Free Fatty Acid Receptors 2 and 3 as Microbial Metabolite Sensors to Shape Host Health: Pharmacophysiological View

The role of the gut microbiome in human health is becoming apparent. The major functional impact of the 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 are absorbed into circulation to impact distant cells/organs. Short-chain fatty acids (SCFAs) are the major microbial metabolites that are produced in the gut through the fermentation of non-digestible fibers. SCFAs are known to function through various mechanisms, however, their signaling through free fatty acid receptors 2 and 3 (FFAR2/3; type of G-coupled protein receptors) is a new therapeutic approach. FFAR2/3 are widely expressed in diverse cell types in human and mice, and function as sensors of SCFAs to change several physiological and cellular functions. FFAR2/3 modulate neurological signaling, energy metabolism, intestinal cellular homeostasis, immune response, and hormone synthesis. FFAR2/3 function 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 discuss 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 a drug target to prevent human diseases.


FFAR2 Expression
During the initial days of FFAR2 discovery, it was found to be intensively expressed in human immune cells such as peripheral blood mononuclear cells (PBMCs) and polymorphonuclear cells (PMNs) with maximum expression in neutrophils [53][54][55][56][57]. However, more recent studies and our own data show that FFAR2 is expressed in human fungiform taste buds [58], dendritic cells (DCs) derived from bone marrow [59], liver [55], heart [55], pancreatic islet of Langerhans [60,61], spleen [55], fetal membranes and placenta [62], L-cells in the large intestine [35,63], brain parenchyma [64], neuronal cell line-SK-N-SH, and the human breast cancer cell line (MCF-7) [65]. Additionally, FFAR2 expressed in colonic epithelia and mucosa but not in the colonic muscle and submucosal regions [66]. Its expression in muscle remains controversial [55,67]. Although FFAR2 expression is seen in brain parenchyma, comprehensive studies on its expression in the brain are needed to define its importance in brain functions [64].
Comprehensive expression analysis of FFAR2 and FFAR3 in different tissues and cell lines of mice and humans are presented in Table 1 and FFAR2 is observed at the C-terminal end. The C-terminal of the receptors consists of most conserved minimotifs and short peptides that regulate receptor binding efficiency, posttranslational modification and trafficking with unique biochemical and physiological properties [130]. The detailed information on C-termini biophysical properties of FFAR2/3 are out of context and yet to be studied in a more comprehensive manner.

Structures of FFAR2 and FFAR3
Multiple emerging sources indicate that FFAR2/3 can be novel targets to prevent and/or treat several human diseases [2,39,60,[113][114][115][116]. However, lack of knowledge in their structure and precise understanding of their interactions with ligands leads to a delay in gaining attention to be considered as novel therapeutic targets. However, growing understanding using evolved computational in-silico analyses and their significant role in several human diseases such as obesity, diabetes, IBD, and aging, FFAR2/3 are emerging as potential therapeutic targets [117,118]. Homology modeling of FFAR2 and FFAR3 along with gene mutagenesis, structural conformation, and protein-ligand interaction is developed and their importance is discussed in the following section.

FFAR2
The crystal structure of FFAR2 is not yet determined and the structure was predicted using human β 2 -adrenergic receptor as template. [50]. Human FFAR2 comprises 330 amino acids (AAs) that are arranged in a 7-TM structure [117,129]. Structurally, the third TM of FFAR2 contains cysteine residue at the top and an arginine residue at the bottom [100,118], and a conserved domain of GPCR family-Glu-Arg-Tyr motif [100]. The active site of human FFAR2 consists of Tyr 90 , Ile 145 , Arg 180 , Arg 255 , and Glu 166 [46,52]. The small carboxylic acids (SCAs) bind to this binding pocket of Tyr 90 , Ile 145 , and Glu 166 [52]. Arg 180 and Arg 255 are positively charged orthosteric sites which interact with negatively charged glutamine residue (Glu 171 ) to stabilize the protein structure for proper binding with ligands [46]. Similar to humans, mouse FFAR2 is also a 7-TM protein made of 330 AAs [129] and shows 81.69% nucleotide level and 84.85% protein level similarity to it. The mouse FFAR2 receptor sequence superposition and pairwise alignment with human FFAR2 is shown in Figure 3A-C The active binding site of mice FFAR2 is comprised of Trp 75 , Gln 148 , Tyr 238 , Arg 65 , Arg 180 , Tyr 90 , and Arg 255 according to our in-silico analysis. Both mouse and human FFAR2 show the protein sequential similarity from the 95-111 AAs position except at the 105 th position where mouse FFAR2 consists of methionine whereas human FFAR2 is of isoleucine. The significant change in the secondary structure of human and mice FFAR2 is observed at the C-terminal end. The C-terminal of the receptors consists of most conserved minimotifs and short peptides that regulate receptor binding efficiency, posttranslational modification, and trafficking with unique biochemical and physiological properties [130]. The detailed information on C-termini biophysical properties of FFAR2/3 are out of context and yet to be studied in a more comprehensive manner.  The FFAR2-like protein in chicken contains 367 AAs, encoded with the gene of 1105 bp nucleotides as per National Center for Biotechnology Information (NCBI)'s latest update. The chicken FFAR2 paralog homology model shows four active AA residues at the positions His 140 , Arg 180 , His 242 , and Arg 255 [91]. These residues are further supported by Thr 201 , Glu 113 , and His 115 , and associated with proteinligand interactions [91]. The homology models and chromosomal location of other laboratory animal species (Mesocricetus auratus, Cavia porcellus, and Oryctolagus cuniculus) are not yet known and studies are needed to comprehend their location, structure, and functionalities. The FFAR2-like protein in chicken contains 367 AAs, encoded with the gene of 1105 bp nucleotides as per National Center for Biotechnology Information (NCBI)'s latest update. The chicken FFAR2 paralog homology model shows four active AA residues at the positions His 140 , Arg 180 , His 242 , and Arg 255 [91]. These residues are further supported by Thr 201 , Glu 113 , and His 115 , and associated with protein-ligand interactions [91]. The homology models and chromosomal location of other laboratory animal species (Mesocricetus auratus, Cavia porcellus, and Oryctolagus cuniculus) are not yet known and studies are needed to comprehend their location, structure, and functionalities.

