SCFAs act as extracellular signaling molecules by binding to their cognate G-protein-coupled receptors (GPCRs), FFA2 and FFA3, which can bind all the three SCFAs but with discrete efficacies (Table 1) and G-protein coupling profiles. Due to their coexpression in tissues, shared endogenous ligands, and lack of selective synthetic ligands, defining their physiological roles has been challenging. However, recent studies using novel rodent models have provided an increased appreciation of the roles of these receptors in various metabolic and immune states [12].

Both FFA2 and FFA3 are expressed in islets, predominantly in the β cells in both rodents and humans [34]. Pioneering work from the Layden Laboratory along with others has established the role of these receptors in the regulation of β cell function and mass [38,39,40,41,42,43,44]. Most of these effects are based upon distinct G-protein coupling preferences of FFA2 and FFA3. Upon SCFA binding, FFA2 can couple with either Gα_q/11 and Gα_i/o, thus exerting stimulatory or inhibitory effects on cellular function, respectively. FFA3, on the other hand, couples almost exclusively with Gα_i/o, with an inhibitory tone in its signaling [36]. Accordingly, in the islet, these receptors have opposing effects on insulin secretion: in both human and mouse islets, FFA3 inhibits insulin secretion in a Gα_i/o-dependent manner [38,42,45], whereas FFA2 activation may increase [39,44,46,47] or decrease insulin secretion [45], depending upon whether it couples to Gα_q/11 or Gα_i/o. Variance observed in FFA2 activity suggests that under any given condition, the effect of FFA2 activation on insulin secretion depends upon its preferred G-protein coupling [39,44]. This calls for the development of G-protein-biased ligands for FFA2. In fact, orthosteric FFA2 agonists SCA14, SCA15, and ZINC03832747 mediate the Gα_q/11-dependent increase in mouse islets or β cell insulin secretion in contrast to the allosteric agonists CMTB and CPTB that decrease insulin secretion via Gα_i/o [39,48].

Mediation of β cell function by these receptors projects a similar profile in vivo. Whole-body deletion of FFA3 improves insulin secretion and glucose tolerance both under high fat diet induced metabolic stress [42,43,45] and a regular diet [42,43]. Correspondingly, β-cell-specific FFA3 overexpression deteriorates glucose responsiveness in mice [42]. These effects appear to be a β cell secretory phenotype, as no changes in insulin sensitivity have been observed [42,43,45]. Additionally, gene expression analysis of islets from FFA3 knockout mice [39] or β-cell-specific FFA3 overexpression mice [42] revealed complementary changes (i.e., downregulation in knockout and upregulation in overexpression model) in genes related to inflammation and immune response (such as IL1β, IL1α, CD80), besides changes in genes of calcium response and glucose utilization pathways.

Evaluation of the in vivo roles of FFA2, similar to the ex vivo data, has yielded conflicting results. Mice globally lacking FFA2 exhibited fasting hyperglycemia, reduced insulin secretion, and glucose intolerance under dietary metabolic stress [39,44]. In contrast, another study has reported a phenotype of improved glucose tolerance and enhanced insulin secretion in FFA2 knockout mice [45]. Additionally, in this same study, FFA2 and FFA3 double knockout or FFA3 knockout in combination with β-cell-specific FFA2 knockout improved glucose tolerance and insulin secretion under metabolic stress. These conflicting data may arise from differences in the G-protein coupling of activated FFA2, roles of FFA2 in other metabolically active tissues, impact of gut microbiome, and/or duration of metabolic stress, besides receptor-independent effects of SCFAs [49,50,51].

SCFA receptors, specifically FFA2, also modulate β cell mass [40,41,44]. FFA2 is required for the prenatal establishment of β cell mass, as FFA2 knockout mouse neonates and 21-day-old weanlings exhibit impaired β cell mass at birth and throughout adulthood [41]. Under conditions of dietary metabolic stress [44] and pregnancy [40], when β cells are compensating for insulin resistance, this deficiency in β cell mass is magnified. FFA2 activation, as a matter of fact, increases β cell proliferation [41,44], enhances the expression of genes involved in β cell differentiation [44], and reduces cytokine- and palmitate-induced β cell apoptosis [46,47]. FFA3, on the other hand, as a Gα_i/o-coupling receptor, may restrict β cell mass [52]. However, FFA3 knockout mice islets have been reported to be smaller with reduced proliferation and number of β cells [42], an effect not seen in a later study [43]. Similarly, β-cell-specific FFA3 overexpression in mice shows compensatory increased β cell proliferation and area [42]. Collectively, these data highlight the role of SCFA receptors FFA2 and FFA3 in modulating β cell function and mass. Importantly, defects in these two features are fundamental to the pathology of T1D.

