Signaling through Free Fatty Acid Receptor 4 Attenuates Cardiometabolic Disease

Abstract

A surge in the prevalence of obesity and metabolic syndrome, which promote systemic inflammation, underlies an increase in cardiometabolic disease. Free fatty acid receptor 4 is a nutrient sensor for long-chain fatty acids, like ω3-polyunsaturated fatty acids (ω3-PUFAs), that attenuates metabolic disease and resolves inflammation. Clinical trials indicate ω3-PUFAs are cardioprotective, and this review discusses the mechanistic links between ω3-PUFAs, free fatty acid receptor 4, and attenuation of cardiometabolic disease.

Introduction

With the increasing prevalence of metabolic syndrome, driven primarily by obesity and type 2 diabetes, afflicting 34.3% in the United States (1), attendant cardiometabolic diseases such as heart failure with preserved ejection fraction (HFpEF), coronary heart disease (CHD), myocardial infarction, and stroke are on the rise (1). Furthermore, obesity and metabolic syndrome provoke systemic inflammation that may drive worsening cardiometabolic disease (2, 3). On the other hand, ω3-polyunsaturated fatty acid (ω3-PUFA) supplementation might attenuate metabolic syndrome (4–6), and recent success with icosapent ethyl [a nonoxidized preparation of the ω3-PUFA eicosapentaenoic acid (EPA)] to improve cardiovascular outcomes (7, 8) has renewed interest into the mechanistic underpinnings of ω3-PUFA-mediated cardioprotection. Free fatty acid receptor 4 (Ffar4) is a nutrient sensor for endogenous long-chain fatty acids, including but not limited to ω3-PUFAs, that attenuates metabolic disease and resolves inflammation (9, 10). Interestingly, we also recently demonstrated that Ffar4 is cardioprotective (11). Collectively, these findings suggest that Ffar4 could be the mechanistic basis for ω3-mediated cardioprotection. However, as we will discuss in this review, it is not that simple. Several complex questions regarding Ffar4 physiologic function in the heart, as well as ligand specificity and activation of downstream signaling pathways in the context of cardiometabolic disease, remain unanswered, including the following:

Q1) What is the physiologic function of Ffar4 in the context of cardiometabolic disease?

Q2) Ffar4 is not just a receptor for ω3-PUFAs but binds to most, if not all, long-chain saturated, monounsaturated, and polyunsaturated fatty acids (SFAs, MUFAs, and PUFAs). Previous studies in vitro indicate that SFAs, MUFAs, and PUFAs have similar potency at Ffar4, but SFAs have lower efficacy (12–15). However, the physiologic significance of this difference in FA efficacy at Ffar4 in relation to dietary FA composition is unclear.

Q3) PUFAs, including ω3-PUFAs and ω6-PUFAs, have relatively similar potency and efficacy at Ffar4, and neither function as a biased ligand (12). If this is correct, how does Ffar4 distinguish between ω3- and ω6-PUFA-mediated signaling and can ω3-mediated cardioprotection be explained by signaling through Ffar4?

Free Fatty Acid Receptors

The idea that fatty acids are signaling molecules, more than just an energy source or components of phospholipid bilayers, is a relatively new concept that originated with the finding that endogenous FAs bind to and activate a family of G protein-coupled receptors (GPCRs), the free fatty acid receptors (Ffar). There are currently four recognized free fatty acid receptors, Ffar1–4. Ffar2 and Ffar3, originally identified as GPR43 and GPR41, respectively, are receptors for endogenous short chain fatty acids containing 1–6 carbons (C1–C6) (16–18), have been reviewed elsewhere (9, 10), and will not be considered further here. Ffar1 and Ffar4, originally identified as GPR40 and GPR120, respectively, are receptors for long-chain fatty acids containing ≥10 carbons (C10–C24) (13, 19–21). Ffar1 is expressed in several cell types/tissues as reviewed elsewhere (9, 10). Interestingly, Ffar1 is expressed in the human heart (11, 19) and is upregulated in heart failure (HF) (11) but is not detected in rodent hearts (20, 22). Understanding the role of Ffar1 in the human heart will be critical for understanding the role of FA signaling in the context of cardiovascular disease. However, there are currently no reports on the function of Ffar1 in the heart, and therefore, Ffar1 will not be considered further here.

