Recent studies have elucidated a profound role for FFA

Recent studies have elucidated a profound role for FFA4 in modulation of metabolism and energy utilization as well as endocrine and immune function, and as a consequence, have thrust FFA4 to the forefront of drug discovery efforts. For example, activation of FFA4 by fatty BLU 9931 or synthetic ligands has been shown to elicit incretin hormone release from the gastrointestinal tract, modulate anti-inflammatory effects in macrophages, enhance hepatic glucose uptake and decrease steatosis, and improve insulin resistance and sensitivity [6], [7], [8], [9], [10]. The relative importance of FFA4 in modulating metabolic homeostasis is highlighted in FFA4−/− animals, which develop obesity, glucose intolerance, hepatic steatosis, and demonstrate impaired glucose metabolism and heightened lipogenesis compared to control animals when fed high fat diets [11]. Moreover, FFA4 expression is elevated in obese humans compared to lean controls, and a non-synonymous mutation (R270H) of the human receptor inhibits its function, enhances inflammation and hepatic steatosis, and increases the risk of obesity in European populations, further underscoring the significance of the receptor in human metabolism and endocrine regulation [11]. The goal of this research update is to summarize the most recent and seminal advances in our understanding of the structure, function, and regulation of FFA4, and to highlight agents that are known to modulate its activity.
FFA4 gene, protein, and alternative splice variants The gene for the human FFA4 was initially cloned from a genomic DNA fragment [6] and was shown to contain 1134 nucleotides encoding for a 377 amino acid protein with structural topology predictive of GPCRs [3], [6], [12]. Interestingly, in humans, two gene products arise due to alternative splicing of exon 3, an effect that can form both the longer 377 amino acid protein, commonly referred to as FFA4-‘Long’ (FFA4-L), as well as a shorter transcript of 1086 nucleotides that encodes for a 361 amino acid protein, referred to as FFA4-‘Short’ (FFA4-S) [12], [13] (A). Notably, FFA4-L contains an additional 16 amino acid sequence within intracellular loop 3 (ICL3), a GPCR domain that is typically involved in protein interactions, downstream signaling, and desensitization. Cloning of the mouse and rat orthologs showed high sequence homology of 98% between the rodent amino acid sequences, and 85% and 86% between the rat and human and mouse and human FFA4-S proteins, respectively (B) [13]. An important species distinction has shown that FFA4-L exists only in humans as no evidence of the longer transcript has been demonstrated to date in rodents or non-human primates [13]. As will be discussed in detail below, the long isoform has signal transduction effects that are distinct from those of the short and its tissue distribution is sparse compared to FFA4-S.
FFA4 signal transduction
FFA4 expression and tissue-specific physiological functions Hirasawa and colleagues initially demonstrated dense expression of FFA4 by RT-PCR in human lung and gastrointestinal tract, as well as adrenal gland [6]. Since, FFA4 expression has been shown to be fairly ubiquitously distributed, with much of the literature focusing on expression of the receptor in tissues that function as dietary sensors, for example on the tongue or GI tract, or in tissues that modulate immune function or metabolic homeostasis (Table 1; Fig. 2).
FFA4 ligands
Future directions Over the last decade, free-fatty acid receptors have emerged as important contributors to metabolic, gastrointestinal, and immune-system homeostasis and function, and have also been shown to serve in many cases, as dietary sensors that modulate such functional responses. These receptors have served to demonstrate that many of the effects of fatty acids that have long-been known to be beneficial in man are actually receptor-mediated. In this context, the nutriceutical value of FFA4 agonism by endogenous PUFA cannot be overstated, especially given the massive human public consumption of PUFA supplements, including fish oil and flax seed oil, which are enriched in the n-3 PUFA DHA/EPA and ALA, respectively. However, not all PUFA supplements are made equally and many are actually enriched with contaminating n-6 fatty acids, which can dysregulate the metabolic and in vivo beneficial effects of both n-3 and n-6 PUFA. The consequence of dietary n-3:n-6:n-9 ratios in man is well established, but the effect of dietary intake of these various fatty acids on activation and efficacy of FFA4 remains unstudied, as are the outcomes of chronic nutrient-based agonism of FFA4 on receptor desensitization, internalization, and cell-surface expression in vivo. Given that FFA4 is degraded from the cell surface fairly rapidly following its agonism in cells [15], it will be of interest to gauge what effect this effect may have on dietary supplementation of PUFA in vivo. This is an effect that should also be of concern for drug discovery efforts, as receptor desensitization following agonism with small molecules can circumvent effectiveness; yet, this lies in stark contrast to many published studies described here which show that long term administration of dietary PUFA actually increases FFA4 expression. Mechanisms of such regulation will need to be further characterized for better understanding on de novo synthesis, degradation, or recycling of FFA4 protein in a given tissue.