FFAR3
Human FFAR3 structure was also predicted based on the crystal structure of the human β 2 -adrenergic receptor [50]. Human FFAR3 is made of 346 AAs and has 52% of AA sequence similarity with mice FFAR2 [117,129]. Similar to FFAR2, FFAR3 also contains an arginine at the bottom of the third TM domain [100,118] which contains Glu-Arg-Phe motif of GPCR family class A [100]. AA residues Phe 96 , Tyr 151 , and Leu 171 are involved in specific ligand binding [52] with SCAs including SCFAs. The presence of positively charged Leu 171 residues provides stabilization to negatively charged arginine residues at the second extracellular loop (EL2) [46]. The olymorphism of FFAR3 can be considered for detailed comprehensive genetic and pharmacophysiological study against various diseases [94,131]. Mouse FFAR3 is 319 AAs long [129] and has similarity of 80.41% (nucleotide level) and 76.66% (protein level) with human FFAR3. Our, in-silico analysis between human and mouse FFAR3 proteins showed two substitutions at A103S and A107T site, and significant differences in human and mouse FFAR3 secondary structure was found at the C-terminal end. A superimposed structure and pairwise alignment of human and mice FFAR3 is shown in Figure 3D-F Detail information on chromosomal location and structural analyses of FFAR3 protein from other species are not available, and need further comprehensive studies.

Comparative Structural Analyses of FFAR2 and FFAR3
The structure-activity relationship (SAR) study showed that the endogenous binding site volume of human homology FFAR3 (105 Å 3 ) is twice more than the volume of FFAR2 (41 Å 3 ) [51]. SAR helps in determining the chemical structure of a receptor, its relationship with the chemical compounds associated with any biological activity, and chemical structural modification in the receptors to increase the biological activity of the compound [132]. Along with SAR, solvent accessible surface area (SASA) also helps in determining the molecular interaction of a biomolecule with the surrounded solvent to judge its biological effect on the organism [133]. SASA analysis of human and mice FFAR2/3 revealed that both have significantly higher hydrophobic residues than hydrophilic residues [52]. Human FFAR2 receptors have higher SASA hydrophobicity by 39 Å 2 and higher aromatic SASA value by 63 Å 2 as compared to human FFAR3 [52]. Based on virtual docking of different allosteric compounds to these receptors, it was found that FFAR2 pockets have larger volumes (553 Å 3 ) and surface areas (510 Å 2 ) as compared to FFAR3 binding pockets [117]. However, the volume of the FFAR3 binding cavity (385 Å 3 ) is larger than FFAR2 (332 Å 3 ) [52] whereas the depth of FFAR2 pockets is less than FFAR3 pockets by 2 Å [117]. Therefore, focus must be given to the compounds with diverse small-part SCAs (having lipophilic tails such as branched, cyclic, and unsaturated structures) for proper binding with FFAR2/3. According to Tikhonova et al. [52], prediction for the receptor subtype with preferably selective binding residues between FFAR2/3 are Tyr 90 , Ile 145 , and Glu 166 in FFAR2 and Phe 96 , Tyr 151 , and Leu 171 . Thus, SAR information shows that FFAR2 and FFAR3 are lying in very close proximity to each other and can interact with the same chemical compound to compensate for each other's biological response [99]. Thus, more intensive and precise study must be done to determine the individual biological function of each receptor and its binding to a particular ligand.

FFAR2 Interaction with SCFA
SCFAs are the orthosteric ligand of FFAR2/3 as they bind to endogenous binding sites [51]. SCFAs' carboxylic group interacts with the arginine groups of third, fifth, and/or sixth TM domain of FFAR2 for efficient binding [51]. This SAR data explained that FFAR2 prefers flat, unsaturated moieties within the SCAs [51]. As a result, FFAR2 mostly binds to the ligands with sp 2 -or sp-hybridized α-carbon [51]. That means carbon atoms of SCAs form covalent bonds with either two or one hydrogen (H) atoms for interaction with FFAR2. This concept has been further justified by Tikhonova and Poerio [52] by showing that the FFAR2-selective binding with tiglic acid as an orthosteric ligand (binding of the ligand at the endogenous site) forms a network-intensive H-bond, while leaving a small binding cavity in FFAR2. However, substitutional mutation of histidine at His 140 and His 242 residues to alanine in the fourth and sixth TM domains decrease the binding potential of SCFAs to FFAR2 [128,134]. Using site-specific mutagenesis revealed that arginine (Arg 180 , Arg 255 ) mutation at the top of either five and/or seven TM helix is important for facilitating the interactions of SCFAs with human and mice FFAR2 [100] (Figure 4). Through FFAR2 signaling, acetate moved to the peripheral tissue to regulate lipogenesis, cholesterol metabolism, and control central appetite [135]. Moreover, propionate is responsible for maintaining the whole body's energy metabolism by controlling satiety signaling via FFAR2 [17,24]. Activated FFAR2 signaling by propionate treatment to human breast mesenchymal-like MDA-MB-231 and MDA-MB-436 cell inhibited the Hippo-Yap pathway to reduce metastatic [126]. In addition, FFAR2 signaling mediated by butyrate treatment to human enteroendocrine cell lines such as NCI-H716 (colorectal cell line) and HuTu-80 (duodenal cell line) increase Peptide YY (PYY) gut hormonal synthesis [125].    [26,125]. In mice as well, the activated   [26,125]. In mice as well, the activated FFAR2 receptor regulates biological functions such as hormonal synthesis [125], systemic inflammation [18,45], lipid metabolism [87], and adipogenesis [74,75] in maintaining body homeostasis. Detailed studies on the binding of SCFAs with FFAR2 of other species organisms are needed.
Therefore, in both humans and mice, SCFAs are associated with activation of FFAR2 in regulating biological functions such as incretin hormonal synthesis [24], metabolic syndrome [25,28,36], and occurrence of autoimmune diseases [20,136] in host. Therefore, these findings provide opportunities to study in detail which biological functions are regulated by FFAR2 and simultaneously screen the synthetic molecules for effective activation of FFAR2 for effective biological response by either inhibiting the mutation or changing the structural form of the receptor.

