Free fatty acid receptor 3 is a key target of short chain fatty acid: What is the impact on the sympathetic nervous system?

Nervous system (NS) activity participates in metabolic homeostasis by detecting peripheral signal molecules derived from food intake and energy balance. High quality diets are thought to include fiber-rich foods like whole grain rice, breads, cereals, and grains. Several studies have associated high consumption of fiber-enriched diets with a reduced risk of diabetes, obesity, and gastrointestinal disorders. In the lower intestine, anaerobic fermentation of soluble fibers by microbiota produces short chain fatty acids (SCFAs), key energy molecules that have a recent identified leading role in the intestinal gluconeogenesis, promoting beneficial effects on glucose tolerance and insulin resistance¹. SCFAs are also signaling molecules that bind to specific G-protein coupled receptors (GPCRs) named Free Fatty Acid Receptor 3 (FFA3, GPR41) and 2 (FFA2, GPR43). However, how SCFAs impact NS activity through their GPCRs is poorly understood. Recently, studies have demonstrated the presence of FFA2 and FFA3 in the sympathetic NS of rat, mouse and human^{2, 3}. Two studies have showed that FFA3 activation by SCFAs increases firing and norepinephrine (NE) release from sympathetic neurons^{3, 4}. However, the recent study from the Ikeda Laboratory² revealed that activation of FFA3 by SCFAs impairs N-type calcium channel (NTCC) activity, which contradicts the idea of FFA3 activation leading to increased action potential evoked NE release. Here we will discuss the scope of the latter study and the putative physiological role of SCFAs and FFAs in the sympathetic NS.     

Selective orthosteric free fatty acid receptor 2 (FFA2) agonists: identification of the structural and chemical requirements for selective activation of FFA2 versus FFA3

Free fatty acid receptor 2 (FFA2; GPR43) is a G protein-coupled seven-transmembrane receptor for short-chain fatty acids (SCFAs) that is implicated in inflammatory and metabolic disorders. The SCFA propionate has close to optimal ligand efficiency for FFA2 and can hence be considered as highly potent given its size. Propionate, however, does not discriminate between FFA2 and the closely related receptor FFA3 (GPR41). To identify FFA2-selective ligands and understand the molecular basis for FFA2 selectivity, a targeted library of small carboxylic acids was examined using holistic, label-free dynamic mass redistribution technology for primary screening and the receptor-proximal G protein [(35)S]guanosine 5'-(3-O-thio)triphosphate activation, inositol phosphate, and cAMP accumulation assays for hit confirmation. Structure-activity relationship analysis allowed formulation of a general rule to predict selectivity for small carboxylic acids at the orthosteric binding site where ligands with substituted sp(3)-hybridized α-carbons preferentially activate FFA3, whereas ligands with sp(2)- or sp-hybridized α-carbons prefer FFA2. The orthosteric binding mode was verified by site-directed mutagenesis: replacement of orthosteric site arginine residues by alanine in FFA2 prevented ligand binding, and molecular modeling predicted the detailed mode of binding. Based on this, selective mutation of three residues to their non-conserved counterparts in FFA3 was sufficient to transfer FFA3 selectivity to FFA2. Thus, selective activation of FFA2 via the orthosteric site is achievable with rather small ligands, a finding with significant implications for the rational design of therapeutic compounds selectively targeting the SCFA receptors.     

Mutational analysis of G-protein coupled receptor – FFA2

FFA2 (GPR43) is a receptor for short-chain fatty acids (SCFAs), acetate, and propionate. FFA2 is predominantly expressed in islets, a subset of immune cells, adipocytes, and the gastrointestinal tract which suggest a possible role in inflammatory and metabolic conditions. We have previously described the identification and characterization of novel phenylacetamides as allosteric agonists of FFA2. In the current study, we have investigated the molecular determinants contributing to receptor activation with the endogenous and synthetic ligands as well as allosteric interactions between these two sites. The mutational analysis revealed previously unidentified sites that may allosterically regulate orthosteric ligand’s function as well as residues potentially important for the interactions between orthosteric and allosteric binding sites.     

Identification of diarylsulfonamides as agonists of the free fatty acid receptor 4 (FFA4/GPR120)

The exploration of a diarylsulfonamide series of free fatty acid receptor 4 (FFA4/GPR120) agonists is described. This work led to the identification of selective FFA4 agonist 8 (GSK137647A) and selective FFA4 antagonist 39. The in vitro profile of compounds 8 and 39 is presented herein.     