Ffar4 is expressed in several cells/tissues including intestinal I, K, and L enteroendocrine cells, pancreas, brain, lung, immune cells, and taste buds (9, 10). More importantly, Ffar4 is expressed in tissues with direct relevance to cardiometabolic disease, including the rodent and human heart, with expression in both cardiac myocytes and fibroblasts (11, 22), macrophages (11, 14, 22, 23), and both white and brown adipocytes (24–26). Based on this expression pattern, this review will focus exclusively on Ffar4 in the context of cardiometabolic disease.

Ffar4 Physiology: Attenuation of Metabolic Disease and Inflammation

Ffar4 is a G_q-coupled receptor that activates both G_q- and βArrestin2 (βArr2)-mediated signaling pathways (13, 14, 27), although Ffar4 also activates G_i-mediated signaling pathways in pancreatic δ-cells (28). Interestingly, humans express a short and long isoform of Ffar4; Ffar4S, homologous to Ffar4 in other species, and Ffar4L, differentiated by a 16 amino acid insertion in the third intracellular loop (27). The Ffar4S and Ffar4L isoforms differentially activate G_q and βArr2, with the Ffar4S activating both, whereas the Ffar4L activates only βArr2 (27). However, Ffar4L expression is rather limited in humans, and the physiologic significance of the Ffar4L isoform is not entirely clear (29, 30).

Ffar4 regulates several metabolic and inflammatory physiological functions important in cardiometabolic disease. In the gut, Ffar4 regulates hormone secretion, increasing the secretion of glucagon-like peptide-1 (13, 31–33), cholecystokinin (34), and gastric inhibitory polypeptide (35) from enteroendocrine cells, while attenuating ghrelin secretion (36). In the pancreas, Ffar4 expression and function in specific islet cell types are still debated. Nonetheless, existing studies suggest that Ffar4 in α-cells regulates glucagon secretion (15), Ffar4 in β-cells regulates insulin secretion, possibly through regulation of PDX1 (37–40), Ffar4 in δ-cells regulates somatostatin secretion (28, 41), and Ffar4 in PP cells regulates pancreatic peptide secretion (42). In macrophages, Ffar4 signals through βArr2 to activate TAB1, inhibit TAK1, and prevent NF-κB signaling, ultimately inducing an M2-like phenotype to attenuate adipocyte inflammation and reduce insulin resistance (14, 23). Further, Ffar4-mediated activation of cytosolic phospholipase A2α (cPLA2α)-cyclooxygenase (COX2) signaling and production of oxylipins can attenuate proinflammatory signaling in macrophages (43–45). Ffar4 may also have a role in the taste sensation of fat (46). In adipose, Ffar4 promotes adipogenesis (24, 25, 47). In brown adipose tissue, Ffar4 induces browning, increases FA oxidation, and attenuates obesity in mice (48). In mouse models of obesity/type 2 diabetes, loss of Ffar4 worsens metabolic disease, with evidence of insulin resistance, glucose intolerance, adipocyte dysfunction, and fatty liver, but with little or no effect on weight gain (14, 25, 49, 50), although others have suggested that Ffar4 has no effect on weight gain or metabolic function (51). Conversely, activation of Ffar4 with synthetic ligands (TUG-891, compound A, or compound 34) improves metabolic dysfunction and insulin resistance in mice (23, 48, 52). In humans, Ffar4 is expressed in adipose tissue, and its expression is increased in obesity (25). Interestingly, in a European cohort, the Ffar4 R270H inactivating polymorphism is associated with morbid obesity (25), but in a different Danish cohort, there was no association between R270H and metabolic disease (51).

Q1: Ffar4 Attenuates Cardiovascular Disease

In the heart, we demonstrated that Ffar4 functions as a cardioprotective nutrient sensor (11, 53). When challenged with pathologic pressure overload induced by transverse aortic constriction or a diet regimen designed to induce cardiometabolic disease, ventricular remodeling was more severe in mice with systemic deletion of Ffar4 (Ffar4KO) (11, 53). Although we previously found that Ffar4 attenuated transforming growth factor β1-induced profibrotic signaling in vitro, we surprisingly did not observe more fibrosis in Ffar4KO mice in vivo (22), suggesting loss of Ffar4 in cardiac myocytes might explain the worsened outcomes. In the context of pressure overload, analysis of the transcriptome of cardiac myocytes isolated from Ffar4KO hearts identified transcriptional deficits in genes associated with cPLA2α signaling and oxylipin (oxidatively modified FAs) synthesis (11). This deficit in Ffar4-cPLA2α signaling led to specific reductions in EPA-derived oxylipins in both high-density lipoproteins (HDL) and the hearts from Ffar4KO mice, suggesting a more proinflammatory state in the heart (11, 53). Specifically, in the context of cardiometabolic disease in Ffar4KO mice, HDL and cardiac levels of the EPA-derived, proresolving oxylipin 18-hydroxyeicosapentaenoic acid (18-HEPE) were decreased (53). Simultaneously, levels of arachidonic acid (AA)-derived, proinflammatory oxylipin 12-hydroxyeicosatetraenoic acid (12-HETE), previously associated with worsened remodeling in cardiometabolic disease (54), were increased in HDL and hearts from Ffar4KO mice (53). This alteration in inflammatory oxylipin levels suggests that activation of Ffar4-cPLA2α signaling in cardiac myocytes could be an important and novel cardioprotective signaling pathway.