In addition to their role within pancreatic islets, SCFA and their receptors are suggested to participate in the secretion of incretin hormones. In the upper intestine, SCFA concentrations range from 0.1 to 1 mM and are largely produced by oral microbiota [53]. By contrast, luminal SCFAs in the colon can reach levels of up to 100 mM due to the fermentation of dietary fibers via the gut microbiota. Within the intestine, SCFA receptors FFA2 and FFA3 are thought to act as sensors of these metabolites, and many important actions are carried out through this signaling.

Secreted by enteroendocrine cells (EECs) embedded within the intestinal epithelium, incretin hormones are peptide hormones that stimulate the release of insulin in response to nutrient intake, thereby lowering the level of circulating blood glucose [54]. Additionally, incretin hormones facilitate numerous postprandial metabolic functions, including lowering food intake, gastric emptying, and increasing cardiac output [55]. There are two primary incretin hormones: glucagon-like peptide-1 (GLP-1) and gastric inhibitory polypeptide (GIP). While both hormones carry out their various functions through the binding of their specific receptors (GLP-1R and GIPR, respectively) on the surface of various tissues, they contribute to the regulation of glucose metabolism in distinct mechanisms. Although both stimulate insulin release through Gα/cAMP at β cells, in islet α cells, GLP-1 suppresses glucagon, while GIP increases it [56]. Both hormones also protect β cell mass by inhibiting apoptosis. GLP-1 and GIP play important roles in the control of glucose levels after a meal via the physiological response known as the “incretin effect.” This occurs when higher levels of glucose-stimulated insulin secretion are observed when glucose is administered orally rather than intravenously, an effect that is lost in type 2 diabetes but preserved in T1D [57].

FFA2 and FFA3 are broadly expressed within EECs throughout the gastrointestinal tract. EECs are divided into subtypes based on the peptide hormone they express and secrete [58]. Using immunohistochemical analysis and a Ffar2-red fluorescent protein (RFP) reporter mouse, FFA2 has been found to colocalize with peptide YY (PYY)/GLP-1 containing L cells in rodents and humans [59,60,61,62]. Using in situ hybridization and a Ffar3-RFP reporter mouse, FFA3 expression has been confirmed in several types of intestinal cells, including PYY-positive cells [59,63,64]. However, due to the concentrated expression of FFA3 in enteric ganglia and sympathetic ganglia, it is uncertain if its effects on EEC function arise from its expression in enterocytes or are secondary to its mediation of enteric neuronal function [59,65,66]. Further, transcriptomic analysis has found high coexpression of both receptors with gip, indicating their possible involvement in mediating GIP secretion [67].

Several studies have shown that stimulation of FFA2 by SCFA results in an increased secretion of GLP-1 from EECs in the intestine. Primary intestine cells harvested from global FFA2 knockout mice showed reduced GLP-1 release in vitro, and another study by the same group found that propionate was able to stimulate GLP-1 release in vivo only in the wild-type mice [68,69]. Besides GLP-1, studies have also documented the role of FFA2 in mediating GIP [44] and PYY secretion, with the latter in both humans and mice [44,70,71]. Controversy exists for this role of FFA2, however, with some studies reporting no difference in basal- and glucose-stimulated GLP-1 levels in FFA2 knockout mice compared to control mice [40,45].

For FFA3, while there is a paucity of data regarding its role in gut hormone secretion, FFA3 knockout mice have reduced GLP-1 and PYY secretion, and primary colonic cultures derived from these mice display impaired secretory response upon SCFA stimulation [63,69]. Predictably, FFA3-specific agonist enhances GLP-1 release from primary colonic cultures [59]. FFA3 is also implicated in the inhibition of GIP secretion, an effect more likely with the predominant Gα_i/o coupling of the receptor [72].

More research is needed to ascertain the respective roles of FFA2 and FFA3 in the regulation of incretin hormones in the intestine. This includes the development of potent selective ligands and tissue-specific knockout mouse models. Some progress has already been made in this direction. Selective and potent human FFA2 inverse agonists have been developed and shown to stimulate GLP-1 secretion in the human EEC line, NCI-H716 [73]. Through a chemogenetic knock-in strategy, mice with designer receptors exclusively activated by the designer drugs (DREADD) variant of human FFAR2 replacing the mouse Ffar2 locus have been generated. DREADD activation in these mice has been shown to augment GLP-1 secretion in colonic crypt cultures and in vivo [74].

SCFAs can regulate immune cell function via two major processes, either through their cognate GPCRs such as FFA2 and FFA3 or by modulating histone deacetylase (HDAC) activity [75]. Here, we emphasize the first role. With the highest SCFA concentration, a rich and diverse population of immune cells with the majority expressing FFA2 and few, such as dendritic cells expressing FFA3, the gut is an important site where SCFAs can impact the immune cells through these receptors.

