Free fatty acid receptor 3 (FFAR3, also termedGPR41)protein is aG protein coupled receptor (i.e., GPR or GPCR) that in humans is encoded by theFFAR3gene (i.e.,GPR41 gene).[5] GPRs reside on cell surfaces, bind specific signaling molecules, and thereby are activated to trigger certain functional responses in their parent cells. FFAR3 is a member of thefree fatty acid receptor group of GPRs that includesFFAR1 (i.e., GPR40),FFAR2 (i.e., GPR43), andFFAR4 (i.e., GPR120).[6] All of these FFARs are activated byfatty acids. FFAR3 and FFAR2 are activated by certainshort-chain fatty acids (SC-FAs), i.e., fatty acids consisting of 2 to 6carbonatoms[7] whereas FFFAR1 and FFAR4 are activated by certain fatty acids that are 6 to more than 21 carbon atoms long.[8][9][10]Hydroxycarboxylic acid receptor 2 is also activated by a SC-FA that activate FFAR3, i.e.,butyric acid.[11]
The humanFFAR3 gene is located next to theFFAR2 gene atlocus 13.12 on the long (i.e., "q") arm ofchromosome 19 (location abbreviated as 19q13.12).
Studies have reported that humans express FFAR3 in their: (a)enteroendocrineL cells andK cells of theintestines;[10] (b)endothelium of blood vessels in thefrontal cortex of the brain,[12] pancreatic β-cells,[13] andadipose. i.e., fat, tissue (but not in mouse adipose tissue);[14] (c) the vascular endothelium of themyometrium, the epithelium of theamnion,chorion andplacenta, and certainimmune cells in these tissues of pregnant women;[15] (d) thehippocampus of the brain;[16] (e)sympathetic ganglia, i.e.,autonomic ganglia of thesympathetic nervous system;[17][18] (f) certain types of immune cells, i.e., bloodmonocytes (but not mouse monocytes),basophils,[19][20][21]dendritic cells derived from human monocytes isolated fromwhole blood, and the tissues containing these blood cells, i.e., thebone marrow,spleen,lymph nodes, andthymus;[22] and (g)alveolar macrophages, andmacrophages in various other tissues;[19] and (h) certainimmortalised cell lines, i.e., MCF-7 breast cancer,[23] HCT116 colorectal cancer,[24] HEK293 embryonic kidney,[25][21]U937leukemicpromonocyte,THP-1 leukemic monocyte, EoL-1 leukemiceosinophil,Jurcat leukemicT lymphocyte, MOLT-4 Tlymphoblast leukemic, andHL60acute myeloid leukemia cells (but only when the HL60 cells are pre-treated withphorbol 12-myristate 13-acetate to promote theircellular differentiation).[21] As noted, the expression of FFAR3 in the cells and tissues of animals are not always the same as those in humans.
Free fatty acid receptor 3 is a member of theG protein-coupled receptor (GPCR) superfamily, characterized by its seven transmembranealpha-helices. FFAR3 shares significant sequence similarity withFFAR2 but exhibits distinct structural features that influence its ligand specificity and signaling. The receptor's orthosteric binding pocket is formed by transmembrane helices 3, 4, and 5, with key conserved residues such as Arg-185 (5.39), Arg-255 (7.35), His-140 (4.56), and His-242 (6.55) contributing to the binding and recognition ofshort-chain fatty acids (SCFAs) likeacetate,propionate, andbutyrate. Notably, FFAR3's binding cavity is more hydrophilic compared to its close relative FFAR2, which affects its ligand interactions. The second extracellular loop is important in modulating ligand selectivity and receptor activation. Additionally, the presence of a His-45 (2.40) is predicted to coordinate allosteric modulators.[26][27]
The human FFAR3 and FFAR2 proteins consist of 346 and 330 amino acids, respectively,[17] and share about a 40%amino acidsequence homology.[28] The two FFARs have been found to form aheteromer complex (i.e., FFAR3 and FFAR2 bind to each other and are activated together by a SC-FA). When stimulated by a SC-FA, the cells expressing both FFAR3 and FFAR2 may form this heterodimer and thereby activatecell signaling pathways and mount responses that differ from those of cells expressing only one of these FFARs.[25] The formation of GPR43-GPR41 heterodimers has not been evaluated in most studies and may explain otherwise conflicting results on the roles of FFAR3 and FFAR2 in cell function.[10][29][30] Furthermore, SC-FAs can alter the function of cells independently of FFAR3 and FFAR2 by altering the activity of cellularhistone deacetylases which regulate thetranscription of various genes or by altering metabolic pathways which alter cell functions.[31][19] Given these alternate ways for SC-FAs to activate cells as well as the ability of SC-FAs to activate FFAR2 or, in the case of butyric acid, hydroxycarboxylic acid receptor 2, the studies reported here focus on those showing that the examined action(s) of an SC-FA is absent or reduced in cells, tissues, or animals that have no or reduced FFAR3 activity due respectively toknockout (i.e., removal or inactivation) orknockdown (i.e., reduction) of the FFAR3 protein gene, i.e., theFfar3 gene in animals orFFAR3 gene in humans.