In atherosclerosis, leukocyte-specific deletion of Ffar4 (Leuk-Ffar4KO) had no effect on atherosclerosis progression in low-density lipoprotein (LDL) receptor KO mice, while dietary supplementation with ω3- or ω6-PUFAs had a similar ability to attenuate atherosclerosis progression in wild-type (WT) and Leuk-Ffar4KO mice (55). On the other hand, Ffar4 synthetic ligands (TUG-891 or GW9508, which is a mixed Ffar1/4 ligand) attenuated atherosclerosis progression by increasing M2-like, anti-inflammatory macrophages and reducing M1-like, proinflammatory macrophages (56, 57).

Ffar4-Cytosolic Phospholipase A2α Signaling in Cardiometabolic Disease

As noted, our recent studies suggest that Ffar4-mediated cardioprotection requires activation of cPLA2α and production of 18-HEPE (11). cPLA₂α, a member of the Group IV family of phospholipase A2 enzymes, cleaves PUFAs, traditionally thought to be AA, from the sn2-acyl bond in membrane phospholipids (58). cPLA2α is activated by increased calcium and phosphorylation at several sites, with phosphorylation at Ser505 by MAPK considered the most important (59). Once released from phospholipids, ω6-PUFAs like AA or linoleic acid (LA), but also ω3-PUFAs including EPA and docosahexaenoic acid (DHA), can be further metabolized intracellularly by lipoxygenases, COX, CYP_hydroxylases, and CYP_epoxygenases to produce oxylipins. Oxylipins, including, for example, leukotrienes and prostaglandins, mediate several pro- or anti-inflammatory responses or promote the resolution of inflammation (60). In macrophages, for instance, Ffar4-mediated activation of cPLA₂α-COX2 signaling and production of oxylipins can attenuate proinflammatory signaling (43–45).

In cardiac myocytes cultured from WT but not Ffar4KO mice, we found that the Ffar4 agonist TUG-891 directly and specifically increased the production of 18-HEPE (11). Furthermore, TUG-891 and 18-HEPE each attenuated cardiac myocyte death induced by oxidative stress in wild-type cardiac myocytes, and 18-HEPE rescued cell death in Ffar4KO cardiac myocytes (11). In support of our findings, increased 18-HEPE levels in macrophages from fat-1 transgenic mice are associated with attenuated ventricular remodeling following pressure overload, but more importantly, direct injection of 18-HEPE was also cardioprotective (61). In addition, EPA supplementation increased 18-HEPE levels and attenuated atherosclerosis in mice (62).

To date, no receptor has been identified for 18-HEPE that might explain these cardioprotective effects; however, 18-HEPE is the precursor for E-series resolvins (RvE), which signal through the GPR ChemR23 (63, 64). RvE1 is a member of a family of specialized proresolving mediators (SPMs), which are derived from ω3-PUFAs and as the name implies, mediate the resolution of inflammation. The larger SPM family includes E-series resolvins derived from EPA and D-series resolvins, maresins, and protectins derived from DHA (65). In the heart, RvE1 attenuates ischemic injury both ex vivo (66) and in vivo. (67). Collectively, these studies suggest that 18-HEPE has cardioprotective effects, but whether this is a direct effect of 18-HEPE or if these protective effects are mediated through RvE1 is not clear, nor has it been demonstrated that ChemR23 is expressed in cardiac myocytes.

