Omega-3 Fatty Acids and FFAR4

The deorphanization of the free fatty acid (FFA) receptors FFA1 (GPR40), FFA2 (GPR43), FFA3 (GPR41), GPR84, and GPR120 has made clear that the body is capable of recognizing and responding directly to nonesterified fatty acid of virtually any chain length. Colonic fermentation of dietary fiber produces high concentrations of the short-chain fatty acids (SCFAs) acetate, propionate and butyrate, a process which is important to health. The phylogenetically related 7-transmembrane (7TM) receptors free fatty acid receptor 2 (FFA2) and FFA3 are activated by these SCFAs, and several lines of evidence indicate that FFA2 and FFA3 mediate beneficial effects associated with a fiber-rich diet, and that they may be of interest as targets for treatment of inflammatory and metabolic diseases. FFA2 is highly expressed on immune cells, in particular neutrophils, and several studies suggest that the receptor plays a role in diseases involving a dysfunctional neutrophil response, such as inflammatory bowel disease (IBD). Both FFA2 and FFA3 have been implicated in metabolic diseases such as type 2 diabetes and in regulation of appetite. More research is however required to clarify the potential of the receptors as drug targets and establish if activation or inhibition would be the preferred mode of action. The availability of potent and selective receptor modulators is a prerequisite for these studies. The few modulators of FFA2 or FFA3 that have been published hitherto in the peer-reviewed literature in general have properties that make them less than ideal as such tools, but published patent applications indicate that better tool compounds might soon become available which should enable studies critical to validate the receptors as new drug targets.

Free fatty acid receptor 4 (FFAR4)/GPR120 comprises a receptor for medium‐ and long‐chain fatty acids. We previously identified phytosphingosine (PHS) as a novel ligand of FFAR4. Although many natural FFAR4 ligands have carboxyl groups, PHS does not, thus suggesting that binding to FFAR4 is driven by a completely different mechanism than other natural ligands such as α‐linolenic acid (ALA). To test this hypothesis, we performed docking simulation analysis using a FFAR4 homology model based on a protein model derived from the crystal structure of activated turkey beta‐1 adrenoceptor. The docking simulation revealed that the probable hydrogen bonds to FFAR4 differ between various ligands. In particular, binding was predicted between R264 of the FFAR4 and the oxygen of the carboxylate group in ALA, as well as between E249 of the FFAR4 and the oxygen of the hydroxy group at the C4‐position in PHS. Alanine substitution at E249 (E249A) dramatically reduced PHS‐induced FFAR4 activation but demonstrated a weaker effect on ALA‐induced FFAR4 activation. Kinetic analysis and K m values clearly demonstrated that the E249A substitution resulted in reduced affinity for PHS but not for ALA. Additionally, we observed that sphingosine, lacking a hydroxyl group at C4‐position, could not activate FFAR4. Our data show that E249 of the FFAR4 receptor is crucial for binding to the hydroxy group at the C4‐position in PHS, and this is a completely different molecular mechanism of binding from ALA. Because GPR120 agonists have attracted attention as treatments for type 2 diabetes, our findings may provide new insights into their development.