L cells are enteroendocrine cells, i.e., specialized cells that secretehormones directly into the circulation. L cells reside in theepithelium of thegastrointestinal tract, particularly theterminal ileum andcolon. They are stimulated tosecretePYY (also termed peptide YY) andGLP-1 (also termed glucagon-like peptide-1) by the SC-FAs that accumulate inside the intestines after feeding. L cells express FFAR3 and/or FFAR2.[32]Ffar3 andFfar2 gene knock out mice show reduced secretions of GLP-1 and PYY.[33]
Leptin is apeptidehormone released by adipose tissue that triggerssatiety and thereby tends to reduce or stop further food intake and the development of obesity. It also plays a role in female reproductive function,lipolysis (e.g., the breakdown oftriglycerides into their component free fatty acids andglycerol), the growth of fetuses, inflammation, andangiogenesis (i.e., the formation of new blood vessels from pre-existing blood vessels).[34] While studies have suggested that the SC-FA-induced activation of FFAR3 leads to the secretion of leptin from the white adipose tissue of intact animals and the fat tissue isolated from human tissues, other studies have suggested that FFAR2 rather than FFAR3 is responsible for the SC-FA-induced release of leptin from fat tissue. A systematic review of the published studies on this issue concluded that SC-FA-induced activation of FFAR3 is likely responsible for the SC-FA-induced release of leptin from cultured fat tissue taken from animals. However, the data were insufficient to support a role for FFAR3 in the release of leptin from cultured human fat tissues or the fat tissue of intact animals. The role of FFAR3 stimulation of leptin release in appetite suppression and obesity needs further study.[35]
In a high-fat diet-induced obesity model offatty liver disease (i.e., excessive buildup of fat in the liver), mice fed a diet that increased intestinal levels of SC-FAs showed reductions in their livers' synthesis oflipids,triglyceride levels, and weights. These reductions did not occur inFfar3 gene knockout mice but did occur inFfar2 gene knocked-out mice. These results indicate that the SC-FA-induced activation of FFAR3 suppresses the liver's accumulation of fatty acids that underlies the development of fatty liver disease in this mouse model.[36]
Other studies have found thatFfar3 gene knockout mice showed less weight gain than wild-type mice under standard laboratory conditions, but this difference was lost in mice reared under germ-free conditions (i.e., which causes the mice to have lower intestinal and tissue levels of SC-FAs).[28] These findings indicated that the activation of FFAR3 but not FFAR2 by SC-FAs protects against developing fatty liver disease in mice.[28][37]
Individuals withtype 2 diabetes, which accounts for 90% of all diabetes cases, have decreases in the proliferation, maturation, and activity of theirpancreatic isletinsulin-secretingbeta-cells as well as the potency of insulin's actions. These decreases result in reduced insulin secretion,hyperglycemia, and the many other afflictions associated with this disorder.