Interestingly, ChemR23 is expressed in macrophages (68), smooth muscle cells (62), endothelial cells (69), and adipocytes (70), and based on direct effects of 18-HEPE to prevent cardiac myocytes cell death (11) and RvE1 infusion to attenuate postinfarction remodeling (71), we speculate that ChemR23 might also be expressed in cardiac myocytes. ChemR23 is a dual ligand receptor, and in addition to binding E-series resolvins (63, 64), ChemR23 binds chemerin, a macrophage chemoattractant (72). In macrophages, ChemR23 expression is restricted to naïve and M1-like macrophages, which respond to chemerin produced in inflamed tissue to recruit macrophages, while RvE1 promotes repolarization of M1-like macrophages toward a more resolving M2-like phenotype (73). Further, the balance between the two ChemR23 ligands, chemerin and RvE1, might dictate functional outcomes (74).

Our recent results also indicate that Ffar4 is required for an adaptive response to cardiometabolic disease, based on our recent results using a model of HFpEF secondary to metabolic syndrome (53). Generally, patients with HFpEF are older, female, and often have comorbidities associated with metabolic syndrome, including hypertension, obesity, and type II diabetes (75–77). Clinically, HFpEF patients have been separated phenotypically, and in a predominant subset of patients HFpEF is associated with metabolic syndrome (78). Mechanistically, it has been suggested that systemic, nonresolving inflammation associated with metabolic syndrome may promote HFpEF (75–77). In that regard, we recently reported that in response to a diet designed to induce cardiometabolic disease in mice (high-fat/high-carbohydrate diet with N^ω-nitro-l-arginine methyl ester in the drinking water), total CD64⁺ macrophages were increased in ventricles from wild-type mice. More importantly, in Ffar4KO mice, in which the 12-HETE/18-HEPE ratio was dramatically increased in response to this cardiometabolic disease diet, we found a further increase in CD64⁺ macrophages, suggesting a worsened inflammatory response in the Ffar4KO heart (53). Of note, the increase in CD64⁺ macrophages in the hearts of WT mice subjected to the cardiometabolic disease diet agrees with the hypothesis that HFpEF remodeling is driven by systemic inflammation secondary to comorbidities associated with HFpEF (75–77). Regarding Ffar4, the greater increase in CD64⁺ macrophages in the hearts of Ffar4KO mice in this context suggests that Ffar4-mediated cardioprotective effects are potentially mediated through the production of SPMs that regulate macrophage function and attenuate inflammation in the heart in the context of cardiometabolic disease.

Q2: Ffar4 Signaling Induced by Dietary Long-Chain Fatty Acids

Ffar4 binds to and is activated by long-chain SFAs, MUFAs, and PUFAs. To date, several studies have indicated that all FAs activate Ffar4 with similar potencies, in the low-micromolar range (1–30 µM), while SFAs function as partial agonists relative to PUFAs (12–15). In a comprehensive comparison of 6 SFAs, 10 MUFAs, and 20 PUFAs (12), using an in vitro assay to assess the ability of individual FAs to activate Ffar4-βArr2 signaling, all FAs showed roughly similar potency [e.g., SFA: palmitic acid (PA) EC₅₀: 5.01 µM; ω6-PUFA: LA EC₅₀: 4.57 µM, AA EC₅₀: 12.02 µM; and ω3-PUFA: EPA EC₅₀: 4.57 µM, DHA EC₅₀: 4.27 µM]. However, SFAs generally showed lower efficacy to activate Ffar4-βArr2 signaling (e.g., SFA: PA E_max: 33%; ω6-PUFAs: LA E_max: 105%, AA E_max: 83%; and ω3-PUFAs: EPA E_max: 101%, DHA E_max: 87%). Furthermore, in this study, there was no evidence of biased agonism for a limited number of FAs that were analyzed for activation of G_q-mediated activation of Ca²⁺ signaling (12). In summary, multiple studies clearly indicate that SFAs are partial agonists (12–15), but the in vivo physiologic significance, where SFAs, MUFAs, and PUFAs can potentially all compete to bind Ffar4, remains to be determined.