FFA2 has been reported to affect neutrophil chemotaxis in gut inflammation models, where a deficiency in FFA2 increases neutrophil infiltration to sites of inflammation [76,77]. Accordingly, engagement of FFA2 with acetate mitigates such a response [76,77]. Similar FFA2-dependent neutrophil-driven responses are seen in pulmonary and joint inflammation models [77]. Neutrophil FFA2 engagement by SCFAs in the presence of allosteric modulators can also activate NADPH oxidase and enhance the production of reactive oxygen species, which is deemed necessary for phagocytic activity [77,78]. FFA2-derived neutrophil responses are required for the regulation of inflammatory responses. As recently shown, FFA2 activity promotes coordination between neutrophils and colonic group 3 innate lymphoid cells (ILC3). In neutrophils, inflammasome activation helps in pathogen clearance with the concomitant enhancement of IL1β production, where IL1β leads to IL22 production from ILC3, driving gut repair mechanisms [79]. FFA2 can also promote ILC3 expansion and function independent of neutrophils [80]. Immunomodulation by neutrophil FFA2, thus, appears to strike a balance between pro- and anti-inflammatory effects, potentially in a disease-centric manner.

SCFAs through their HDAC inhibitory activity are considered to be the main players in maintaining the regulatory T cell (Treg) pools [75]. These effects are, in part, mediated through FFA2. It is suggested that FFA2 exerts immune suppression by regulating the number, function, and differentiation of Tregs [81,82]. FFA2 also modulates gut homeostasis by modifying immunoglobulin A (IgA) production [83] and through direct effects on inflammasome activation in intestinal epithelial cells [84].

The role of FFA3 in immune regulation is less explored, likely due to its limited expression in immune cells. It has been suggested to be involved in the resolution of lung inflammation through effects on macrophage and dendritic cell populations [85] and in promoting thymic Treg differentiation in mouse offspring [86]. The function and expansion of CD8⁺ T cells can also be regulated by FFA3, and this has been suggested to aid the resolution of influenza infection [87]. Both FFA3 and FFA2 have been suggested to enhance T cell memory [88], with the engagement of both receptors by butyrate appearing to mediate this effect. However, as the mouse isoform of FFA2 shows a low affinity for butyrate [31], the use of synthetic ligands is required to further delineate the role of the two receptors in this process.

Obliterating the GM in mice wipes off some of the physiological effects discussed above. For instance, in germ-free (GF) mice, antigen-activated T cells fail to transition to memory cells [88]. As the GM does not directly interact with the host cells except at the gut mucosal surfaces, these effects are likely indirect, being mediated via GM-derived factors such as SCFAs. The GM-derived SCFAs acting through their receptors FFA2 and FFA3 project the link, GM→SCFAs→FFA2 and FFA3.

Highlighting this relationship, whole-body FFA2 and FFA3 knockout mice have different gut microbiota profiles as compared to wild-type mice [44,63,82,84,89]. As expected, differences in fecal SCFA profiles accompany these differences in GM profiles due to adaptation to receptor deficiency. More direct evidence for roles of FFA2 and FFA3 in the GM→SCFAs→Receptor link is provided by immune function and GM studies. Both GF and FFA2 knockout mice exhibit a dysregulated immune response to induced colitis, gout, and arthritis [77,90]. While this response is mitigated by acetate supplemented drinking water in GF mice [77], FFA2 knockout mice remain refractory to acetate treatment [81].

Likewise, high-fiber diets that tend to increase GM function and SCFA levels in wild-type mice fail to promote gut homeostasis, alleviate food allergy, and prevent respiratory viral infection in FFA2 knockout mice [82,84,91]. Similar findings have been reported for FFA3. High fiber diet mediated protection against allergic airway disease and influenza virus is not observed in FFA3 knockout mice [85,87].

Metabolic studies have also highlighted the importance of the GM→SCFAs→FFA2 and FFA3 relationship. Reduced adiposity and PYY levels in FFA3 knockout mice are GM dependent, with the effect being lost in GF FFA3 knockout mice [63]. Similarly, GM-derived SCFAs mediate suppression of GIP secretion in FFA3 dependent manner, an effect lost in GF, antibiotic-treated mice (pseudo-GF), and FFA3 knockout mice [72]. In mice, a low-fiber diet or GF status during pregnancy increases the vulnerability of the offspring to obesity and insulin resistance later in life [92]. This effect could be rescued by propionate treatment or high-fiber feeding but not in absence of FFA3 or FFA2. Furthermore, FFA3 and FFA2 SCFA signaling was found to be responsible for normal embryonic development of neural, pancreatic β cell and intestine tissues. Collectively, these data suggest that the GM modulates metabolic and immune features affecting health via SCFA-FFA2 and SCFA-FFA3 axes.