Discovery of novel free fatty acid receptor 1 agonists bearing triazole core via click chemistry

The free fatty acid receptor 1 (FFA1/GPR40) is a novel antidiabetic target based on particular mechanism in enhancing glucose-stimulated insulin secretion. Most of reported FFA1 agonists, however, have been suffered from relatively high lipophilicity and molecular weight. Aiming to develop potent agonists with improved physicochemical property, 25 compounds containing triazole scaffold and various carboxylic acid fragments were synthesized via the click chemistry. Among them, the optimal lead compound 26 with relatively low lipophicity (Log D_7.4 = 1.95) and molecular weight (Mw = 391.78) exhibited a considerable FFA1 agonistic activity (36.15%). In addition, compound 26 revealed a significant improvement in the glucose tolerance with a 21.4% and 14.2% reduction of glucose AUC_0–2h in normal ICR mice and type 2 diabetic C57BL/6 mice, respectively. All of these results demonstrated that compound 26 was considered to be a promising lead compound suitable for further optimization.     

Similarities and differences between the responses induced in human phagocytes through activation of the medium chain fatty acid receptor GPR84 and the short chain fatty acid receptor FFA2R

GPR84 is a recently de-orphanized member of the G-protein coupled receptor (GPCR) family recognizing medium chain fatty acids, and has been suggested to play important roles in inflammation. Due to the lack of potent and selective GPR84 ligands, the basic knowledge related to GPR84 functions is very limited. In this study, we have characterized the GPR84 activation profile and regulation mechanism in human phagocytes, using two recently developed small molecules that specifically target GPR84 agonistically (ZQ16) and antagonistically (GLPG1205), respectively. Compared to our earlier characterization of the short chain fatty acid receptor FFA2R which is functionally expressed in neutrophils but not in monocytes, GPR84 is expressed in both cell types and in monocyte-derived macrophages. In neutrophils, the GPR84 agonist had an activation profile very similar to that of FFA2R. The GPR84-mediated superoxide release was low in naïve cells, but the response could be significantly primed by TNFα and by the actin cytoskeleton disrupting agent Latrunculin A. Similar to that of FFA2R, a desensitization mechanism bypassing the actin cytoskeleton was utilized by GPR84. All ZQ16-mediated cellular responses were sensitive to GLPG1205, confirming the GPR84-dependency. Finally, our data of in vivo transmigrated tissue neutrophils indicate that both GPR84 and FFA2R are involved in neutrophil recruitment processes in vivo.In summary, we show functional similarities but also some important differences between GPR84 and FFA2R in human phagocytes, thus providing some mechanistic insights into GPR84 regulation in blood neutrophils and cells recruited to an aseptic inflammatory site in vivo.     

Role of free fatty acid receptors in the regulation of energy metabolism

Free fatty acids (FFAs) are energy-generating nutrients that act as signaling molecules in various cellular processes. Several orphan G protein-coupled receptors (GPCRs) that act as FFA receptors (FFARs) have been identified and play important physiological roles in various diseases. FFA ligands are obtained from food sources and metabolites produced during digestion and lipase degradation of triglyceride stores. FFARs can be grouped according to ligand profiles, depending on the length of carbon chains of the FFAs. Medium- and long-chain FFAs activate FFA1/GPR40 and FFA4/GPR120. Short-chain FFAs activate FFA2/GPR43 and FFA3/GPR41. However, only medium-chain FFAs, and not long-chain FFAs, activate GPR84 receptor. A number of pharmacological and physiological studies have shown that these receptors are expressed in various tissues and are primarily involved in energy metabolism. Because an impairment of these processes is a part of the pathology of obesity and type 2 diabetes, FFARs are considered as key therapeutic targets. Here, we reviewed recently published studies on the physiological functions of these receptors, primarily focusing on energy homeostasis.     

Perspectives on the therapeutic potential of short-chain fatty acid receptors

There is rapidly growing interest in the human microbiome because of its implication in metabolic disorders and inflammatory diseases. Consequently, understanding the biology of short chain fatty acids and their receptors has become very important for identifying novel therapeutic avenues. GPR41 and GPR43 have been recognized as the cognate receptors for SCFAs and their roles in metabolism and inflammation have drawn much attention in recent years. GPR43 is highly expressed on immune cells and has been suggested to play a role in inflammatory diseases such as inflammatory bowel disease. Both GPR41 and GPR43 have been implicated in diabetes and obesity via the regulation of adipose tissue and gastrointestinal hormones. So far, many studies have provided contradictory results, and therefore further research is required to validate these receptors as drug targets. We will also discuss the synthetic modulators of GPR41 and GPR43 that are critical to understanding the functions of these receptors. [BMB Reports 2014; 47(3): 173-178]     