In humans, dietary SFAs include capric acid (10:0), lauric acid (12:0), myristic acid (14:0), PA (16:0), and stearic acid (SA; 18:0). Furthermore, nutritional status likely induces oscillations in the Ffar4 ligand pool. One potential source of ligands is circulating nonesterified fatty acids (NEFAs), which are prevalent in the fasting state. In this pool, SFAs like PA and SA circulate at ∼100 µM (20-fold higher than their respective EC₅₀), while LA, a PUFA, circulates at 76 µM (16-fold higher than its EC₅₀) (79). However, other PUFAs circulate at concentrations less than the limit of quantitation, which is approximately <1 µM for most PUFAs (79). Interestingly, feeding induces strong suppression of circulating NEFAs (79, 80). For example, PA is suppressed by 85%, potentially shifting the Ffar4 ligand pool away from SFAs. PUFAs are likely to be provided from another pool, and the pool with the largest PUFA content is HDL (81). Endothelial lipase delivers localized, HDL-derived NEFAs (82); however, no studies have explored the role lipoproteins play in Ffar4 activation.

Postprandial, chylomicrons are prevalent and deliver FAs to tissues expressing lipoprotein lipase (83), in which case the potential Ffar4 ligand pool would likely reflect recent meal content (84). In metabolic diseases like obesity, de novo lipogenesis is increased, increasing PA levels and exacerbating obesity (85). However, the fasting NEFA content is only minimally altered, with small increases in PA and small decreases in SA (79), but the relative abundances of SFAs to LA are altered throughout the insulin-dependent response.

In mice, the predominant SFAs are PA and SA, and in the mouse heart, PA and SA together account for roughly 46% of the total FA content in cardiac myocytes (PA: ∼21%; SA: ∼25%), while ω6-PUFAs account for roughly 30% (AA: ∼10%; LA: ∼14%; others: ∼6%) and ω3-PUFAs account for roughly 11% (EPA: ∼0.7%; DHA: ∼9%) (22).

Therefore, in obesity and metabolic disease, which are characterized by increased dietary intake of SFAs, particularly PA, which functions as a partial agonist, higher levels of SFAs could hypothetically attenuate the protective Ffar4 signaling in cardiometabolic disease. In this context though, it is important to consider that the delivery Ffar4 ligand pool is likely to oscillate diurnally based on nutritional status and dietary pattern.

ω3-PUFAs and Cardiovascular Disease

From the original identification of Ffar4 as a receptor of long-chain FAs, there has been interest in the idea that Ffar4 is the mechanistic basis for the protective effects of ω3-PUFAs. Furthermore, recent clinical trials have strengthened the argument that ω3-PUFAs are cardioprotective.

Coronary Heart Disease

Several clinical trials indicate that ω3-PUFAs, EPA, and DHA improve cardiovascular outcomes and reduce the risk of CHD and sudden death (7, 86–91). Recent success in trials with high-dose EPA (4 g/day icosapent ethyl) has further strengthened the assertion that ω3-PUFAs, and specifically EPA, are cardioprotective. The Reduction of Cardiovascular Events with Icosapent Ethyl-Intervention Trial (REDUCE-IT) demonstrated that high-dose EPA (4 g/day icosapent ethyl) significantly improved cardiovascular outcomes in patients with high triglycerides despite statin use (7). Following REDUCE-IT, the Effect of Vascepa in Improving Coronary Atherosclerosis in People with High Triglycerides Taking Statin Therapy Trial (EVAPORATE) found that EPA (4 g/day icosapent ethyl) reduced coronary plaque volume in patients with atherosclerosis and high triglycerides despite statin use (8). These beneficial effects of w3-PUFAs in CHD are bolstered by several recent meta-analyses (92–94). Furthermore, there are multiple studies in mice that demonstrate ω3-PUFAs attenuate atherosclerosis, generally through attenuation of inflammation (55, 62, 95–107).

However, not all trials have shown a beneficial effect of ω3-PUFAs (108–111). More recently, the Vitamin D and Omega-3 Trial (VITAL) found no improvement in primary cardiovascular outcomes with low-dose ω3-therapy (1 g/day Macro/Lovaza, EPA, and DHA ethyl esters) (112), while the Statin Residual Risk Reduction with Epanova in High CV Risk Patients with Hypertriglyceridemia (STRENGTH) trial reported similarly negative results with a high-dose of ω3-PUFAs (4 g/day Epanova, EPA, and DHA carboxylic acids) (113). In VITAL, the low dose of ω3-PUFAs proved ineffective, but an inverse correlation between ω3-PUFA dose and efficacy might explain negative results with low-dose ω3-PUFA interventions (114). Interestingly, a recent analysis comparing REDUCE-IT and STRENGTH concluded that the lack of a positive outcome in STRENGTH might be partially explained through the control oils (mineral vs. corn) but that there is an additional benefit in REDUCE-IT that is likely through a specific effect of EPA (115). In summary, the balance of evidence from clinical trials and animal studies indicates that ω3-PUFAs reduce the risk of CHD and sudden death.