FFAR3 Interaction with SCFA
SAR data showed that human FFAR3 receptors prefer saturated or ali-cyclic moieties of SCAs for ideal binding [51]. Histidine at 4-TM (His 140 ) and 6-TM (His 242 ) is important in deciding the binding efficacy of SCFAs in human FFAR3, as indicated by mutagenesis studies replacing these AAs with alanine [128,134]. SCFAs via FFAR3 regulate various biochemical, cellular, and physiological function such as metastasis, hormone synthesis, gut motility, adipogenesis, lipolysis, apoptosis and others [119,121,123,126]. Detailed in-silico analysis on the binding efficiency of SCFAs with FFAR3 in mice, rodent or any other species needs to be studied.
Although the interactions of SCFAs with FFAR2/3 are similar, they still show a degree of selectiveness in these interactions [11,50]. In addition, these complex interactions can be resolved by designing alternative compounds that show higher efficacy and selectiveness for binding [51]. However, more comprehensive studies are required to define the biological functions of FFAR2/3 independent of compensative effects. There is also critical need to develop specific compounds for activating FFAR2/3 with higher efficacy than SCFAs to exploit their therapeutic potential [50]. The following section describes a few examples of synthetic compounds that bind with FFAR2/3.

FFAR2 Interaction with Synthetic Ligands
Synthetic ligands such as CATPB and GLPG0974 act as allosteric antagonists to the FFAR2 receptor by reducing Ca 2+ and the phosphorylated extracellular signal-regulated kinase (ERK)1/2 pathway [137][138][139]. For the first time, CATPB interaction with human and mouse FFAR2 are shown in Figure 4. Synthetic ligands such as Compound 1 (Cmp1), ZINC03832747, compound 44, phenylacetamide 58, and Euroscreen compound series are orthosteric agonist of human FFAR2 [128,140]. Cmp1 is also an orthosteric agonist for mice FFAR2 [128,137]. Along with mutation at His 242 site to alanine, mutation within the binding pockets at His 140 , Val 179 , Tyr 90 , Tyr 165 , and Tyr 238 residual sites to alanine significantly reduced agonist property of Cmp1 for mice FFAR2 [141], as shown in Figure 5A.

DEFINITIONS:
Orthosteric ligands. The ligand which binds to a receptor at an endogenous active site. Allosteric ligands. The ligand which binds to a receptor other than an endogenous site. Allosteric agonist ligands. Allosteric ligand that activate the receptors in the absence of orthosteric ligands by binding other than on an active site. Ago-allosteric ligands. The ligand binds allosterically to activate a receptor in the absence of an orthosteric ligand equal to an allosteric agonist and also activates the receptor in the presence of an orthosteric ligand as a positive allosteric modulator (potentiate agonist-mediated receptor response). Inverse agonist. An inverse agonist is a ligand that binds to the same receptor as an agonist but induces a pharmacological inhibitory response. Inverse agonist. The ligand binds to the receptor as an agonist but develops pharmacological function opposite to that of agonist. However, in-silico studies remain unknown [51,140,142]. 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, Tyr 246 , Met 80 , and His 182 provide supports to these ligand-binding grooves [91].

FFAR3 Interaction with Synthetic Ligands
Well-known human FFAR3 agonist 1-MCPC forms H-bonds at different binding residues to activate the FFAR3 receptor shown in Figure 5C [52,125]. Pertussis toxin (PTX) is a human FFAR3 inhibitor known to inhibit the FFAR3 receptors' pharmacological and biological function via p38 and the c-Jun N-terminal kinase (JNK) pathway [124]. Similarly, based on biological phenomena, AR420626 and cyclopropanecarboxylic acid are selective allosteric agonists, and AR399519 and CF3-MQC are antagonists for mouse FFAR3, however detailed in-silico analysis has yet to be done [3,51,147]. To the best of our knowledge, so far no studies have directly addressed interactions of synthetic ligands with human or rodent FFAR3, therefore this opens opportunities to study the topic in detail using dry and wet lab technologies. The CFMB (previously known as phenylacetamide 1) [142], AMG-7703 [143], and tiglic acid [125] are allosteric agonists (that bind to other than orthosteric sites and activate receptor activity) of human FFAR2. CFMB forms H-bond at Ile 66 [143] with human FFAR2. 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 [143]. 2CTAP, BTI-A-404, and BTI-A-292 are inverse agonists (a ligand binds to the receptor as an agonist but inhibits its pharmacological response) of human FFAR2 and reduced Ca 2+ level via Gα q signaling [120,144]. However, detailed information on the 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 positive allosteric modulators) and also activates the human FFAR2 receptor of its own (like an allosteric agonist) [120,143,145,146]. The ago-allosteric modulator 4-CMTB binding interaction with human FFAR2 receptor is shown in Figure 5B. CFMB, phenylacetamide 2, and phenylacetamide 58 are allosteric agonists to mice FFAR2, but only demonstrated through biological phenomenons. However, in-silico studies remain unknown [51,140,142]. 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, Tyr 246 , Met 80 , and His 182 provide supports to these ligand-binding grooves [91].

