechanistic insights into FABP action demonstrated that these lipid chaperones serve as upstream and critical modulators of many lipid-signaling cascades. FABPs are abundant cytoplasmic proteins that reversibly bind hydrophobic ligands such as saturated and unsaturated long chain fatty acids, plus eicosanoids such as hydroxyeicosatetraenoic acid, leukotrienes and prostaglandins [46,47]. FABPs may actively facilitate the transport of lipids to specific compartments in the cell: such as to the mitochondria for oxidation; to the lipid droplet for storage; to enzymes for mediating activity; to the nucleus for lipid-mediated transcriptional regulation; or outside the cell to signal in an autocrine or paracine fashion. Overexpression and anti-sense studies in cultured cells have suggested potential roles in fatty acid import, storage, and export, as well as cholesterol and phospholipid metabolism [47]. Furthermore, FABPs are involved in the conversion of fatty acids to eicosanoid intermediates and in the stabilization of leukotrienes [48–50,51•]. In addition, aP2 has been shown to modify hormone-sensitive lipase activity through direct protein–protein interaction [52]. Finally, movement of FABPs into the nucleus and interaction with nuclear hormone receptors is possible, and this mechanism might potentially deliver ligands to this protein family [53–55]. Overall, FABPs act to sequester or distribute ligands to regulate signaling processes and enzymatic activity. Clear evidence on the specific impact of FABPs on cell biology and lipid metabolism in complex systems had been lacking until FABP-deficient mice models were created.

aP2^−/− model and atherosclerosis

Adipocyte/macrophage FABP, also designated aP2 or FABP4, is an important contributor to the maintenance of systemic glucose metabolism and adipocyte biology [14,17,18]. As a result of alterations in insulin sensitivity and serum lipids in aP2-deficient mice, we investigated the role of aP2 in atherosclerosis in the apolipoprotein E^−/− model with the goal of exploring the link between insulin resistance and atherosclerosis in an experimental model. Strikingly, mice deficient in aP2 exhibited as much as 88% reduction in vascular lesions compared with aP2^+/+ controls on the apolipoprotein E^−/− background, independent of any effects on insulin signaling or serum lipids [12]. The extent of protection from atherosclerosis by aP2 deficiency exceeds most, if not all, reported models of anti-atherogenic activity. The aP2 deficiency-mediated protection from atherogenesis in the apolipoprotein E^−/− background persists even when these animals are kept on a hypercholesterolemic Western diet [9]. It thus appeared that additional, and probably inflammatory, pathways are also targeted by FABPs in the regulation of atherosclerosis.

At this stage, we and others observed the presence of aP2 in macrophages, and have shown that expression can be induced in monocytes by phorbol myristate acetate-induced differentiation and activation, lipopolysaccharide/Toll receptor activation, PPAR-γ agonists, oxidized LDL, and rapamycin inhibitor treatments, and decreased by treatment with a cholesterol-lowering statin [12,56–59,60•]. Parallel patterns also exist in humans, with a similar relationship to atherosclerotic lesions (unpublished observations and Damcott et al. [61]). On the basis of these findings, we designed experiments to test the impact of macrophage aP2 deficiency in atherosclerosis directly. Bone marrow transplantation studies demonstrated that despite the adipocyte being the major site of aP2 expression, macrophage-specific action of aP2 is the predominant contributor to vascular lesion formation [12]. At the cellular level, aP2-deficient macrophages have increased free fatty acids and decreased cholesterol and cholesterol esters [13••]. In a gain of function model, the overexpression of aP2 in human macrophage cell lines drives the accumulation of cholesterol esters and foam cell formation [62]. We hypothesized that the modulation of intracellular fatty acids by aP2 leads to changes in lipid-mediated signaling pathways that modify cholesterol metabolism and inflammatory responses in the macrophage. Our data show that aP2 is a critical regulator of the PPAR-γ–LXR-α–ABCA1 pathway and contributes to foam cell formation [11,13••]. PPAR-γ activity is elevated in aP2^−/− macrophages with stimulation of downstream targets including LXR-α, ABCA1, and apolipoprotein E expression [13••]. This has dramatic consequences on cholesterol trafficking in macrophages, with the lack of aP2 resulting in enhanced efflux of cholesterol [11,13••]. In parallel, aP2 coordinates the inflammatory activity of macrophages [11,13••]. In aP2^−/− macrophages, several inflammatory signaling responses are suppressed, including cytokine and chemokine secretion, such as TNF-α and pro-inflammatory enzyme production and function including inducible nitric oxide synthase and cyclo-oxygenase 2 [13••]. We have also demonstrated that aP2 deficiency results in modified inflammatory responses and the inhibition of the IKK–NF-κB pathway, well upstream of transcriptional activity [11,13••]. Consequently, the overall reduction in foam cell formation and modified inflammatory responses of aP2^−/− macrophages is highly beneficial against the formation of atherosclerosis.