Heart Failure

To date, only one clinical trial, Gruppo Italiano Per Lo Studio Della Sopravvivenze Nell’Insufficienza Cardiaca-Heart Failure (GISSI-HF), has examined ω3-PUFAs in HF. Specifically, GISSI-HF reported that low-dose ω3-PUFAs (1 g/day, Omacar/Lovaza) reduced total mortality by 9%, and cardiovascular mortality and hospitalizations by 8% (116). Subsequently, several smaller clinical trials have reported beneficial effects of ω3-PUFAs in HF, demonstrating improved left ventricular ejection fraction (117–120) and reduced markers of inflammation (119, 120). Interestingly, the Effect of Omega-3 Acid Ethyl Esters on Left Ventricular Remodeling After Acute Myocardial Infarction (OMEGA-REMODEL) trial examined the effects of high-dose ω3-PUFAs (4 g/day Omacar/Lovaza) on postinfarct remodeling when patients are at risk of developing HF. OMEGA-REMODEL found that ω3-PUFAs improved left ventricular function, reduced fibrosis in the sparred myocardium, and reduced markers of inflammation and that patients with the highest quartile of ω3-index had the best outcomes (121). However, the Omega-3 Fatty Acids in Elderly with Myocardial Infarction Trial, which was similar to OMEGA-REMODEL, failed to show a benefit, possibly due to the much older patient population (122). In addition, several studies in multiple species, including humans, have demonstrated that ω3-PUFAs attenuate HF and reduce cardiac fibrosis (61, 121, 123–130). Of note, in all the animal studies mentioned, as well as the aforementioned trials, which either did not specify or only enrolled patients with heart failure reduced ejection fraction (HFrEF), no animal studies or clinical trials have specifically examined ω3-PUFAs in HFpEF. However, we recently found that in the Multi-Ethnic Study on Atherosclerosis (MESA) cohort that high plasma EPA and DHA individually were associated with reduced risk of both HFrEF and HFpEF (131). In short, studies in humans and animal models indicate a likely benefit of ω3-PUFAs in HFrEF and a potential benefit in HFpEF.

Metabolic Syndrome

Currently, only one ancillary study from a major ω-PUFA clinical trial has examined outcomes associated with metabolic syndrome. The Japan EPA Lipid Intervention Study (JELIS) found that EPA (1.8 g/day, EPA ethyl ester) substantially reduced the risk for major coronary events by 53% (132). REDUCE-IT did not list metabolic syndrome but did report that in subgroup analysis, participants with high triglycerides/low HDL had lower risk for composite vascular infarct events (7), which is consistent with risk-lowering among metabolic syndrome subjects, particularly those with lipid-oriented pathology. The cause of hypertriglyceridemia in insulin resistance is primarily reduced very-low-density lipoprotein synthesis (133, 134), a result of increased FA flux from the adipocytes to the liver, which itself is proposed to be attenuated by Ffar4 activation in resident macrophages, suppression of cytokines, and consequent reduction in intracellular adipocyte lipolysis (i.e., hormone-sensitive lipase), increased extracellular lipolysis (i.e., lipoprotein lipase), or both (134). Patients with metabolic syndrome also have a poor capacity to retain lipids in response to a glucose challenge (79), and an improvement in FA sequestration by adipocytes would both reduce FA delivery to the liver as well as reduce Randle cycle inhibition of glucose utilization. The triglyceride-lowering effect of ω3-PUFAs when administered to patients with metabolic syndrome is inversely proportional to the increase in erythrocyte ω3-PUFAs (135). Finally, in the context of high-fat feeding, several animal studies show that ω3-PUFAs prevent weight gain (136–142), but more importantly, some studies indicate that ω3-PUFAs can reverse diet-induced obesity (143–146). Concurrent with beneficial effects on weight gain, ω3-PUFAs also improved insulin sensitivity in diet-induced obesity (136, 139, 141–143, 145, 146). However, improved insulin sensitivity in response to ω3-PUFAs is not commonly reported in humans, where the focus is on the glycemic response.