FFAR3 Interaction with Synthetic Ligands
Well-known human FFAR3 agonist 1-MCPC forms H-bonds at different binding residues to activate the FFAR3 receptor shown in Figure 5C [52,125]. Pertussis toxin (PTX) is a human FFAR3 inhibitor known to inhibit the FFAR3 receptors' pharmacological and biological function via p38 and the c-Jun N-terminal kinase (JNK) pathway [124]. Similarly, based on biological phenomena, AR420626 and cyclopropanecarboxylic acid are selective allosteric agonists, and AR399519 and CF 3 -MQC are antagonists for mouse FFAR3, however detailed in-silico analysis has yet to be done [3,51,147]. To the best of our knowledge, so far no studies have directly addressed interactions of synthetic ligands with human or rodent FFAR3, therefore this opens opportunities to study the topic in detail using dry and wet lab technologies.
Interestingly, in many instances, FFAR2 and FFAR3 activities are interchangeable and/or compensatory, this is because of similar chemical and structural characteristics. For example, substation of FFAR2 amino acid residues such as Glu 166 , Leu 183 , and Cys 184 with corresponding FFAR3 residues such as Leu, Met, and Ala using site-directed mutagenesis favored the binding of FFAR3 agonists 1-MCPC and 3-pentenoic acid, while these compounds were not able to bind wild type FFAR2 [51]. However, in humans as well as in mice, the important source of SCFAs (known orthosteric ligands of FFAR2/3) is the host gut microbiota, which drives the next step to discuss the role of gut microbiota in SCFAs production in regulating the pharmacological and physiological function of the host body through FFAR2/3 signaling.
FFAR2 signaling activation led to increase immunoglobulin (Ig)A (first line of defense against pathogens at the mucosal surfaces) production to protect intestinal epithelium against foreign pathogenic microbe invasion [115,158]. Sun et al. [153] showed that activation of FFAR2 signaling increases cathelicidin-related antimicrobial peptide (CRAMP) production from pancreatic endocrine cells as protection against type 1 diabetes (T1D) [153]. In addition, butyrate-mediated activation of FFAR2 signaling in mice chondrocyte exhibit anti-inflammatory activity by inhibiting the phosphorylation of NFκB (nuclear factor kappa-light-chain-enhancer of activated B cells), MAPK, AMPK-α (5' adenosine monophosphate-activated protein kinase), and the PI3K (Phosphatidylinositol 3-kinase)/Akt (Protein Kinase B) pathway [159]. Moreover, FFAR2 activation by SCFAs reduce IECs graft-versus-host disease by activating nucleotide-binding oligomerization domain-like receptor protein 3 inflammasome (associated with IECs repairing by IL-18 secretion and maintaining integrity) [160]. FFAR2 also induces neutrophil chemotaxis through activation of P13Kγ, Rac2 (Rho family GTPase), p38-MAPK, and extracellular signal-regulated kinases (ERK) signal transduction pathway [161]. On the other hand, FFAR2 KO mice with chronic DSS-induced colitis phenotype shows a decrease in invasion of PMNs and cytokine keratinocyte chemoattractant synthesis as compared to wild-type (WT) mice [42,43,45]. Additionally, FFAR2 KO mice reveal decrease in adaptive inflammatory response as compare to WT littermate in gout pathology [39]. This debatable immune modulation by FFAR2 signaling needs further studies to understand its precise mechanism(s) and their importance in different disease pathologies.  -SCFAs such as Acetate, propionate, and butyrate administration have no effect on insulin and glucagon secretion regardless of glucose level.
-CFMB (FFAR2 agonist) has a 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. [28,168] 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.   -SCFAs triggered Ca 2+ elevation in L-cells with enhanced GLP-1 and PYY secretion through G q -mediated pathway, implicating FFAR2 signaling involvement.
-Synthetic phenylacetamide agonist of FFAR2, CFMB, mobilizes more intracellular Ca 2+ in L-cells and elevates GLP-1 hormone secretion, in the presence of DPPIVi but not in its absence in mice. [11,22,162,170] 10 Colonic Mucosa -FFAR2 express in the colonic mucosa -Withdrawal of ceftriaxone antibiotic leads to reduction in SCFA concentration and increase in increased number of conditionally pathogenic Enterobacteria, E. coli, Clostridium, Staphylococcus spp., and hemolytic bacteria in colonic gut.
-FFAR2 immune regulation mechanism get hamper with increase in cytokine concentration in colonic mucosa.
-Increase histopathology condition of colitis with goblet cell dysfunction, colonic dilatation and wall thickening, ultimate leads to IBD. [78]

FFAR3 in Immune Regulation
FFAR3 signaling activated by acetate and propionate reduces the production of pro-inflammatory cytokine (Tumor Necrosis Factor alpha [TNF-α]) secretion [42], and enhances anti-inflammatory chemokines (C-X-C motif ligand 1 (CXCL-1) and CXCL-2) via enhancing the extra-cellular ERK1/2, p38-MAPK, PI3K, or mTOR (mammalian target of rapamycin) signaling [44,164,173]. In addition, FFAR3 expression increases on soluble fiber administration with a decrease in macrophages, eosinophils, neutrophils migration, and exhaled nitric oxide synthesis (eNOS) against asthma [40], so FFAR3 signaling enhances adaptive immune response. Moreover, in influenza infected mice, FFAR3 pathway increases anti-viral immunity activity on dietary fermentable fibers and SCFAs administration [114]. In addition, FFAR3 pathway stimulated by propionate reduces the lungs' allergic inflammation and total amount of IgE (antibody associated with allergic reaction) concentration in the serum [6]. Moreover, FFAR3 KO mice show lower immune response against Citrobacter rodentium infection with delayed in expression of interferon gamma (INFγ) (critical cytokine for innate and adaptive immunity against infection) through rapidly accelerated fibrosarcoma which activates the MAPK/ERK pathway [44]. However, single-cell RNAseq of eosinophilic esophagitis patient T-cell exhibits higher expression of FFAR3 with increased Th2 cytokine (that exacerbate allergies) production [41,174]. These observations indicate that FFAR3 signaling is involved in differential immune response of allergic reactions.
In mice macrophages (Raw 264.7), activation of FFAR3 signaling by SCFAs reduces the proinflammatory cytokines and increases nitric oxide synthase (iNOS) secretion [48]. Moreover, in human umbilical vein endothelial cells (HUVEC), FFAR3 mediated signaling reduces the LPS or TNFα stimulated atherosclerosis by inhibiting the proinflammatory cytokines and vascular cell adhesion molecule-1 synthesis on propionate and butyrate treatment [62,175]. However, another study reported that butyrate treatment in femoral bone marrow derived macrophages develops an anti-microbial effect through histone deacetylases inhibitor (HDACi) pathway independent of FFAR3 [176]. There, epigenetic or FFAR2 immune response compensates FFAR3 immune signaling. However, in ruminant Capra hircus fed with a high concentrate diet (60%) increases LPS and SCFAs production that activate FFAR2/3 signaling to produce cytokines and chemokines which in turn lead to cecal inflammation [177]. These results indicated that the role of FFAR3 signaling in regulating inflammation is controversial, and it may be disease/context dependent, hence further studies are needed to comprehend the role of FFAR3 signaling in immune modulation in a disease-specific manner.
Overall, these observations indicate that both FFAR2/3 are closely associated with complex mechanism of immune response [42,70,161,178,179], and cell specific responses in different diseases remain to be elucidated.
NCI-H716 cells through downregulation of ERK, p38 MAPK, and NF-κB pathways [144]. These results profoundly indicate that FFAR2 signaling regulates GLP-1 and PYY secretion and may pave the way to consider FFAR2 as a therapeutic target against diabetes, because GLP-1 increase is beneficial in regulation of blood glucose levels. However, in a rat study, FFAR2 agonist CFMB had no effect on colonic GLP-1 hormonal synthesis [183], indicating that either CFMB is not agonist for rat FFAR2 and or it plays different role in rat intestines compared to that of humans and mice. Figure 7. Role of diet-derived SCFAs activated FFAR2/3 signaling in regulation of energy balance through (A) Regulating the food intake by modulating gut-brain axis (B) Maintaining homeostasis by decreasing the fat accumulation and increasing the energy expenditure in adipose tissues, manipulating rate of gluconeogenesis in liver, and increasing insulin secretion and beta-cell function in the pancreas (C) Maintaining intestinal cellular homeostasis by increasing gut transit, mucus production, tight junction protein expression, and gut hormone synthesis and secretion.
In addition, FFAR2 signaling also involved 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 the 5-HT4 receptor, and muscarinic M1 and M3 receptors [171], which therefore balances the acidity coming from stomach in the duodenum.