Short-chain fatty acid mitigates adenine-induced chronic kidney disease via FFA2 and FFA3 pathways

Short-chain fatty acids (SCFAs), including acetate, butyrate, and propionate, are produced when colonic bacteria in the human gastrointestinal tract ferment undigested fibers. Free fatty acid receptor 2 (FFA2) and FFA3 are G-protein-coupled receptors recently identified as SCFA receptors that may modulate inflammation. We previously showed through in vitro experiments that SCFAs activate FFA2 and FFA3, thereby mitigating inflammation in human renal cortical epithelial cells. This study used a murine model of adenine-induced renal failure to investigate whether or not SCFAs can prevent the progression of renal damage. We also examined whether or not these FFA2 and FFA3 proteins have some roles in this protective mechanism in vivo. Immunohistochemical analyses of mouse kidneys showed that FFA2 and FFA3 proteins were expressed mainly in the distal renal tubules and collecting tubules. First, we observed that the administration of propionate mitigated the renal dysfunction and pathological deterioration caused by adenine. Consistent with this, the expression of inflammatory cytokines and fibrosis-related genes was reduced. Furthermore, the mitigation of adenine-induced renal damage by the administration of propionate was significantly attenuated in FFA2^−/− and FFA3^−/− mice. Therefore, the administration of propionate significantly protects against adenine-induced renal failure, at least in part, via the FFA2 and FFA3 pathways. Our data suggest that FFA2 and FFA3 are potential new therapeutic targets for preventing or delaying the progression of chronic kidney disease.     

Identification of a free fatty acid receptor,FFA2R,expressed on leukocytes and activated by short-chain fatty acids

Short-chain fatty acids (SCFAs) have long been known to exert cellular effects on blood leukocytes. Acetate, propionate, and butyrate represent the most capable SCFA, inducing calcium mobilization which subsequently regulates leukocyte function in the immune system. We have cloned the previously described putative orphan G-protein coupled receptor, GPR43, and have functionally identified SCFA as the activating ligands. Acetate and propionate were found to be the two most potent ligands, although butyrate, formate, and valerate (in this order of potency) also were able to induce receptor activation. Both the human and mouse receptor homologues were found to share the same pattern of ligand activation. This finding, together with a high degree of amino acid sequence similarity between the mouse and human homologues, indicates an evolutionary conserved function. Upon ligand stimulation, the receptor mobilized intracellular calcium in both a recombinant system as well as in human granulocytes. We found the human gene to be predominantly expressed in peripheral blood leukocytes and, to a lesser extent, in spleen. We suggest the designation FFA(2)R to this second receptor activated by free fatty acids. The first-described FFAR, now named FFA(1)R, is activated by medium- to long-chain free fatty acids.     

Design,synthesis and biological evaluation of indane derived GPR40 agoPAMs

GPR40 (FFAR1 or FFA1) is a G protein-coupled receptor, primarily expressed in pancreatic islet β-cells and intestinal enteroendocrine cells. When activated by fatty acids, GPR40 elicits increased insulin secretion from islet β-cells only in the presence of elevated glucose levels. Towards this end, studies were undertaken towards discovering a novel GPR40 Agonist whose mode of action is via Positive Allosteric Modulation of the GPR40 receptor (AgoPAM). Efforts were made to identify a suitable GPR40 AgoPAM tool molecule to investigate mechanism of action and de-risk liver toxicity of GPR40 AgoPAMs due to reactive acyl-glucuronide (AG) metabolites.     

Novel free fatty acid receptor 1 (GPR40) agonists based on 1,3,4-thiadiazole-2-carboxamide scaffold

Free fatty acid receptor 1 (FFA1), previously known as GPR40 is a G protein-coupled receptor and a new target for treatment of type 2 diabetes. Two series of FFA1 agonists utilizing a 1,3,4-thiadiazole-2-caboxamide scaffold were synthetized. Both series offered significant improvement of the potency compared to the previously described 1,3,4-thiadiazole-based FFA1 agonists and high selectivity for FFA1. Molecular docking predicts new aromatic interactions with the receptor that improve agonist potency. The most potent compounds from both series were profiled for in vitro ADME properties (plasma and metabolic stability, Log D, plasma protein binding, hERG binding and CYP inhibition). One series suffered very rapid degradation in plasma and in presence of mouse liver microsomes. However, the other series delivered a lead compound that displayed a reasonable ADME profile together with the improved FFA1 potency.     

