FFAR2/3 as Microbial Metabolite Sensors to Shape Host Health: Pharmacophysiological View

Abstract: Role of gut microbiome in human health is becoming apparent. The major functional impact of gut microbiome is transmitted through the microbial metabolites that are produced in the gut and interact with host cells either in the local gut environment or get absorbed in the circulation to impact distant cells/organs. Short chain fatty acids (SCFAs) are the major microbial metabolites that are produced in the gut through fermentation of non-digestible fibers. SCFAs are known to function through various mechanism, however, their signaling through free-fatty acid receptor 2 and 3 (FFAR2/3; type of G-coupled protein receptors) is new therapeutic approach. FFAR2/3 are widely expression in diverse cell types in human and mice, and functions as sensors of SCFAs to change several physiological and cellular functions. FFAR2/3 modulates neurological signaling, energy metabolism, intestinal cellular homeostasis, immune response and hormone synthesis. FFAR2/3 functions through Gi and/or Gq signaling, that is mediated through specific structural features of SCFAs-FFAR2/3 bindings and modulating specific signaling pathway. In this review, we discussed the wide-spread expression and structural homologies between human and mice FFAR2/3, and their role in different human health conditions. This information can unlock opportunities to weigh the potential of FFAR2/3 as drug target to prevent human diseases.

The CFMB (previously known as phenylacetamide 1) [147], AMG-7703 [148], and tiglic acid [136] allosteric agonists (that bind other than orthosteric site and activate receptor activity) [ Histidine residue at site (His 242 ) in human FFAR2 serves as a key residual site to classify whether a ligand will show allosteric or orthosteric activity [148]. 2CTAP, BTI-A-404 and BTI-A-292 are inverse agonist (a ligand bind to the receptor as an agonist but inhibit its pharmacological response) of human FFAR2 and reduced Ca 2+ level via Gαq signaling [150,151]. But detailed information on structural and molecular interactions of 2CTAP, BTI-A-404, BTI-292, and GLPG0974 with human FFAR2 are not available.
4-CMTB is an ago-allosteric modulator ligand for human FFAR2 as it increases the binding efficacy of SCFAs (like a positive allosteric modulators) and also activate the human FFAR2 receptor of its own (like a allosteric agonist) [148][149][150]152]. The ago-allosteric modulator, 4-CMTB binding interaction with human FFAR2 receptor is shown in Figure 5.2. The CFMB, phenylacetamide 2 and phenylacetamide 58 are allosteric agonist to mice FFAR2, but only demonstrated through biological phenomenon, however insilico studies remain unknown [128,145,147]. The chicken FFAR2 homology model has shown that four active residues are responsible for binding of vorapaxar ligand to FFAR2 receptor [91]. Three more AAs at Tyr 246 , Met 80 and His 182 provide supports to this ligand-binding grooves [91].

FFAR3
A well-known human FFAR3 agonist, 1-MCPC forms H-bonds at different binding residues to activate FFAR3 receptor shown in Figure 5.3 [125,136]. Pertussis Toxin (PTX) is a human FFAR3 inhibitor known to inhibit the FFAR3 receptors pharmacological and biological function via p38 and JNK (c-Jun N-terminal kinase) pathway [153]. Similarly, based on biological phenomenon, AR420626 and cyclopropanecarboxylic acid are selective allosteric agonist, and AR399519 and CF3-MQC are antagonist for mice FFAR3, however detail in-silico analysis yet to be done [2,128,154]. To the best of our knowledge, so far no studies have directly addressed interactions of synthetic ligands with human nor rodent FFAR3, therefore this opens opportunities to study such in detail such interactions using dry and wet lab technologies.