G protein-coupled receptors have been well described to contribute to the regulation of glucose-stimulated insulin secretion (GSIS). The short-chain fatty acid-sensing G protein-coupled receptor, free fatty acid receptor 2 (FFAR2), is expressed in pancreatic β-cells, and in rodents, its expression is altered during insulin resistance. Thus, we explored the role of FFAR2 in regulating GSIS. First, assessing the phenotype of wild-type and Ffar2(-/-) mice in vivo, we observed no differences with regard to glucose homeostasis on normal or high-fat diet, with a marginally significant defect in insulin secretion in Ffar2(-/-) mice during hyperglycemic clamps. In ex vivo insulin secretion studies, we observed diminished GSIS from Ffar2(-/-) islets relative to wild-type islets under high-glucose conditions. Further, in the presence of acetate, the primary endogenous ligand for FFAR2, we observed FFAR2-dependent potentiation of GSIS, whereas FFAR2-specific agonists resulted in either potentiation or inhibition of GSIS, which we found to result from selective signaling through either Gαq/11 or Gαi/o, respectively. Lastly, in ex vivo insulin secretion studies of human islets, we observed that acetate and FFAR2 agonists elicited different signaling properties at human FFAR2 than at mouse FFAR2. Taken together, our studies reveal that FFAR2 signaling occurs by divergent G protein pathways that can selectively potentiate or inhibit GSIS in mouse islets. Further, we have identified important differences in the response of mouse and human FFAR2 to selective agonists, and we suggest that these differences warrant consideration in the continued investigation of FFAR2 as a novel type 2 diabetes target.

Free fatty acid receptor 2 promotes cardiomyocyte hypertrophy by activating STAT3 and GATA4

Free fatty acid receptor 3 (FFA3) is a recently-deorphanized G-protein-coupled receptor. Its ligands are short-chain fatty acids (SCFAs), which are key nutrients derived from the gut microbiome fermentation process that play diverse roles in the regulation of metabolic homeostasis and glycemic control. FFA3 is highly expressed within the intestine, where its role and its effects on physiology and metabolism are unclear. Previous in vivo studies involving this receptor have relied on global knockout mouse models, making it difficult to isolate intestine-specific roles of FFA3. To overcome this challenge, we generated an intestine-specific knockout mouse model for FFA3, Villin-Cre-FFA3 (Vil-FFA3). Model validation and general metabolic assessment of male mice fed a standard chow diet revealed no major congenital defects. Because dietary changes are known to alter gut microbial composition, and thereby SCFA production, an obesogenic challenge was performed on male Vil-FFA3 mice and their littermate controls to probe for a phenotype on a high-fat, high-sugar "Western diet" (WD) compared with a low-fat control diet (CD). Vil-FFA3 mice versus FFA3fl/fl controls on WD, but not CD, were protected from the development of diet-induced obesity and exhibited significantly less fat mass as well as smaller adipose depositions and adipocytes. Although overall glycemic control was unchanged in the WD-fed Vil-FFA3 group, fasted glucose levels trended lower. Intestinal inflammation was significantly reduced in the WD-fed Vil-FFA3 mice, supporting protection from obesogenic effects. Furthermore, we observed lower levels of gastric inhibitory protein (GIP) in the WD-fed Vil-FFA3 mice, which may contribute to phenotypic changes. Our findings suggest a novel role of intestinal FFA3 in promoting the metabolic consequences of a WD, including the development of obesity and inflammation. Moreover, these data support an intestine-specific role of FFA3 in whole body metabolic homeostasis and in the development of adiposity.NEW & NOTEWORTHY Here, we generated a novel intestine-specific knockout mouse model for FFA3 (Vil-FFA3) and performed a comprehensive metabolic characterization of mice in response to an obesogenic challenge. We found that Vil-FFA3 mice fed with a Western diet were largely protected from obesity, exhibiting significantly lower levels of fat mass, lower intestinal inflammation, and altered expression of intestinal incretin hormones. Results support an important role of intestinal FFA3 in contributing to metabolism and in the development of diet-induced obesity.

AimsMolecular imaging of the free fatty acid receptor 1 (FFAR1) would be a valuable tool for drug development by enabling in vivo target engagement studies in human. It has also been suggested as a putative target for beta cell imaging, but the inherent lipophilicity of most FFAR1 binders produces high off-target binding, which has hampered progress in this area. The aim of this study was to generate a suitable lead compound for further PET labeling.MethodsIn order to identify a lead compound for future PET labeling for quantitative imaging of FFAR1 in human, we evaluated tritiated small molecule FFAR1 binding probes ([3H]AZ1, [3H]AZ2 and [3H]TAK-875) for their off-target binding, receptor density and affinity in human pancreatic tissue (islets and exocrine) and rodent insulinoma.Results[3H]AZ1 showed improved specificity to FFAR1, with decreased off-target binding compared to [3H]AZ2 and [3H]TAK-875, while retaining high affinity in the nanomolar range. FFAR1 density in human islets was approximately 50% higher than in exocrine tissue.ConclusionsAZ1 is a suitable lead compound for PET labeling for molecular imaging of FFAR1 in humans, due to high affinity and reduced off-target binding.