Q3: ω3-PUFA Supplementation and Activation of Ffar4 in Cardiometabolic Disease

Currently, the evidence suggests that both Ffar4 and ω3-PUFAs are cardioprotective, but the question remains as to whether Ffar4 is the mechanistic basis of ω3-cardioprotection. With the original identification of Ffar4 as a receptor for long-chain FAs, DHA, among other PUFAs, was identified as a full agonist (13). Since that time, several studies, starting with Ref. 14, have suggested that Ffar4 is required for the beneficial effects of ω3-PUFAs. Furthermore, the recent success of high-dose EPA (icosapent ethyl) to reduce cardiovascular risk in REDUCE-IT (7) and EVAPORATE (8) has raised questions related to the mechanism of ω3-PUFAs and particularly EPA-mediated cardioprotective effects. In fact, ω3-PUFAs have multiple mechanisms of action, including activation of Ffar1/4, activation of peroxisome proliferator-activated receptors, generation of oxylipins (18-HEPE for example), and direct effects on membrane structure, as we have previously reviewed (147). Therefore, it is entirely possible that ω3-PUFAs have a multitude of beneficial effects that have been and will continue to be difficult to define using traditional reductionist approaches and will likely require a systems-based approach that will consider multiple variables simultaneously.

As we have reviewed, supplementation with ω3-PUFAs is certainly beneficial for cardiovascular outcomes, possibly because ω3-PUFAs are much more efficacious activators of Ffar4 signaling than SFAs (12), However, ω6-PUFAs, which are typically more abundant but less well-understood than ω3-PUFAs in the heart (148), have similar potency and efficacy to ω3-PUFAs at Ffar4 (12). The most practical distinction between PUFA classes is that ω3-PUFAs are more easily modified by dietary intake relative to ω6-PUFAs. However, the similar potency and efficacy of ω3- and ω6-PUFAs implies that Ffar4 does not distinguish between ω3- and ω6-PUFAs based on ligand binding or activation of proximal signaling events. If so, how might Ffar4 mediate the cardioprotective effects of ω3-PUFAs? We hypothesize that activation of Ffar4-cPLA2α-mediated signaling and production of specific ω3-derived oxylipins and SPMs, such as 18-HEPE, and/or suppression of ω6-proinflammatory oxylipins, such as 12-HETE, might explain the cardioprotective effects of ω3-PUFAs. In support of this hypothetical paradigm, our recent results indicate that 1) in cardiac myocytes, Ffar4 specifically and uniquely induces production of the EPA-derived, proresolving, cardioprotective oxylipin 18-HEPE (11); 2) in experimental models of HF in Ffar4KO mice, 18-HEPE levels are decreased in HDL, suggesting systemic production of 18-HEPE was decreased and that Ffar4 induces production of 18-HEPE not just in the heart, but other cells/tissues as well (11, 53); 3) in a model of cardiometabolic disease in Ffar4KO mice, cardiac-specific 18-HEPE levels were decreased, while 12-HETE levels were increased, which correlated with increased CD64⁺ macrophages in the heart and worsened ventricular remodeling (53); and 4) dietary supplementation with ω3-PUFAs specifically increases ω3-PUFA levels at the expense of ω6-PUFAs in cardiac myocytes, and increasing ω3-PUFAs levels would in theory increase substrate availability for the production of ω3-derived oxylipins (22). Although intriguing, this model will need to be validated in vivo.

Ffar4 Synthetic Ligands in Cardiometabolic Disease

As already mentioned studies in mice indicate that highly potent, Ffar4 synthetic ligands attenuate metabolic disease and improve insulin resistance (23, 48, 52), as well as attenuate atherosclerosis progression (56, 57). These limited findings suggest the translational potential of Ffar4 synthetic ligands, which is a significant area of research (for review, see Ref. 10). However, based on our proposal that Ffar4 induces the production of ω3-PUFA-derived oxylipins, like 18-HEPE, it remains to be determined how tissue-specific ω3-PUFA status might affect Ffar4 ligand efficacy.

Conclusions

The goal of this review was to address the current unknowns regarding Ffar4, ω3-PUFAs, and their interaction in the context of cardiometabolic disease. Based on the studies reviewed, we propose the following hypotheses to explain the function of Ffar4 in cardiometabolic disease (FIGURE 1):

1. An increase in dietary low-efficacy SFAs, which is common in patients with cardiometabolic disease, might result in attenuated Ffar4 signaling, which could worsen cardiometabolic outcomes.

2. Ffar4 mediates a GPR-signaling amplification cascade by regulating the synthesis of GPR ligands that modulate cardiac myocyte and macrophage function. Therefore, ω3-PUFA supplementation might increase the availability of EPA and DHA as precursors for Ffar4-mediated production of cardioprotective SPMs, like 18-HEPE/RvE1.