Free fatty acids inhibit serum deprivation-induced apoptosis through GPR120 in a murine enteroendocrine cell line STC-1

Free fatty acids (FFAs) provide an important energy source and also act as signaling molecules. FFAs are known to exert a variety of physiological responses via their G protein-coupled receptors (GPCRs), such as the GPR40 family. Recently, we identified a novel FFA receptor, GPR120, that promotes secretion of glucagon-like peptide-1 (Hirasawa, A., Tsumaya, K., Awaji, T., Katsuma, S., Adachi, T., Yamada, M., Sugimoto, Y., Miyazaki, S., and Tsujimoto, G. (2005) Nat. Med. 11, 90-94). Here we showed that FFAs inhibit serum deprivation-induced apoptosis of murine enteroendocrine STC-1 cells, which express two types of GPCRs, GPR120 and GPR40, for unsaturated long chain FFA. We first found that linolenic acid potently activated ERK and Akt/protein kinase B (Akt) in STC-1 cells. ERK kinase inhibitors significantly reduced the anti-apoptotic effects of linolenic acid. Inhibitors for phosphatidylinositol 3-kinase (PI3K), a major target of which is Akt, significantly reduced the anti-apoptotic effects. Transfection of STC-1 cells with the dominant-negative form of Akt also inhibited the anti-apoptotic effect. These results suggested that the activation of ERK and PI3K-Akt pathways is required for FFA-induced anti-apoptotic effects on STC-1 cells. Transient transfection of STC-1 cells with GPR120 cDNA, but not GPR40 cDNA, enhanced inhibition of caspase-3 activation. RNA interference experiments showed that reduced expression of GPR120, but not GPR40, resulted in reduced ERK activation and reduced effects of FFAs on caspase-3 inhibition. Collectively, these results demonstrated that FFAs promote the activation of ERK and PI3K-Akt pathways mainly via GPR120, leading to the anti-apoptotic effect of STC-1 cells.     

Extracellular Ionic Locks Determine Variation in Constitutive Activity and Ligand Potency between Species Orthologs of the Free Fatty Acid Receptors FFA2 and FFA3

Free fatty acid receptors 2 and 3 (FFA2 and FFA3) are G protein-coupled receptors for short chain free fatty acids (SCFAs). They respond to the same set of endogenous ligands but with distinct rank-order of potency such that acetate (C2) has been described as FFA2-selective, whereas propionate (C3) is non-selective. Although C2 was confirmed to be selective for human FFA2 over FFA3, this ligand was not selective between the mouse orthologs. Moreover, although C3 was indeed not selective between the human orthologs, it displayed clear selectivity for mouse FFA3 over mouse FFA2. This altered selectivity to C2 and C3 resulted from broad differences in SCFAs potency at the mouse orthologs. In studies to define the molecular basis for these observations, marked variation in ligand-independent constitutive activity was identified using a [³⁵S]GTPγS assay. The orthologs with higher potency for the SCFAs, human FFA2 and mouse FFA3, displayed high constitutive activity in this assay, whereas the orthologs with lower potency for the agonist ligands, mouse FFA2 and human FFA3, did not. Sequence alignments of the second extracellular loop identified single negatively charged residues in FFA2 and FFA3 not conserved between species and predicted to form ionic lock interactions with arginine residues within the FFA2 or FFA3 agonist binding pocket to regulate constitutive activity and SCFA potency. Reciprocal mutation of these residues between species orthologs resulted in the induction (or repression) of constitutive activity and in most cases also yielded corresponding changes in SCFA potency.     

Allosteric rescuing of loss-of-function FFAR2 mutations

FFAR2 (GPR43) is a receptor for short-chain fatty acids (SCFAs), acetate and propionate. In the current study, we investigate the molecular determinants contributing to receptor activation by endogenous ligands. Mutational analysis revealed several important residues located in transmembrane domains (TM) 3, 4, 5, 6, and 7 for acetate binding. Interestingly, mutations that abolished acetate activity, including the mutation in the well-conserved D(E)RY motif, could be rescued by a recently identified synthetic allosteric agonist. These findings provide additional insight into agonist binding and activation which may aid in designing allosteric ligands for targeting receptor function in various diseases.     

Discovery of novel pyrrole-based scaffold as potent and orally bioavailable free fatty acid receptor 1 agonists for the treatment of type 2 diabetes

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The free fatty acid receptor 1 (FFA1) has gained significant interest as a novel antidiabetic target. Most of FFA1 agonists reported in the literature bearing a common biphenyl scaffold, which was crucial for toxicity verified by the researchers of Daiichi Sankyo. Herein, we describe the systematic exploration of non-biphenyl scaffold and further chemical modification of the optimal pyrrole scaffold. All of these efforts led to the identification of compound 11 as a potent and orally bioavailable FFA1 agonist without the risk of hypoglycemia. Further molecular modeling studies promoted the understanding of ligand-binding pocket and might help to design more promising FFA1 agonists.     