Short-chain fatty acids (SCFAs) produced by fermentation from prebiotics not only provide energy but also activate cell membrane receptors, thereby contributing to the maintenance of homeostasis in the human body. Recently, free fatty acid receptor 2 (FFAR2), which uses SCFAs as ligands, was found to exert oncoprotective effects on several types of neoplasia. This study examined whether SCFAs have oncoprotective effects on uterine cervical neoplasia. Immunohistochemical analysis revealed that FFAR2 was expressed in atypical cells and cancer cells of cervical neoplasia. Moreover, reverse transcription polymerase chain reaction showed that FFAR2 was expressed in a human cervical cancer cell line, HeLa. We also found that SCFAs inhibited the proliferation of HeLa cells, and a FFAR2 antagonist, GLPG0974, used to suppress the binding of SCFAs significantly restored the cell viability of HeLa cells blocked by acetic acid treatment. These results suggest that ingestion of prebiotics and the resulting production of SCFAs may play an oncoprotective role against uterine cervical neoplasia via FFAR2 expression.

The free fatty acid receptor 3 (FFA3) is a nutrient sensor of gut microbiota-generated nutrients, the short-chain fatty acids. Previously, we have shown that FFA3 is expressed in β-cells and inhibits islet insulin secretion ex vivo. Here, we determined the physiological relevance of the above observation by challenging wild-type (WT) and FFA3 knockout (KO) male mice with 1) hyperglycemia and monitoring insulin response via highly sensitive hyperglycemic clamps, 2) dietary high fat (HF), and 3) chemical-induced diabetes. As expected, FFA3 KO mice exhibited significantly higher insulin secretion and glucose infusion rate in hyperglycemic clamps. Predictably, under metabolic stress induced by HF-diet feeding, FFA3 KO mice exhibited less glucose intolerance compared with the WT mice. Moreover, similar islet architecture and β-cell area in HF diet-fed FFA3 KO and WT mice was observed. Upon challenge with streptozotocin (STZ), FFA3 KO mice initially exhibited a tendency for an accelerated incidence of diabetes compared with the WT mice. However, this difference was not maintained. Similar glycemia and β-cell mass loss was observed in both genotypes 10 days post-STZ challenge. Higher resistance to STZ-induced diabetes in WT mice could be due to higher basal islet autophagy. However, this difference was not protective because in response to STZ, similar autophagy induction was observed in both WT and FFA3 KO islets. These data demonstrate that FFA3 plays a role in modulating insulin secretion and β-cell response to stressors. The β-cell FFA3 and autophagy link warrant further research.

Free fatty acid receptor-4 regulates T-cell-mediated allogeneic reaction through activating an aryl hydrocarbon receptor pathway.

Free fatty acid receptor 4 (FFA4) is encouraged by extended-chain fatty acids and has gained recognition as a promising agent for the treatment of diabetes mellitus (DM) treatment. Research indicates that FFA4 functions as a receptor for detecting unsaturated long-chain fatty acids, shedding light on the cellular pathways through which these fatty acids influence various biological functions. These mechanisms include the secretion of digestive tract peptide hormones and the control of glucose level. Activation of FFA4 is linked to enhanced insulin susceptibility and decreased release of glucagon-like peptide 1 from enteroendocrine cell types, making it an appealing target for developing therapies for overweight and diabetes. Agonists of FFA4 have demonstrated potential in enhancing satiety, stimulating incretin release, mitigating inflammatory metabolic responses, and boosting insulin sensitivity. As a result, drugs targeting FFA4 are anticipated to become available soon. This review explores FFA4’s role in managing key metabolic processes, such as adipogenesis, lipid metabolism, and glucose regulation. It also assesses the potential of FFA4-based therapies for treating DM, emphasizing the progress and future directions in this evolving field of metabolic research.