FFAR2/3 in gut hormonal synthesis
FFAR2/3 signaling significantly contribute in gut hormone homeostasis through gut-hepatic [4,178] and gut-brain [2,157,179,180] axis regulate metabolic functions (Figure 7). increase is beneficial in regulation of blood glucose levels. However, in a rat study, FFAR2 agonist CFMB has no effect on colonic GLP-1 hormonal synthesis [184], indicating that either CFMB is not agonist for rat FFAR2 and or it plays differential role in rat intestine compared to human and mice.
In addition, FFAR2 signaling also involve in the GI tract buffering, especially on the conjunction of stomach and duodenum where acid of stomach poured down in the duodenum. A study showing that FFAR2 agonist phenylacetamide 1 increases the duodenal HCO3 -secretion via activating 5-HT4 receptor, and muscarinic M1 and M3 receptors [185], therefore balances the acidity coming from stomach in the duodenum. interactive action with targeted agonist and antagonist would help in exploring exact mechanism of action of FFAR2/3 against various gut hormonal comorbidities like obesity and T2D.

FFAR2/3 in intestinal epithelial integrity and inflammation
Emerging evidence indicate that the FFAR2/3 signaling significantly contribute in nutrient absorption [61,75] and helps to maintain the intestinal epithelial integrity [7,23] (Figure 7), as described below. Also, FFAR2 KO-NOD mice have higher rate of T1D development as compare to FFAR2 WT-NOD mice [175]. However, acetylated high-amylose maize starch administration to FFAR2WT-NOD mice shows protection against diabetes but such effect was no seen in FFAR2 KO-NOD mice [175].
Furthermore, butylated high-amylose maize starch administration to FFAR2 KO-NOD mice show protection against diabetes due to increase in the population of CD4+Foxp3+ Treg cells in the colon [175]. At molecular level, via epigenetic-histone modification butyrate convert the naive Fox3 -T-cells to Fox3 + Treg cells through overexpression of FoxP3 protein, IL-10 and Helios transcription factor to provide protection against T1D (or autoimmune activity) by increasing the number of autoreactive T cells and Treg cells [175]. In human intestinal PBMC, FFAR2 agonist butyrate reduces gut permeability and protection against LPS-induced pro-inflammatory (IL-1β and TNFα) production [7,191]. model [193,194]. However, contradictory findings reported by Hatanaka et al. [195] showing that the FFAR2 signaling promotes occurrence of GIT tumorigenesis. This controversial results of FFAR2 in intestinal integrity is might be due to (i) epigenetic changes induced by SCFAs and/or (ii) compensatory response by FFAR3 signaling. Further, precise mechanistic studies to develop full understanding about the role of FFAR2 signaling in intestinal integrity are warranted.

FFAR3
FFAR3 maintains intestinal integrity by activating the cytokines and chemokines through MEK-ERK pathway [43]. In FFAR3 -/-mice, inflammatory response was significantly reduces as compare to their WT [176]. Grape seed proanthocyanidins reduces the diarrhea occurrence by improving intestinal integrity and by shifting towards SCFAs-producing microbes (Lactobacillaceae and clostridium) in young swine model [196]. SCFAs also decreases intestinal permeability by increasing Ocln and FFAR3 mRNA expression in swine intestine [196]. The SCFAs treatment shows potential inhibitor action against LPS-induced pro-inflammatory (IL-1β, IL-6 and TNFα) production by activatingFFAR3, tested in FFAR3 KO mice signaling [7,176,191]. However, FFAR3 KO mice on treatment with TNBS shows reduce in immune response along with suppression of neutrophil infiltration [176], so apart from FFAR3 signaling intestinal inflammatory action is regulated by some other mechanism.

FFAR2/3 in neurophysiology
After the deorphanization, many research groups have reported that neither FFAR2 nor FFAR3 are expressed in CNS [8,102]. But recently it has been reported that low expression of FFAR2 is detected in the CNS which is limited to glia and neurons of the caudate, but FFAR2 can also be detected in cortical neurons and pituitary gland [197]. FFAR3 is expressed in peripheral nervous system (PNS), particularly sympathetic neurons of the superior cervical ganglion as a vasoconstriction phenotypic effect [22,102,103].