Studies in the past have reported that the activation of FFAR3 reduced the insulin secreted by (1) human and mouse beta cellsin vivo, (2) cultured human and murine beta cell-containingpancreatic islets, and (3) cultured beta cell lines.[17][38] These studies showed that acetic acid-induced inhibition of insulin secretion by mouse pancreatic islets did not occur in islets that had both of theirFfar3 andFfar2 genes knocked out but had no effect on insulin secretion in islets that had only one of the two genes knocked out.[38]
An early study showed that theintravenous injection of propionic acid into mice induced a brief (<5 min)hypotensive response as defined by drops in theirmean arterial pressures. This response was reduced in mice that had one of their twoFfar3 genes knocked out and absent in mice that had bothFfar3 genes knocked out.[39][40]
A subsequent study reported thatFfar3 gene knockout mice developed abnormally highpulse pressures (i.e.,systolic minus diastolic blood pressures) as well as increased amounts of cardiaccollagen andelastinconnective tissue and increased cardiac stiffness as evidenced by a reduced rate of heart muscle relaxation measured bypressure-volume loop analysis tau levels.[41]
Studies have shown that compared towild type mice,Ffar3 gene knockout mice have:a) significantly smaller-sizedsympathetic nervous system ganglia as judged by measurements of this systems' largest ganglia, thesuperior cervical ganglion;b) significantly slowerheart rates; andc) significantly lower norepinephrine levels in their blood plasma. (Norepinephrine is aneurotransmitter that is released by sympathetic nervous systemneurons and among other actions increases heart rate and total bodyenergy expenditure.) Furthermore, the treatment of wild type mice with propionic acid increased their heart rates but did not do so inFfar3 gene knockout mice.[42][18] Finally, the offspring ofFfar3-gene knockout mice had slower heart rates (as well as lower body temperatures) than the offspring of wild type mice.[10] These findings indicate that FFAR3 regulates heart rates and energy expenditure in mice. Studies are needed to determine if it does so in humans.[42][18]
Certain bacteria in thegastrointestinal tractferment fecal fiber into SC-FAs andexcrete them as waste products. The excreted SC-FAs enter the gastrointestinal walls, diffuse into theportal venous system, and ultimately flow into thesystemic circulation. During this passage, they can activate the FFAR3 on cells in the intestinal wall as well as throughout the body.[42] This activation may: suppress the appetite for food and thereby reduce overeating and the development of obesity;[34][43] inhibit theliver's accumulation of fatty acids and thereby the development offatty liver diseases;[36] decreaseblood pressure and thereby the development ofhypertension and hypertension-related cardiac diseases;[39] modulateinsulin secretion and thereby the development and/or symptoms oftype 2 diabetes;[38] reduceheart rate andblood plasmanorepinephrine levels and thereby lower total bodyenergy expenditures;[42] and suppress or delay the development of allergic asthma.[44]
The specific types of bacteria in the intestines can be modified to increase the number which make SC-FAs by using foods that stimulate the growth of these bacteria (i.e.,prebiotics), preparations of SC-FA-producing bacteria (i.e.,probiotics), or both methods (seesynbiotics).[45] Individuals with disorders that are associated with low levels of the SC-FA-producing intestinal bacteria may show improvements in their conditions when treated with prebiotics, probiotics, or synbiotics while individuals with disorders associated with high levels of SC-FAs may show improvements in their conditions when treated with methods, e.g.,antibiotics, that reduce the intestinal levels of these bacteria.[42][46] (For information on these treatments seeDisorders treated by probiotics andDisorders treated by prebiotics). In addition, drugs are being tested for their ability to act more usefully, potently, and effectively than SC-FAs in stimulating or inhibiting FFAR3 and thereby for treating the disorders that are inhibited or stimulated, respectively, by SC-FAs.[47]
The SC-FAs that activate FFAR3 includeproprionic, butyric,acetic,[32]valeric[42]caproic,[46] andformic acids.[37] (Confusingly, butyric acid also activates hydroxycarboxylic acid receptor 2[37] andβ-hydroxybutyric acid has been reported to stimulate or inhibit FFAR3.[48]) FFAR2 is activated by many of these same SC-FAs but differs from FFAR3 in its relativebinding affinities for them. In humans, the binding affinity ranking of FFAR3 is: propionic = butyric = valeric > acetic > formic acids (acetic[32] and formic[37] acids have very low binding affinities for, and therefore must be at extremely high levels to activate, FFAR3); FFAR2's relative binding affinity ranking for these SC-FAs is: acetic = propionic > butyric > valeric = formic acids.[37] AR420626 (a derivative of an older compound 1-MCPC)[17][49] has been reported to be a selective activator of FFAR3[50] but has also been reported to inhibit the activation of FFAR3.[42] Its actions require further characterizations.[42] AR399519 and CF3-MQC have been reported to inhibit the activation of mouse FFAR3; the actions of these agents also require further characterizations.[17]