FIGURE 1.

Free fatty acid receptor 4 (Ffar4) mediates a G-protein receptor (GPR)-signaling amplification cascade in cardiac myocytes and macrophages A: Ffar4 is expressed in cardiac myocytes and macrophages. ChemR23 (RvE1) and GPR31 (12-HETE) are expressed in macrophages, and expression is postulated in cardiac myocytes (gray text). Ffar4 ligands include high-efficacy polyunsaturated fatty acid (PUFAs; solid line, ω3-PUFAs: green; ω6-PUFAs: purple), and low-efficacy saturated fatty acid (SFAs; dashed line). In cardiac myocytes, Ffar4 induces synthesis of 18-HEPE and potentially RvE1 (gray text), which is cytoprotective. In cardiometabolic disease, Ffar4 increases cardiac 18-HEPE level and decreases 12-HETE levels, which we hypothesize is cardioprotective. In macrophages, Ffar4 and ChemR23 induce an M2-like proresolving phenotype, whereas GPR31 induces an M1-like proinflammatory phenotype. Finally, we postulate cellular cross talk between myocytes and macrophages based on cell-specific oxylipin production might be achieved through oxylipin export to HDL and subsequent release through binding to HDL receptor scavenger receptor B1 (SR-B1) and local release. SA, stearic acid; PA, palmitic acid. B: in cardiac myocytes, Ffar4 signals through either G_q- and β-Arr2 to activate cytosolic phospholipase A2α (cPLA2α). cPLA2α cleaves PUFAs from the Sn2 position in membrane phospholipids at the outer nuclear membrane [eicosapentaenoic acid (EPA): green; arachidonic acid (AA): purple; or other PUFAs: gray]. Released PUFAs are further oxidatively metabolized by lipoxygenases (LOX), cyclooxygenases (COX), and CYP_hydrolases/CYP_epoxygenases (CYP). EPA is metabolized by COX or CYP enzymes to 18-HEPE (red), whereas AA is metabolized by 12-lipoxygenase to make 12-HETE (blue), which again is inhibited by Ffar4 in the context of cardiometabolic disease. Oxylipins have 4 fates: 1) remain free in the cell (nonesterified); 2) be reesterified into the membrane (sequestration); 3) be exported as a free oxylipin, a low-frequency event; or 4) be exported into HDL.

In support of these hypotheses, our recent work using in vivo models of pressure overload HF and cardiometabolic disease has demonstrated that Ffar4 is cardioprotective (11, 53). Interesting, investigations into the pharmacological function of Ffar4 indicate that while SFAs, MUFAs, and PUFAs have similar potency to activate Ffar4, SFAs have lower efficacy (12–15). As we have argued, the Ffar4 ligand pool is likely to change based on nutritional status and meal content, implying that Ffa4-mediated signaling could oscillate. This also implies that increased consumption of SFAs might inhibit cardioprotective Ffar4 signaling. A reasonable caveat is that current knowledge on Ffar4 pharmacology is based on in vitro studies of single FA ligands, and validation of these findings in vivo, albeit difficult, is required. However, the success of synthetic Ffar4 ligands to attenuate metabolic disease in animal models (23, 48, 52) does hold promise for targeting Ffar4 in cardiometabolic disease in humans. Based on the success of recent clinical trials, there is also renewed interest in ω3-PUFA-mediated cardioprotective effects (7, 8). Mechanistically, Ffar4 might mediate the beneficial effects of ω3-PUFAs, but ω3- and ω6-PUFAs have similar potency and efficacy, suggesting that discrimination between ω3- and ω6-PUFAs is not at the level of the receptor itself. Regarding the mechanistic basis for the cardioprotective effects of Ffar4, we found that in cardiac myocytes, Ffar4-cPLA2α signaling induces the production of the cytoprotective oxylipin 18-HEPE, and we affirmed the importance of Ffar4-cPLA2α-18-HEPE signaling axis in vivo. Coming full circle, we have hypothesized that Ffar4 mediates ω3-cardioprotection by activation of cPLA2α signaling and production of ω3-derived oxylipins and SPMs, which will require additional validation. In total, the evidence presented here indicates that Ffar4 attenuates cardiometabolic disease, but much work remains to define the mechanisms behind these protective effects and how ω3-PUFAs factor into this paradigm.