Free fatty acid receptor 2 (FFA2), also known as GPR43, is activated by short-chain fatty acids (SCFAs) that are mainly produced by the gut microbiota through the fermentation of undigested carbohydrates and dietary fibers. FFA2 currently appears to be a potential target in the management of obesity, diabetes, inflammatory diseases, and cancer. In the study, a series of novel phenylthiazole-carboxamido acid derivatives has been synthesized and evaluated as potential orthosteric FFA2 ligands for the study of structure-activity relationships. Compound 6e was found to exhibit the twofold potent agonistic activity in the stable hFFA2-transfected CHO-K1 cells (EC50 = 23.1 μm) as that of positive control propionate (EC50 = 43.3 μm). We also reported the results of mutagenesis studies based on the crystal structure of hFFA1 bound to TAK-875 at 2.3 Å resolution to identify important residues for orthosteric agonist 6e inducing FFA2 activation.

FFAR4 (free fatty acid receptor 4) has emerged as a target for preventing cardiovascular disease through its ability to control macrophage inflammation and foam cell formation. Previous studies have shown that FFAR4 activation can protect against the accumulation of arterial plaque buildup in atherosclerotic animal models. The goal of our study is to test the hypothesis that FFAR4 deficiency will increase atherosclerotic plaque development in apoE-/- mice. Male and female apoE-/-/Ffar4-/- mice and their apoE-/- controls were fed a Western diet for 8 or 16 weeks to assess early and advanced atherosclerotic lesions, respectively. At the end of each study, atherosclerotic plaque severity was determined by analyzing the aortic sinus lesion area of the heart and the en face lesion area of the aortic arch. Following 8 weeks of Western diet feeding, lesions from apoE-/-/Ffar4-/- male and female mice had 33% and 22% decreases, respectively, in the aortic sinus lesion area with no changes in the aortic arch lesion area. After 16 weeks of Western diet feeding, the lesions showed no changes in the area or volume of the aortic sinus between apoE-/-/Ffar4-/- mice and apoE-/- controls. However, male apoE-/-/Ffar4-/- mice had a 27% increase in the plaque lesion area in the aortic arch compared with apoE-/- controls. Despite similar sizes of lesions in the aortic sinus, apoE-/-/Ffar4-/- mice had larger necrotic cores compared with the apoE-/- control mice. In fact, male and female mice had 43% and 37% increases in the necrotic lesion area, respectively. These data suggest a novel role for FFAR4 in reducing necrotic core lesion formation and support a protective role for FFAR4 in stabilizing atherosclerotic plaques.

ABSTRACTIntroduction: The activation of free fatty acid receptor 1 (FFAR1) induces insulin secretion in a glucose-dependent manner, and thereby is considered as an attractive anti-diabetic target. The clinical studies provided a lot of evidence that FFAR1 agonists improved glucose control in T2DM without the risk of hypoglycemia. The field of FFAR1 agonists is extremely competitive with many patent applications filed in recent years identifying potent candidates.Area covered: The present review summarizes patent applications (2016–2019) filing for FFAR1 modulators, including FFAR1 partial/full agonists, atypical agonists, and multiple target agonists, along with in vitro and in vivo evaluation.Expert opinion: The clinical studies of FFAR1 agonists have proved their potential for the improvement of glucose control. However, there are a few issues still to be solved in this field since TAK-875 terminated in Phase III studies due to liver toxicity. The biggest challenge on the development of FFAR1 agonists may not be the identification of a highly potent compound, but finding out the exact mechanisms of hepatotoxicity and avoid it. Moreover, the further exploration of chemical spaces on FFAR1 full agonists and multi-targeted agonists, as well as corresponding clinical studies, will be expected and might open up new directions in this field.