FFAR3 in Gut Hormone Synthesis
Tolhurst et al. [11] also reported that GLP-1 hormone synthesis is regulated by FFAR3 signaling mediated through SCFA. GLP-1 and PYY significantly reduced in FFAR3 KO mice as compared to their Figure 7. Role of diet-derived SCFAs activated FFAR2/3 signaling in regulation of energy balance through (A) Regulating the food intake by modulating gut-brain axis (B) Maintaining homeostasis by decreasing the fat accumulation and increasing the energy expenditure in adipose tissues, manipulating rate of gluconeogenesis in liver, and increasing insulin secretion and beta-cell function in the pancreas (C) Maintaining intestinal cellular homeostasis by increasing gut transit, mucus production, tight junction protein expression, and gut hormone synthesis and secretion.

FFAR2 in Gut Hormone Synthesis and Secretion
FFAR2 activation increases GLP-1 and PYY synthesis in human, rodents and guinea pig L-cells [63,76,181,182]. Tolhurst et al. [11] for the first time reported that activated FFAR2 signaling increases GLP-1 hormonal synthesis from L-cells of mice with an increase in Ca 2+ levels. In addition, FFAR2 KO mice show a decrease in GLP-1 and insulin secretion leads to impair glucose tolerance even under SCFA treatment [11]. However, inulin (a prebiotics that promotes SCFA production) feeding increases L-cell population in HFD-fed mice and protects against obesity/T2D, while such effects of inulin disappeared in FFAR2 KO mice [24], suggesting that FFAR2 is required for acetate action to prevent HFD-induced obesity/T2D. The activated FFAR2 controls blood glucose by increasing PYY and GLP-1 [11]. In addition, FFAR2 agonist (CFMB) treatment to mice intestinal organoid directs more PYY and GLP-1 secretions with reduced cyclic adenosine monophosphate (cAMP) levels [24,172]. The novel FFAR2 antagonists such as CATPB, BTI-A-404, and BTI-A-292 decreases the GLP-1 hormonal synthesis from NCI-H716 cells through downregulation of ERK, p38 MAPK, and NF-κB pathways [144]. These results profoundly indicate that FFAR2 signaling regulates GLP-1 and PYY secretion and may pave the way to consider FFAR2 as a therapeutic target against diabetes, because GLP-1 increase is beneficial in regulation of blood glucose levels. However, in a rat study, FFAR2 agonist CFMB had no effect on colonic GLP-1 hormonal synthesis [183], indicating that either CFMB is not agonist for rat FFAR2 and or it plays different role in rat intestines compared to that of humans and mice.
In addition, FFAR2 signaling also involved 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 HCO 3 secretion via activating the 5-HT 4 receptor, and muscarinic M 1 and M 3 receptors [171], which therefore balances the acidity coming from stomach in the duodenum.

FFAR3 in Gut Hormone Synthesis
Tolhurst et al. [11] also reported that GLP-1 hormone synthesis is regulated by FFAR3 signaling mediated through SCFA. GLP-1 and PYY significantly reduced in FFAR3 KO mice as compared to their WT littermates [4]. However, FFAR3 agonist (AR420626) increases GLP-1 release in mice colonic crypts [3]. In addition, co-administration of maltose and miglitol (α-glucosidase inhibitor) to mice increases plasma SCFA and GIP levels via FFAR3. This effect was not seen in FFAR3 KO mice [184]. Such effect of dietary fibers on GLP-1 levels was not seen in antibiotic treated, germ-free and FFAR3 KO mice [184]. Activation of FFAR3 signaling increases the GLP-1 [11], while AR420626 (FFAR3 agonist) and AR399519 (FFAR3 antagonist) treatment to rats and AR420626 (FFAR3 agonist) treatment to mice intestinal organoid shows no effect on the synthesis of PYY and GLP-1 hormonal synthesis [24,183]. The exact reason behind these discrepant results is not known. Moreover, FFAR3 agonist (AR420626) treatment reduces enteropathy (ulcer formation and gastrointestinal bleeding) symptoms induced by indomethacin in rats by increasing duodenal HCO 3 and GLP-2 hormonal synthesis whereas FFAR3 antagonist (CF 3 -MQC) counteract the AR420626 effect by reducing the enteropathy condition [147], indicating that the mucosal protective effect of AR420626 was mediated by FFA3 activation. Even so, increased synthesis of PYY and GLP-1 hormone by Roux-en-Y gastric bypass (RYGB) surgery leads to overexpression of both FFAR2 and FFAR3 in the intestine [185]. This indicates that incretin hormonal synthesis is associated with FFAR2/3 receptor signaling in response to metabolic syndrome in either direction. Either through genetic mutational study on FFAR2/3 or their interactive action with targeted agonists and antagonists would help in exploring the exact mechanism of action of FFAR2/3 against various gut hormonal comorbidities such as obesity and T2D.