Studies have shown thatFfar3 gene knockout mice fed a high fat diet have significant increases in their food intake and body weights compared towild-type (i.e., genetically unaltered) mice.[42] These and other studies in animals suggest that the activation of FFAR3 and FFAR2 on L cells by SC-FAs triggers the release of PYY and GLP-1, both of which, among various other activities, inhibit gastric emptying and thereby suppress appetite and the development of obesity.[42][43] Further studies are needed to determine if FFAR3 plays a similar role in human satiety and obesity.[42]
Semaglutide, also called Wegovy, is a peptide with a modified GLP-1-like structure. It strongly stimulatesGLP-1 receptors and thereby suppresses appetite and promotes weight loss in obese individuals.[51]
Studies in mice have shown that activation of FFAR3 by short-chain fatty acids (SC-FAs) suppresses liver lipid synthesis, reduces triglyceride accumulation, and decreases liver weight in models of diet-induced obesity. Mice lacking theFfar3 gene fail to exhibit these protective effects, suggesting a critical role for FFAR3 in preventing excessive hepatic fat accumulation. These findings support further research to determine whether FFAR3 functions similarly in humans and whether FFAR3 activators could be developed as potential treatments for human fatty liver diseases, includingnon-alcoholic fatty liver disease.Koh A, De Vadder F, Kovatcheva-Datchary P, Bäckhed F (June 2016)."From Dietary Fiber to Host Physiology: Short-Chain Fatty Acids as Key Bacterial Metabolites".Cell.165 (6):1332–1345.doi:10.1016/j.cell.2016.05.041.PMID 27259147.S2CID 8562345.
In contrast to previous studies, a recent study ofstreptozotocin-induced and high-fat diet-induced murine models of diabetes found that the FFAR3-activating drug, AR420626, increased blood plasma insulin levels and stimulated skeletal muscle to take up glucose and thereby improvedglucose tolerance test results.[52]
Other recent studies have reported that activated FFAR3 may reduce, increase, or have little effect on insulin secretion depending on1) the levels of ambient glucose and FFAR3 activators studied, (2) human or animal species studied, (3) age of the animals studied, and (4) variations in the proportions ofalpha, beta, anddelta cells in the pancreatic islets of humans. The role of FFAR3 in human as well as animal models of insulin secretion and diabetes requires further studies.[13]
In a model where FFAR2 and FFAR3 were both deleted, these animals had an exaggerated response to hypertension, with higher fibrosis in the kidney; this was explained by a breakdown in the gut epithelial barrier and activation of the immune system via LPS/TLR4 binding.[53]
Studies in humans have found that individuals undergoing hemodialysis using dialysis solutions that contain acetic acid often develop hypotension; the role of FFAR3 in this response, if any, was not investigated.[39][40] A study of 69 individuals (55.1% women, mean age 59.8 years) found that arterial stiffness was associated with lower levels of FFAR3 and FFAR2 in circulating bloodimmune cells (particularlyregulatory T cells which are known to be protective in murine models of hypertension[54]).[55]
Overall, the mouse studies suggest that FFAR3 contributes to suppressing hypertension and its subsequent effects on the heart in mice[41] and that SC-FA-activated FFAR3 and/or FFAR2 may have vasodilatory actions and thereby suppress the development of hypertension and hypertension-induced arterial stiffness in humans. Further studies in humans are needed to investigate the latter possibilities.[41][54]
Asthma may be atopic (i.e., symptoms triggered byallergens) or non-atopic (i.e., symptoms triggered by non-allergenic factors such as cold air). The studies reported here relate to allergen-induced asthma. Mice fed a diet that lowers their SC-FAs levels and then given intranasal injections of dust mite extract developeddust mite allergyasthma reactions to the injections. Theirrespiratory tract airways had increased numbers ofeosinophils andgoblet cells as well as excessive levels ofmucus; their lung tissue levels ofinterleukin-4,interleukin-5,interleukin-13, andinterleukin-17A andserumimmunoglobulin E levels were elevated; and theirairway resistance response to abronchial challenge test was high. In contrast, mice fed a diet that increased their SC-FAs levels developed less of these responses to the extract. Furthermore, mice on the SC-FA lowering diet that were given propionic acid also had far less of these responses to the mite extract. And,Ffar3 (but notFfar2) gene knockout mice on the low SF-FA diet did not show rises in their lung airway eosinophil levels in response to the mite extract (this was the only parameter of asthma reported in the knockout studies). These finding implicate propionic acid and FFAR3 in the suppression of asthma allergic reactions to mite extract in mice.[56]