Short-chain fatty acid receptor GPR41-mediated activation of sympathetic neurons involves synapsin 2b phosphorylation

Synapsins are neuronal phosphoproteins that coat synaptic vesicles and are believed to function in the regulation of neurotransmitter release. The signaling mechanism for short-chain free fatty acid (SCFA)-stimulated NE release was examined using primary-cultured mouse sympathetic cervical ganglion neurons. Pharmacological and knockdown experiments showed that activation of sympathetic neurons by SCFA propionate involves SCFA receptor GPR41 linking to Gβγ-PLCβ3-ERK1/2-synapsin 2 signaling. Further, synapsin 2b directly interacts with activated ERK1/2 and can be phosphorylated on serine when SCFA activates sympathetic neurons.     

Structure-based design of free fatty acid receptor 1 agonists bearing non-biphenyl scaffold

The free fatty acid receptor 1 (FFA1) enhances the glucose-stimulated insulin secretion without the risk of hypoglycemia. However, most of FFA1 agonists have a common biphenyl moiety, leading to a relative deprivation in structure types. Herein, we describe the exploration of non-biphenyl scaffold based on the co-crystal structure of FFA1 to increase additional interactions with the lateral residues, which led to the identification of lead compounds 3 and 9. In induced-fit docking study, compound 3 forms an edge-on interaction with Trp150 by slightly rotating the indole ring of Trp150, and compound 9 has additional hydrogen bond and δ-π interactions with Leu135, which demonstrated the feasibility of our design strategy. Moreover, lead compounds 3 and 9 revealed improved polar surface area compared to GW9508, and have considerable hypoglycemic effects in mice. This structure-based study might inspire us to design more promising FFA1 agonists by increasing additional interactions with the residues outside of binding pocket.     

Density distribution of free fatty acid receptor 2 (FFA2)-expressing and GLP-1-producing enteroendocrine L cells in human and rat lower intestine,and increased cell numbers after ingestion of fructo-oligosaccharide

Glucagon-like peptide 1 (GLP-1) is a multifunctional hormone in glucose metabolism and intestinal function released by enteroendocrine L-cells. The plasma concentration of GLP-1 is increased by indigestible carbohydrates and luminal infusion of short-chain fatty acids (SCFAs). However, the triggers and modulators of the GLP-1 release remain unclear. We hypothesized that SCFAs produced by bacterial fermentation are involved in enteroendocrine cell proliferation and hormone release through free fatty acid receptor 2 (FFA2, also known as FFAR2 or GPR43) in the large intestine. Fructo-oligosaccharide (Fructo-OS), fermentable indigestible carbohydrate, was used as a source of SCFAs. Rats were fed an indigestible-carbohydrate-free diet (control) or a 5% Fructo-OS-containing diet for 28 days. FFA2-, GLP-1-, and 5-hydroxytryptamine (5-HT)-positive enteroendocrine cells were quantified immunohistochemically in the colon, cecum, and terminal ileum. The same analysis was performed in surgical specimens from human lower intestine. The coexpression of FFA2 with GLP-1 was investigated both in rats and humans. Fructo-OS supplementation in rats increased the densities of FFA2-positive enteroendocrine cells in rat proximal colon, by over two-fold, relative to control, in parallel with GLP-1-containing L-cells. The segmental distributions of these cells in human were similar to rats fed the control diet. The FFA2-positive enteroendocrine cells were GLP-1-containing L-cells, but not 5-HT-containing EC cells, in both human and rat colon and terminal ileum. Fermentable indigestible carbohydrate increases the number of FFA2-positive L-cells in the proximal colon. FFA2 activation by SCFAs might be an important trigger for produce and release GLP-1 by enteroendocrine L-cells in the lower intestine.     

Exploration of phenylpropanoic acids as agonists of the free fatty acid receptor 4 (FFA4): Identification of an orally efficacious FFA4 agonist

The long chain free fatty acid receptor 4 (FFA4/GPR120) has recently been recognized as lipid sensor playing important roles in nutrient sensing and inflammation and thus holds potential as a therapeutic target for type 2 diabetes and metabolic syndrome. To explore the effects of stimulating this receptor in animal models of metabolic disease, we initiated work to identify agonists with appropriate pharmacokinetic properties to support progression into in vivo studies. Extensive SAR studies of a series of phenylpropanoic acids led to the identification of compound 29, a FFA4 agonist which lowers plasma glucose in two preclinical models of type 2 diabetes.