FFAR2 in Intestinal Epithelial Integrity and Inflammation
FFAR2 signaling contributes to (i) maintaining intestinal integrity which in turn reduces leaky gut, and (ii) regulating the colonic motility through intestinal 5-hydroxytryptamine (5-HT) release [66,76] and gut dysbiosis [66,76]. FFAR2 activation increases the expression of tight junction proteins (tight junction protein 1 [Tjp1], Occludin [Ocln], Claudine [Cldn]1), and mucus-secreting markers such as mucin (Muc)1 and Muc2 to maintain intestinal integrity [186], thereby reducing pro-inflammatory markers (interleukin [IL]-1β and TNF-α). In contrast, a significant decrease in mucin production (Muc2, Muc3, Muc4, and -Muc5b) was observed in the intestine of FFAR2 KO mice further indicating that FFAR2 KO mice have compromised gut barrier functions that were associated with reduced antimicrobial peptide synthesis (Reg3α, Reg3β, and Reg3γ), suggesting higher risk of microbial translocation [156]. Even in chickens, the modulated intestinal microflora by galactooligosaccharides increases the intestinal innate immune response and barrier function along with FFAR2 gene expression, suggesting that FFAR2 receptors are involved in maintaining intestinal immune homeostasis [187]. However, antibiotic treatment reduces FFAR2 expression and increases colonic epithelial permeability and inflammatory cytokines (TNF-α and IL-10) [78] in mice, further suggesting the role of FFAR2 in maintaining intestinal homeostasis. In addition, the FFAR2 KO mice model with dextran sodium sulphate (DSS)-induced colitis exhibits a decrease in colon length, an increased morbidity, an increased daily activity index (DAI), the inflammatory mediator myeloperoxidase, and a decrease in innate immunity markers such as toll-like receptors (TLR2 and TLR4) compared to their control-FFAR2WT littermate [35,71,154,157,183,188]. While FFAR2 agonist reduces body weight gain, DAI, fecal Lipocalin-2 level (biomarker of intestinal inflammation), and pro-inflammatory cytokines (IL-6) and keratinocytes chemoattractant cytokine secretion from colonic mucosa of DSS-induced colitis mice, suggesting that FFAR2 agonism protects against colitis [71].
Also, FFAR2 KO-NOD mice have a higher rate of T1D development as compared to FFAR2 WT-NOD mice [178]. 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 [178]. Furthermore, butylated high-amylose maize starch administration to FFAR2 KO-NOD mice show protection against diabetes due to an increase in the population of CD4+Foxp3+ Treg cells in the colon [178]. At the molecular level, via epigenetic-histone modification butyrate converts 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 [178]. In human intestinal PBMC, FFAR2 agonist butyrate reduces gut permeability and protection against LPS-induced pro-inflammatory (IL-1β and TNFα) production [8,189]. SCFAs reduce colonic inflammation by decreasing the secretion of proinflammatory cytokines (IL-6 and IL-12), and chemokines from the intestinal epithelial cells and/or through increasing IgA and IgG (B cells) production and interacting with DCs in TNBS (2, 4, 6-trinitrobenzene sulfonic-acid-an intestinal inflammatory agent) and C. rodentium infection induced intestinal inflammation in FFAR2 KO mice [44,190]. However, inulin (a dietary fiber) feeding, which increases SCFAs (ligands of FFAR2), also increased the expression of tight junction proteins independent of FFAR2/3 [24]. In addition, activated FFAR2 signaling by natural indigenous fruit black raspberries increases the host immune response in gut of human and colorectal cancer mice (Apc Min/+ ) model [191,192]. However, contradictory findings reported by Hatanaka et al. [193] showed that the FFAR2 signaling promotes occurrence of GIT tumorigenesis. This controversial result of FFAR2 in intestinal integrity might be due to (i) epigenetic changes induced by SCFAs and/or (ii) a 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 in Intestinal Epithelial Integrity and Inflammation
FFAR3 maintains intestinal integrity by activating the cytokines and chemokines through the MEK-ERK pathway [44]. In FFAR3 -/mice, the inflammatory response was significantly reduced as compare to their WT [44]. Grape seed proanthocyanidins reduces the diarrhea occurrence by improving intestinal integrity and by shifting towards SCFAs-producing microbes (Lactobacillaceae and clostridium) in young swine models [194]. SCFAs also decrease intestinal permeability by increasing Ocln and FFAR3 mRNA expression in swine intestine [194]. The SCFAs treatment shows potential inhibitor action against LPS-induced pro-inflammatory (IL-1β, IL-6 and TNFα) production by activating FFAR3, tested in FFAR3 KO mice signaling [8,44,189]. However, FFAR3 KO mice on treatment with TNBS shows reduced immune response along with suppression of neutrophil infiltration [44], 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 [9,101]. However, 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 [33]. FFAR3 is expressed in PNSs, particularly sympathetic neurons of the superior cervical ganglion as a vasoconstriction phenotypic effect [23,101,102]. Neurophysiology   FFAR2 regulates the blood brain barrier (BBB) permeability [150]. 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 Ocln mRNA expression in the frontal cortex and hippocampus [150]. Even FFAR2 KO mice show severe microglia abnormality with increased dendritic lengths, number of segments, branching points, terminal points, and cell volumes as compared to control mice suggesting that FFAR2 regulates microglial maturation and function [9]. However, multiple sclerosis (autoimmune neuro-inflammatory disease associated with CNS) patients and experimental autoimmune encephalitis (EAE) mice models induced by immunization of myelin oligodendrocyte glycoprotein show lower SCFA concentration and a high expression of proinflammatory marker along with FFAR2 and 3 expression [195]. This is further supported by clinical and histological score that the FFAR2/3 KO mice are more resistant to experimental autoimmune encephalitis (EAE) pathogenesis as compared to WT mice [195]. However, administration of SCFAs to EAE mice shows an anti-inflammatory effect by increasing the IL-10+ T-cells and IL-10 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 [195]. As SCFAs also function through inhibition of histone deacetylase and modulating cellular energy flux such as mitochondrial functions, this may be responsible for such effects. However, these pathways are not yet established in EAE pathogenesis.