FFAR2
FFAR2 regulates the blood brain barrier (BBB) permeability [157,179]. Butyrate mediated activation of FFAR2 signaling and colonization of single bacterial strain Clostridium tyrobutyricum (responsible for production of butyrate) and Bacteroides thetaiotaomicron (mainly produce acetate and propionate) in germ-free mice decreases BBB permeability through boosting up Ocln mRNA expression in the frontal cortex and hippocampus [157]. Even FFAR2 KO mice shows severe microglia abnormality with increased dendritic lengths, number of segments, branching points, terminal points and cell volumes as compare to control mice suggesting that FFAR2 regulates microglial maturation and function [8]. However, multiple sclerosis (autoimmune neuro-inflammatory disease associated with CNS) patient and experimental autoimmune encephalitis (EAE) mice model induced by immunization of myelin oligodendrocyte glycoprotein show lower SCFA concentration and high expression of proinflammatory marker along with FFAR2 and 3 expression [198]. Further supported by clinical and histological score that the FFAR2/3 KO mice are more resistant to experimental autoimmune encephalitis (EAE) pathogenesis as compare to WT mice [198]. However, administration of SCFAs to EAE mice shows anti-inflammatory effect by increasing the IL-10+ T-cells and IL-10 Preprints (www.preprints.org) | NOT PEER-REVIEWED | Posted: 3 May 2020 doi:10.20944/preprints202005.0037.v1 expression in CNS tissues to suppress the inflammation. Thus, despite SCFAs beneficial effects on the CNS function, the mechanisms of SCFAs and FFFAR2/3 signaling to protect autoimmune CNS inflammation are not known [198]. As, SCFAs also function through inhibition of histone deacetylase and modulating cellular energy flux such as mitochondrial functions, may be responsible for such effects. However, these pathways are not yet established in EAE pathogenesis.

FFAR3
FFAR3 controls sympathetic neurons which in turn regulates whole body metabolic homeostasis [22]. FFAR3 is expressed in portal neurons and regulates propionate-induced gluconeogenesis via gut-brain axis [156]. FFAR3 expressing neurons in sub-mucosal and myenteric ganglionic plexus of small intestine regulates gut hormonal synthesis [2]. Mostly, in the distal part of small intestine (ileum), the FFAR3expressing neurons reported to be express in substance P and somatostatin enteroendocrine cells derived from the CCK-secretin-GIP-GLP1-PYY-neurotensin lineage [2,180]. These evidence shows FFAR3 signaling just like FFAR2 is a promising therapeutic target for treating gut related disorders such as obesity, T2D, colitis and diarrhea, by honing gut-hormonal synthesis and balancing microbiome-gut-brain axis ( Table 3).

FFAR2/3 in adipogenesis and lipolysis
Several cellular and molecular pathways involved in adipogenesis, lipolysis, glucose homeostasis, insulin sensitivity and energy metabolism are regulated by FFAR2/3 signaling ( Figure   7) [ increases the FFAR2 expression via PPARγ-dependent manner to regulate adipogenesis [205]. In contrast, FFAR2 KO mice fed with HFD show lower body fat mass, improved glucose control, lower plasma lipids, increased body temperature with BAT density and lower WAT inflammationindicating that FFAR2 deletion protects HFD-induced obesity/T2D [206]. Other studies show that acetate and propionate has no effect on adipogenesis in 3T3-L1 cells or mouse models and also no effect on either FFAR2 or FFAR3 expression [114,138]. A human study also reveals that FFAR2 expression in adipose tissues has no correlation with adipogenesis [207]. These observations indicates that the role of FFAR2/3 in adipose biology remain controversial and need further investigations.