A second study investigated the effects that aninulin-rich diet (which raises bodily SC-FA levels) feed to rats had on their offsprings' development of asthma. Pregnant rats were feed a normal or inulin-rich diet for 1 week. Their offspring were injected with ovalbumin on days 21 and 29 after birth, 7 days later challenged with aerosolized ovalbumin, and on the next day examined for their responses to the aerosol. Compared to the offspring of mothers on a normal diet, the offspring of mothers on the inulin diet had lower levels of lung inflammatory cells, lesshistological evidence of allergic lung disease, lower lung tissue levels of immunoglobulin E, interleukin-4, and interleukin-17, and significantly elevated lung levels of FFAR3 (Lung FFAR2 levels were not significantly elevated). These results indicate that a diet promoting the production of SC-FAs in pregnant rats suppresses the development of asthmatic disease in their offspring; this suppression may involve FFAR3.[44] In a similar study, newborn mice were feed breast milk from mothers who had drunk pure water or water containing a SC-FA. After 3 weeks, the newborns were weaned off the mothers' milk, feed plain water, and 3 weeks thereafter sensitized to and challenged by injection of mite extract into theirtracheas. Mothers who drank pure water or water laced with acetic or butyric acid and sensitized to the mite extract had asthma signs after challenge with the extract whereas mothers who drank propionic acid-laced water had far less of these signs. Furthermore,Ffar3 gene knockout mothers who drank propionic acid-laced water and then sensitized to the mite extract had asthma signs similar to these in wild type mothers challenged with the extract. The study also found that the offspring of mothers who drank propionic acid-laced water had fewer eosinophils andT helper cells in their airways than the offsprings of mothers who drank pure water, acetic acid-laced water, or butyric acid-laced water. Propionic acid-laced water did not suppress the development of asthma inFfar3 gene knockout offsprings. These results indicate that ingestion of propionic acid, but not acetic or butyric acid, suppresses the development of allergic asthma in adult as well as newborn rats and does so by a FFAR3-dependent mechanism. The studies also indicate that the milk of pregnant rats who consumed propionic acid-laced but not those who drank pure water reduced the susceptibility of newborn rats to developing allergic asthma by a mechanism dependent on FFAR3 in the mothers as well as the offsprings. These findings support further studies to determine if propionic acid or other FFAR3 activators would be useful for preventing and/or treating asthma in humans.[19][57]
A study of humans living on European farms or in non-farm rural areas reported that the fecal levels of butyric but not acetic acid in 12 month old children who had not develop asthma by the time they entered the first year of school were significantly higher than these levels in children who did develop asthma by the school entry age.[58] A study conducted in Canada reported that the levels of fecal acetic acid (but not butyric or propionic acid) were lower in 3 month old human infants who were predicted to have asthma by school age (based on a Phylogenetic Investigation of Communities by Reconstruction of Unobserved States predictionalgorithm) compared to infants predicted not to do so.[59] Finally, a study conducted in Japan found that the fecal levels of propionic but not acetic or butyric acid trended lower in 1 month old human infants that developed asthma by age 5 than in infants that did not develop asthma. The fecal levels of propionic as well acetic and butyric acid obtained from 1 week-, 1 year, and 5-year-old infants did not show this trend.[57] The different SC-FA implicated in suppressing asthma in these three studies may reflect dietary or other differences between the populations of the three countries. In all events, the studies allow that, based on rodent studies, FFAR3 may mediate these SG-FA actions and, based on human studies, SC-FAs may act to suppress, or at least delay) the onset of, asthma in children. Further studies are needed to determine if FFAR3 is involved in the apparent actions of the cited SC-FAs in the development of asthma in children.[57]