FFAR3 in Neurophysiology
FFAR3 controls sympathetic neurons which in turn regulate whole body metabolic homeostasis [23]. FFAR3 is expressed in portal neurons and regulates propionate-induced gluconeogenesis via gut-brain axis [149]. In FFAR3 KO mice, catecholamine-producing enzyme tyrosine hydroxylase (TH) level is significantly lower which affects the neuronal growth [23]. The heart rate is reduced in FFAR3 KO mice which is associated with decreased norepinephrine release from sympathetic neurons, indicating that FFAR3 signaling regulates sympathetic neuronal functioning [23]. FFAR3 signaling activates G βγ -phospholipase C (PLC)-β3-ERK1/2-synapsin 2-β at serine 426 pathway to enhance norepinephrine release from sympathetic nerve endings [103]. FFAR3-dependent synthesis of norepinephrine releases from synaptic vesicles which helps to modulate energy expenditure of the host body [103]. Further, the treatment of mice with FFAR3 agonist propionate, elevates the heart rate and oxygen consumption by increasing β-adrenergic receptor in ganglions [23]. In addition, FFAR3-signaling inhibit N-type calcium channels in neurons [102,196].
Won et al. [104] suggests FFAR3 signaling activates G βγ signaling pathway and inhibits N-type Ca 2+ channels, which in turn reduces neuronal catecholamine release in rat sympathetic nervous systems. Moreover, in proximal colonic mucosa of rats, FFAR3 is associated with cholinergic-mediated secretory response in enteric nervous system [197]. Thus, FFAR3 is a potential target for treating neurogenic diarrheal disorder by reducing the nicotinic acetylcholine receptor (nAChR) activity [198]. Moreover, on treatment with FFAR3, synthetic agonist AR420626 suppresses nAChR or serotonin mediated motility changes with a consistent effect on the FFAR3-stimulated anti-secretory effect [198]. FFAR3 expressing neurons in sub-mucosal and myenteric ganglionic plexus of small intestine regulates gut hormonal synthesis [3]. Mostly in the distal part of small intestine (ileum), the FFAR3-expressing neurons reported to be expressed in substance P and somatostatin enteroendocrine cells derived from the CCK-secretin-GIP-GLP1-PYY-neurotensin lineage [3,180]. These evidence shows FFAR3 signaling similar to 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 the microbiome-gut-brain axis (Table 3).

FFAR2 in Adipogenesis and Lipolysis
FFAR2 is responsible for energy accumulation in adipose tissues, adipogenesis, and metabolic syndrome disease pathogenesis [113]. In-vitro (differentiated 3T3-L1 cells) and in-vivo (C57BL/6J mice) study on adipocyte has shown that FFAR2 increases adipogenesis [75]. In mice, acetate and propionate administration boosts FFAR2 expression in adipose tissues with reduce plasma FFA levels and decrease lipolysis [31,75,140]. Moreover, when FFAR2 KO mice fed with high fat diet (HFD) show higher energy expenditure, plasma FFA level and higher food intake leads to obesity as compared to WT mice [30,33,117]. However, activated FFAR2 signaling by SCFA administration to diet-induced obese (DIO) mice demonstrates reduced body weight by promoting beige adipogenesis and mitochondrial biogenesis with reduction of Firmicutes: Bacteroidetes ratio along with lower plasma FFA level [19,199]. Moreover, SCFA treatment to adipose-specific FFAR2 KO transgenic (aP2-Gpr43TG) mice induces pro-inflammatory cytokine (TNF-α) in anti-inflammatory M2-type macrophages within the adipose tissue milieu [200]. Apart from SCFAs, ketogenic metabolites aceto-acetate activates FFAR2 which activates ERK1/2 signaling in ketogenic condition (fasting or diabetic) to regulate energy homeostasis and maintains lipid metabolism [201]. During lactation, bovine adipocytes exhibit higher FFAR2 expression, which indicates genetic switch-on of FFAR2 enhanced adipogenesis to compensate for high energy requirement of the animal during lactation [84].    -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. [148,165,198] 12 Colonic Mucosa -FFAR2 express in the colonic mucosa -Withdrawal of ceftriaxone antibiotic leads to reduction in SCFA concentration and increase number of conditionally pathogenic Enterobacteria, E. coli, Clostridium, Staphylococcus spp. and hemolytic bacteria in colonic gut.
-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. [78] 13 Duodenum L-cells -FFAR3 is colocalized with GLP1 and expressed in L cells.
-FFAR3 agonist activity is sensitive to acetylcholinergic (ACh) neurotransmission in rat colon mucosa.
-Pretreatment with serosal PTX along with MQC application restored the CCh response indicating the FFAR3 anti-secretory effect is mediated through G i/o pathway in rat proximal colon. [197] 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. [19,199]   Additionally, in the differentiated 3T3-L1 cells, FFAR2 activation by propionate enhances adipogenesis via peroxisome proliferator-activated receptor gamma 2 (PPAR-γ2) pathway and the presence of FFAR2 siRNA (small interfering ribonucleic acid) which inhibits the adipogenesis process [75]. In addition, in 3T3-L1 cells, FFAR2 allosteric agonist (phenylacetamide 1 and 2) suppresses the adipocyte lipogenesis indicating activated FFAR2 receptors that reduces lipogenesis [140,142]. Moreover, in immortalized brown adipocyte cell lines (IM-BAT), Rosiglitazone (anti-diabetic adipogenic drug) increases the FFAR2 expression via PPARγ-dependent manner to regulate adipogenesis [32]. 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 inflammation-indicating that FFAR2 deletion protects HFD-induced obesity/T2D [33,206]. 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 [113,119]. A human study also reveals that FFAR2 expression in adipose tissues has no correlation with adipogenesis [207]. These observations indicate that the role of FFAR2/3 in adipose biology remain controversial and need further investigations.

FFAR3 in Adipogenesis and Lipolysis
A human multipotent adipose tissue-derived stem (hMADS) model reveals that activated FFAR3 by acetate significantly reduces lipolysis through decreasing hormone-sensitive lipase phosphorylation [15]. In mice, FFAR3 stimulated by gut microbiota derived SCFA increases in leptin production, hepatic lipogenesis, and adipocyte adipogenesis [4]. Under HFD administration, FFAR3 KO male shows high body fat mass, plasma leptin level, and blood glucose level as compare to female littermates [204]. In pigs, 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 [87]. 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 [20]. Furthermore, FFAR3 triggered by SCFAs regulates intestinal gluconeogenesis via cAMP-activated pathway [149] and satiety signaling through gut-brain axis [17,24], thereby controlling whole body energy metabolism. Moreover, butyrate effects to regulate lipolysis depends on FFAR3, as PTX (known FFAR3 antagonist) treated 3T3-L1 (adipocytes) and Raw 264.7 (macrophages) show no effects on lipolysis, while butyrate alone increases lipolysis in these cells [48]. The leptin synthesis and FFAR2 expression found low in adipose tissues of FFAR3 KO mice [74], however, reason for these changes are not known. Overall, these findings indicate that FFAR3 plays a significant, but controversial role in regulating energy metabolism, however, precise mechanism(s) remain elusive and need 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. Therefore, maintaining and preserving beta-cell mass and functions remain critical. Beta-cell proliferation and differentiation is important for maintaining beta-cell population, while beta-cell functions are important for efficiently releasing insulin in response to glucose. In T2D, beta-cell proliferation, differentiation, and functions are deteriorated, which ultimately causes a decrease in insulin secretion and hyperglycemia in long-term diabetics. The coupling effect of FFAR2/3 receptors plays a fundamental role in the regulation of glucose-stimulated insulin secretion (GSIS) [28,39,73,203] and directly or indirectly responsible for β-cell functions in regulating pathology T2D [19,33,208].