FFAR3
Human multipotent adipose tissue-derived stem (hMADS) model reveals that activated FFAR3 by acetate significantly reduces lipolysis through decreasing hormone-sensitive lipase phosphorylation [14]. In mice, FFAR3 stimulated by gut microbiota derived SCFAs increases leptin production, hepatic lipogenesis and adipocyte adipogenesis [3]. Under HFD administration, FFAR3 KO male shows high body fat mass, plasma leptin level and blood glucose level as compare to female littermates [208]. In porcine, stimulated FFAR3 by butyrate administration enhances lipid accumulation and adipogenesis by upregulating glucose uptake and de novo lipogenesis through activation of Akt and AMPK pathways [86]. Moreover, FFAR3 signaling reduces blood pressure of the mice by increasing renin (angiotensin secreted from kidney in controlling blood pressure, and maintaining body fluid and electrolytes level) production [19]. Furthermore, FFAR3 triggered by SCFAs regulates intestinal gluconeogenesis via cAMP-activated pathway [156] and satiety signaling these findings indicates that FFAR3 plays a significant, but controversial role in regulating energy metabolism, however, precise mechanism(s) of remain elusive and needs further investigations.

FFAR2/3 in regulating pancreatic beta-cells proliferation and functions
Pancreatic beta-cells are crucial to regulate blood glucose homeostasis, by producing insulin. Overall, these findings indicate that FFAR2/3 signaling is critical to regulate pancreatic beta-cells either by changing their proliferation, differentiation, insulin synthesis and regulating their functions in terms of GSIS, which in turn maintains better glucose homeostasis, however, the their precise role in regulating proliferation and differentiation are poorly understood. -Cmp1 and CATPB function as an agonist and antagonist for the neutrophil FFAR2 respectively.

Conclusion and Future directions
-Cmp1 and acetate activates the phospholipase C-inositol phosphate 3 (IP3) Ca 2+ signaling while CATPB inhibits it.
-Cmp1 act as a potent activator of the NADPH-oxidase in TNF-α-primed neutrophils with increased release of superoxide.
-Human monocyte FFAR2 reduces inflammatory cytokine expression in response to acetate.
-Increased β-cell contributes to more insulin secretion.
-FFAR2 KO mice gestational glucose tolerance worsened even under antibiotic treatment and further deteriorated during second pregnancy.
-Antibiotic modulation of gut microbiota does not disrupt the contribution of FFAR2 to gestational glucose tolerance.
-FFAR2 act as a novel target for β-cells adaptation to pregnancy-induced insulin resistance during to maintain normal glucose homeostasis.