PurposeTo investigate the relationship between free fatty acid receptor 2 (FFAR2) expression in peripheral blood monocytes and carotid atherosclerosis (CAS).MethodsBetween November 2023 and June 2024, individuals who underwent carotid ultrasound in the inpatient or outpatient settings of Sichuan Provincial People’s Hospital were enrolled in this study. Based on the intima-media thickness (IMT) results from the carotid ultrasound examination, participants were categorized into three groups: the non-CAS group (IMT < 1 mm), the carotid intima-media thickness (cIMT) thickening group (1.0 mm ≤ IMT < 1.5 mm), and the carotid plaque (CP) group (IMT ≥ 1.5 mm with invasion of the lumen or limited intimal thickening higher than 50% of peripheral IMT). The expression levels of FFAR2 in peripheral blood monocytes were detected by flow cytometry. Comparative analyses were conducted on general information and monocyte FFAR2 expression levels across these three groups.ResultsA total of 86 cases were enrolled, comprising 21 cases in the non-CAS group, 21 in the cIMT thickening group, and 44 in the CP group. There were significant statistical differences among the three groups in age, history of hypertension, and Hemoglobin A1c levels (P < 0.05). Specifically, the median (interquartile range) expression levels of FFAR2 in peripheral blood monocytes were 32.04 (17.92, 59.48) × 103, 82.00 (51.19, 99.41) × 103, and 45.03 (26.86, 82.22) × 103 for the non-CAS group, the cIMT thickening group, and the CP group, respectively. The results of inter-group comparisons indicated that there were significant differences in the expression levels of FFAR2 in monocytes between the cIMT thickening group and the non-CAS group (P < 0.001). Meanwhile, significant differences in the expression levels of FFAR2 in monocytes were also observed between the cIMT thickening group and the CP group (P < 0.05).ConclusionPeripheral blood monocyte FFAR2 expression appears to be potentially associated with CAS. The upregulation of peripheral blood monocyte FFAR2 expression levels may play a role as a negative feedback regulator, potentially influencing the progression of carotid atherosclerosis, although further research is needed to confirm this hypothesis.

To evaluate the role of free fatty acid receptor 2 (FFAR2)/G-protein coupled receptor 43 in mediating the effects of the short chain fatty acids (SCFAs) sodium acetate (SA) and sodium propionate (SP) on islet function in vitro, and to identify the intracellular signalling pathways used in SCFA-induced potentiation of glucose-induced insulin secretion. Islets of Langerhans were isolated from wild-type and FFAR2-/- mice and from human donors without diabetes. The effects of SA and SP on dynamic insulin secretion from perifused islets were quantified by radioimmunoassay, signalling downstream of SCFAs was profiled by single-cell calcium microfluorimetry, and measurement of cAMP was performed using a fluorescence assay. Islet apoptosis was induced by exposure to cytokines or sodium palmitate, and the effects of SA and SP in regulating islet apoptosis were assessed by quantification of caspase 3/7 activities. Deletion of FFAR2 did not affect islet morphology or insulin content. SA and SP reversibly potentiated insulin secretion from mouse islets in a FFAR2-dependent manner. SCFA-induced potentiation of insulin secretion was coupled to Gq activation of phospholipase C and protein kinase C, with no evidence of Gi-mediated signalling. SA and SP protected human and mouse islets from apoptosis, and these pro-survival properties were dependent on islet expression of FFAR2. Our results indicate that FFAR2 directly mediates both the stimulatory effects of SA and SP on insulin secretion and their protection against islet apoptosis. We have also shown that SCFA coupling in islets occurs via Gq-coupled intracellular signalling.