FFAR2 in Regulating Pancreatic Beta-Cell Proliferation and Functions
Starting from an early embryonic stage, maternal gut microbiota-SCFA-FFAR2 signaling plays a crucial role in regulating metabolic syndrome, as FFAR2 KO mice embryos have lower insulin and higher glucose level, and are more susceptible to obesity and diabetes in adulthood [209]. Additionally, FFAR2 KO mice on normal chow (NC) shows reduced β-cell mass and develop obesity and T2D characterized with increased glucose intolerance and FFA levels [30,32]. In addition, activation of mouse pancreatic β-cells-MIN6 by FFAR2 agonist (phenylacetamide 58) promotes proliferation and differentiation of β-cells and enhances insulin secretion [32,140]. In contrast, deletion of FFAR2 in Min6 and EndoC-βH1 cells (human pancreatic cell line) using siRNA increases the insulin synthesis [28]. Thus, the role of FFAR2 in regulating beta-cell proliferation, differentiation and their functions remains elusive and further comprehensive studies are needed to elaborate our understandings in this context.

FFAR3 in Regulating Pancreatic Beta-Cell Proliferation and Functions
Gut microbiota changes in obese humans are associated with increased FFAR3/ Gi signaling to inhibit insulin synthesis [54]. These changes are further associated with epigenetic changes in FFAR3 receptor promotors (CpGs) and propensity of obesity and T2D, while lower methylation of FFAR3 promoters is associated with a higher body mass index [27]. However, FFAR3 activation by butyrate increases human β-cell mitochondrial respiration, which may be important to ameliorate beta-cell dysfunctions in T2D [127]. Additionally, in rodents, propionate stimulated FFAR3 signaling decreases the glucose oxidation and ATP/ADP ratio via the Gα i/o pathway [49,210]. Opposite findings have been reported that either globally or pancreatic β-cell specific FFAR3 KO mice show greater insulin secretion and improvement of glucose tolerance [28]. Similar type of results reported in Min6 and EndoC-βH1 cell lines where FFAR3 antagonist (PTX) treatment increases insulin secretion [28].
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.

Conclusions and Future Directions
Dysbiotic gut microbiota with reduced SCFAs are related with suppression of FFAR2/3 signaling-that are known to regulate an array of biological pathways participation energy metabolism, adipogenesis, appetite control, intestinal cellular homeostasis, gut motility, glucose metabolism, and inflammatory response. Alterations in these biological pathways are hallmarks of several human diseases such diabetes, obesity, IBS/IBD, Crohn's disease, atherosclerosis, gout, asthma, cardiovascular diseases, arthritis, hypertension, and colitis, therefore, targeting FFAR2/3 signaling can provide promising therapeutic strategies for these human diseases. The immune cell during metabolic diseases such as obesity and T2D causes chronic inflammatory response which provides an insight crucial mechanism for further disease progression. However, the role of FFAR2/3 signaling in these diseases remain controversial and needs to be further studied for better understanding of their role to devise the therapeutic importance of FFAR2/3 agonist/antagonists. For example, the majority of studies show that activation of FFAR2/3 signaling ameliorates obesity/T2D pathology, however, some studies show the opposite. For example, HFD feeding to FFAR2 KO mice shows improved oral glucose tolerance test (OGTT) and insulin sensitivity along with lower fat mass and increased lean mass compared to wild type (WT) mice [28,30,33], indicating that the deletion of FFAR2 protects HFD-induced obesity/T2D. Similarly, the mRNA and protein expression of FFAR2 has no correlation with insulin secretion in T2D patients [54,204]. Therefore, further studies are critically needed to develop better understanding about the role of FFAR2/3 in regulating metabolic functions, and pathology of obesity/T2D.
The pharmacological modification of these SCFAs receptors by endogenous or synthetic ligands provides an opportunity to counteract these gastrointestinal disorders in humans. However, overlapping expression of FFAR2 and FFAR3 in the same tissues/cells, and their similar affinity to specific endogenous ligands develop puzzled outcomes to understand the role of FFAR2/3 in particular biological functioning. Thus, the future studies must aim to develop highly specific and efficacious small molecules to modulate pharmacological actions of FFAR2/3 signaling, and can display a promising strategy to prevent, manage and/or treat human diseases including diabetes, obesity, Crohn's disease, atherosclerosis, gout, asthma, cardiovascular diseases, arthritis, hypertension, and colitis.
Author Contributions: Conceptualization, project administration, supervision, funding acquisition, writing-review and editing, resources-H.Y.; methodology, writing-original draft preparation, investigation, S.P.M.; FFAR2/3 modeling and bioinformatics analyses-P.K., and feedback on manuscript-S.T. All authors have read and agreed to the published version of the manuscript.

Funding:
We are thankful for the support provided by National Institutes of Health grants R01AG018915 and the Pepper Older Americans for Independence Center (P30AG21332), and the Department of Defense funding W81XWH-18-1-0118 and W81XWH-19-1-0236 (HY), as well funds and services provided from the Center for Diabetes, Obesity and Metabolism, Wake Forest Baptist Medical Center, and the National Center for Advancing Translational Sciences (NCATS), the National Institutes of Health-funded Wake Forest Clinical and Translational Science Institute (WF CTSI) through Grant Award Number UL1TR001420.

Conflicts of Interest:
The authors declare no conflict of interest.