Primary Pancreatic
Islet --SCFAs like Acetate, propionate and butyrate administration has no effect on insulin and glucagon secretion regardless of glucose level.
-CFMB (FFAR2 agonist) has significant effect in increasing the somatostatin and insulin secretion whereas no effect was observed in glucagon synthesis.
-Mediate an inhibition of insulin secretion by coupling to Gi-type G Proteins -Under type-2 diabetic condition acetate concentration increases in pancreatic islet and systemic circulation -FFAR2 antagonist might increase insulin secretion in type-2 diabetes -Double knock-out of FFAR2 and FFAR3 altered the glucose tolerance in diabetic condition. [27,222] 3 Ileum -Bacterial metabolites, propionate, activate ileal mucosal FFAR2 to decrease hepatic glucose production.
-Propionate stimulate GLP-1r dependent neuronal network to regulate glucose production activated through ileal FFAR2 signaling.
-Improved glucose homeostasis in diabetic mice by treating with FFAR2 agonist, acetate and phenylacetamide 1. -Even in FFAR2/3 KO mouse monocyte display elevate cytokine response on treatment with SCFAs.
-SCFA does not act through FFAR2 to modulate mice monocyte inflammatory responses.
-FFAR2 expression in small intestine and colonic L-cells as compare to non-L-cell population.
-SCFAs triggered Ca 2+ elevation in L-cells with enhanced GLP-1 and PYY secretion through Gq-mediated pathway, implicating FFAR2 signaling involvement. -FFAR2 immune regulation mechanism get hamper with increase in cytokine concentration in colonic mucosa.
-The activated mucosal FFAR2 act on the nearby nerve endings at 5-HT3 serotogenic receptors.
-SCFAs stimulate PYY and 5-HT secretion from ileum and colonic endocrine cells by activating FFAR2 receptor.
[75, 185] 13 Stomach -The villi and microvilli of gastric brush cells reveal expression of FFAR2 (at gene and protein level) in the mice stomach. [6,75,183] 14 Lungs -Expressed in the mice lungs.
-SCFAs produced from gut microbiota modulate the blood glucose level.  3 Intestinal K-cells -Secrete glucose-dependent insulinotropic peptide (GIP) in response to glucose. -IGN gene expression increases by butyrate mediated through cAMP pathway but not via Gi-nor Gq pathway.
-Neither Gi-nor Gq-sensitive inhibitors (PTX and U73122) able to reduce the IGN gene expression induced by butyrate. [156] 5 Monocyte -Human monocyte FFAR3 reduces cytokine expression in response to acetate.
-Acetate is responsible for the antilipolytic response luminal and systemic level.
-Rosiglitazone increases the expression of FFAR3.
-Treating with Gi-sensitive PTX inhibitors prevents antilipolytic response develop by acetate.
-Colonic or systemic acetate modulation helps in improving the insulin resistance in human adipocytes via FFAR3 mediated attenuation of hormone-sensitive lipase (HSL) phosphorylation. [14,224] 7 Enteric Neurons -FFAR3 agonist, AR420626 response at colon mucusa showed monophasic reductions in short-circuit currents (Isc) and sensitive to neurotoxin tetrodotoxin (TTX). -Treatment with Gαi/o inhibitor NF023 shows no inhibition of NE release, so FFAR3 response is independent of Gαi/o pathway.
-Further, treatment with siRNA against either PLCβ3 or ERK1/2 decreases the expression NE protein by more than 80%.
-Dietary propionate leads to c-Fos (neuronal activation marker) activation in the hypothalamic region which receives neuronal signal from both parabrachial nucleus (PBN) and dorsal vagal complex (DVC), mostly paraventricular nucleus (PVN), the lateral hypothalamus (LH) and the arcuate nucleus (ARC) of hypothalamus. [156] 7 Intestinal Enteroendocrine Cells -Acetate, propionate and butyrate administration in mice protect against diet-induced obesity and insulin resistance.
-Propionate and butyrate but not acetate induce gut hormones and reduces food intake.
-Butyrate had minor effect in stimulation of GLP-1 through FFAR3.
-FFAR3 KO mice shows normal body weight and glucose homeostasis, indicating some additional mediators are involves in these mechanism.
-FFAR3 KO mice shows impair GLP-1 synthesis with altered in mRNA expression of Glucagon, PYY and active GLP-1 peptide.
-Microbiota is associate with increase SCFA production acting through FFAR3 signaling.
-Through selective FFAR3-agonist, AR420626 showed greatest efficacy of FFAR3 at distal regions of intestine to protect mice from diet induced obesity by preventing a reduction in energy expenditure induced by an HFD. -FFAR2 immune regulation mechanism get hampered with increase in cytokine concentration in colonic mucosa.
-Increased histopathology condition of colitis with goblet cell dysfunction, colonic dilatation and wall thickening, ultimate leads to IBD. [77] 13 Duodenum L-cells -FFAR3 is colocalized with GLP1 and expressed in L cells.
-Pretreatment with serosal PTX along with MQC application restored the CCh response indicating the FFAR3 anti-secretory effect is mediated through Gi/o pathway in rat proximal colon. [200] 17 Adipocytes -A mixture of SCFA reduces plasma FFA in DIO mice along with beige adipogenesis marker.
-Increase in adipose tissues with reduction in colon size.
-Reduces body weight by increasing mitochondrial biogenesis and reducing chronic inflammation. [18,202] 18 Lungs -Expressed in the mice lungs.
-Propionate minimize allergy airway inflammation in mice lungs mediated through FFAR3. [5] 19 Duodenal I-cells -The receptor senses the circulating SCFA in plasma to modulate I-cell functions.
-But unlike the LCFA, SCFAs are not involved in the cholecystokinin synthesis from duodenal I-cells.