mal1^−/− model and atherosclerosis

Interestingly, another minor adipocyte FABP, mal1 (FABP5), is also present in macrophages. In the aP2^−/− model, mal1 expression is dramatically upregulated in the adipocytes; however, this level of compensatory regulation has not been observed in the macrophages [12,17]. In addition, although being the minor isoform in adipocytes, mal1 protein levels are comparable to aP2 in normal macrophages. To address the role of mal1 in the metabolic syndrome, we generated a mouse model with a targeted mutation in the mal1 gene [16]. Unlike aP2-deficient animals, mal1^−/− animals on a high fat diet or in the setting of genetic obesity exhibited only a slight protection from the development of insulin resistance [16]. As mal1 is not markedly regulated upon aP2 deficiency in the macrophages, and the atherosclerosis phenotype appears to be predominantly regulated by aP2 in this cell type, mal1 alone may not be a major player in atherosclerosis but may rather enhance the biology of aP2 (see below). In any case, accumulating evidence demonstrates that there must be as yet undiscovered functional differences between aP2 and mal1. Studies to address these questions and the specific role of mal1 in macrophages and atherosclerosis are currently underway.

aP2-mal1^−/− model and atherosclerosis

Our laboratory has recently generated mice with combined aP2-mal1 deficiency (aP2-mal1^−/−) to remove all FABP activity from adipocytes and macrophages, and address the issue of FABP function in these target cells without compensation as a confounding effect. The aP2-mal1^−/− mouse model has striking resistance to the formation of the metabolic syndrome, with improved parameters for multiple components including decreased hypertension, cholesterol, triglycerides, insulin, and glucose [15••]. On a high fat diet, aP2-mal1^−/− mice exhibit alterations in tissue fatty acid composition and do not develop insulin resistance, type 2 diabetes or fatty liver disease, demonstrating that the protective phenotype of this model far exceeds that of individual FABP mutants [15••]. Similarly, when intercrossed to the apolipoprotein E^−/− model, aP2-mal1^−/− mice develop dramatically less atherosclerosis compared with aP2 null and wild-type mice on the same background [10]. Remarkably, aP2-mal1^−/− animals also have significantly increased survival in the apolipoprotein E^−/− background even when fed a Western-type hypercholesterolemic diet for 12 months, probably because of the increased stability of plaques [10].

To understand how FABPs expressed in only a few tissue depots can have such wide-reaching systemic benefits, an in-depth fatty acid profiling of muscle, liver and adipose tissues was performed. This indicated that in the absence of both FABPs, there are increased short chain fatty acids in the muscle and adipose tissues of aP2-mal1^−/− mice, which favored enhanced insulin receptor signaling, AMP-activated kinase activity, fatty acid oxidation, and insulin-stimulated glucose uptake [15••]. There were also alterations in liver fatty acid composition, which differed from other sites and favored lipid mobilization over storage and suppressed stearoyl coenzyme A desaturase and sterol-regulatory element-binding protein activities, thus reducing hepatosteosis [15••]. This phenomenal mouse model has shed new light on the role of FABPs in regulating intracellular fatty acid profiles and how these alterations are linked to specific biochemical pathways critical in metabolic homeostasis.

Conclusion

FABP-mediated lipid metabolism is closely linked to both metabolic and inflammatory processes through modulating critical lipid-sensitive pathways in target cells; macrophages and adipocytes. The lack of aP2 alters the intracellular lipid milieu such that lipid-sensitive targets including nuclear hormone receptors and inflammatory kinases have broad ranging protective consequences on macrophage biology. The absence of aP2 and mal1 has even more remarkable protection from atherosclerosis and the metabolic syndrome through fatty acid-mediated alterations in kinase and transcriptional activity. The phenotypes observed in the absence of adipocyte/macrophage FABPs illustrate the integrating role played by these proteins in metabolic and inflammatory responses, and establish these genes as a strong example of the ‘thrifty’ gene family. Finally, these models illustrated further networks of bioactive lipid signals between adipocytes and macrophages, and muscle and liver tissues, the identification of which should be highly informative. Bone marrow transplantation and genetic loss of function studies are currently under way to determine the effect of mal1 and aP2-mal1 macrophage and adipocyte-specific deficiency on atherosclerosis to dissect their biology in detail. Further investigation of fatty acid profiles, eicosanoid synthesis, the partitioning of fatty acids into various cellular domains, and the identification of target genes should aid in clarifying the exact function of FABPs in macrophages and other cell types.

Our findings, as well as recent developments in the field, invite a deeper examination of the relevance of fatty acids and eicosanoids in metabolism and disease. There are hundreds of biologically active lipid derivatives that may act as signaling molecules. A fundamental shift in our approach to lipid signaling is necessary so that we no longer simply bulk all fatty acids into one parameter, but consider each one and its metabolites individually. This requires investigation by large scale, high throughput lipomic and metabolomic approaches, coupled with mechanistic and functional studies to address how FABPs expressed in limited tissues can have such wide-ranging effects on atherosclerosis and the metabolic syndrome. These questions notwithstanding, targeting adipocyte/macrophage FABPs, particularly aP2, by small molecule inhibitors offers highly attractive therapeutic opportunities for the management of atherosclerosis and other components of the metabolic syndrome.

Abbreviations

ABC

ATP binding cassette

15d-PGJ₂

15-deoxy-Δ^12,14-prostaglandin J₂

FABP

fatty acid binding protein

IKK

inhibitor of kappa kinase

LXR

liver X receptor

NF-κB

nuclear factor kappa B

PPAR

peroxisome proliferator-activated receptor