Free Fatty Acid Receptor 1 Agonists Research Articles

Role for CCN1 in lysophosphatidic acid response in PC-3 human prostate cancer cells.

Lysophosphatidic acid (LPA) and sphingosine 1-phosphate (S1P) are bioactive phospholipids that act as mitogens in various cancers. Both LPA and S1P activate G-protein coupled receptors (GPCRs). We examined the role of CCN1/CYR61, an inducible matricellular protein, in LPA-induced signal transduction in PC-3 human prostate cancer cells. We found that both LPA and S1P induced expression of CCN1 and CCN2 within 2-4h. CCN1 was induced by 18:1-LPA, but not by 18:0-, 18:2-, or 18:3-LPAs. A free fatty acid receptor-4 agonist inhibited LPA-induced CCN1 induction. CCN1 appeared in the ECM within 2h after LPA addition. LPA caused biphasic activation of Erk MAPK, with an initial peak at 10-20min followed by a later phase after 6h. LPA increased adhesion of PC-3 cells to culture substrates (standard culture plates, fibronectin, or extracellular matrix) at 2h, an intermediate event between early and late LPA signals. Knockdown of CCN1 suppressed LPA-induced adhesion to ECM or fibronectin. ECM from CCN1 knockdown cells was a poor substrate for adhesion, as compared to ECM from control cells. These results suggest that CCN1 contributes to LPA responses in the tumor microenvironment. The LPA-CCN1 axis holds promise for the development of novel therapeutic strategies in cancer.

Bornyl-Containing Derivatives of Benzyloxyphenylpropanoic Acid as FFAR1 Agonists: In Vitro and In Vivo Studies.

Type 2 diabetes mellitus (T2DM) is one of the most common chronic diseases worldwide. Several classes of hypoglycemic drugs are used to treat it, but various side effects limit their clinical use. Consequently, the search for new anti-diabetic agents remains an urgent task for modern pharmacology. In this investigation, we examined the hypoglycemic effects of bornyl-containing benzyloxyphenylpropanoic acid derivatives (QS-528 and QS-619) in a diet-induced model of T2DM. Animals were given the tested compounds per os at a dose of 30 mg/kg for 4 weeks. At the end of the experiment, compound QS-619 demonstrated a hypoglycemic effect, while QS-528 showed hepatoprotection. In addition, we performed a number of in vitro and in vivo experiments to study the presumed mechanism of action of the tested agents. Compound QS-619 was determined to activate the free fatty acid receptor-1 (FFAR1) similarly to the reference agonist GW9508 and its structural analogue QS-528. Both agents also increased insulin and glucose-dependent insulinotropic polypeptide concentrations in CD-1 mice. Our results indicate that QS-619 and QS-528 are probably full FFAR1 agonists.

Hepatoprotective Effect of a New FFAR1 Agonist—N-Alkylated Isobornylamine

Free fatty acid receptor-1 (FFAR1) is one of the possible therapeutic targets in the search for new hepatoprotective drugs. FFAR1 agonists were found to have hypolipidemic, antifibrotic, anti-inflammatory, antiproliferative and antioxidant effects in addition to hypoglycemic action. In this work, we conducted a study of the hepatoprotective effect of the compound QS-528 (previously discovered as an agonist of FFAR1) at doses of 60, 90, 120 and 150 mg/kg on carbon tetrachloride (CCl4)-induced liver injury. At the end of the experiment, a biochemical blood assay demonstrated that the introduction of QS-528 dose-dependently reduces the levels of liver enzymes (AST, ALT and ALKP). Histological and morphometric studies of animals' livers treated with QS-528 at doses of 120 and 150 mg/kg showed a decrease in degenerative/necrotic changes in hepatocytes and an increase in the regenerative activity of the liver. In addition, no toxicity at a single oral dose of 1000 mg/kg and an increase in HepG2 cell viability in vitro were found. Thus, the compound QS-528 was found to exhibit a hepatoprotective effect against CCl4-induced toxic liver damage.

Discovery of a structurally novel, potent, and once-weekly free fatty acid receptor 1 agonist for the treatment of diabetes

Type 2 diabetes mellitus (T2DM) is a lifelong disease that requires long-term medication to control glucose levels, and thereby long-acting drug has been clinically needed for improving medical adherence. The free fatty acid receptor 1 (FFA1) was considered as a promising target for several diseases, such as T2DM, pain and fatty liver. However, no once-weekly FFA1 agonist has been reported until now. Herein, we report the successful discovery of ZLY50, the first once-weekly FFA1 agonist with a completely new chemotype, highly agonistic activity and selectivity on FFA1. Moreover, ZLY50 has enough brain exposure to activate FFA1 in brain, and it is the first orally available FFA1 agonist with analgesic activity. Notably, the long-term anti-diabetic and anti-fatty liver effects of ZLY50 (once-weekly) were better than those of HWL-088 (once-daily), a highly potent FFA1 agonist with far stronger glucose-lowering effect than Phase 3 clinical candidate TAK-875. Further mechanism studies suggested that ZLY50 alleviates fatty liver by regulating the expressions of genes related to lipid metabolism, mitochondrial function, and oxidative stress in liver.

Mechanistic Studies on the Stereoselectivity of FFAR1 Modulators.

Free fatty acid receptor 1 (FFAR1) is a potential therapeutic target for the treatment of type 2 diabetes (T2D). It has been validated that agonists targeting FFAR1 can achieve the initial therapeutic endpoints of T2D, and the epimer agonists (R,S) AM-8596 can activate FFAR1 differently, with one acting as a partial agonist and the other as a full agonist. Up to now, the origin of the stereoselectivity of FFAR1 agonists remains elusive. In this work, we used molecular simulation methods to elucidate the mechanism of the stereoselectivity of the FFAR1 agonists (R)-AM-8596 and (S)-AM-8596. We found that the full agonist (R)-AM-8596 disrupts the residue interaction network around the receptor binding pocket and promotes the opening of the binding site for the G-protein, thereby resulting in the full activation of FFAR1. In contrast, the partial agonist (S)-AM-8596 forms stable electrostatic interactions with FFAR1, which stabilizes the residue network and hinders the conformational transition of the receptor. Our work thus clarifies the selectivity and underlying molecular activation mechanism of FFAR1 agonists.

Roles for CCN proteins in Lysophosphatidic Acid and Sphingosine 1‐Phosphate Signaling in Prostate Cancer Cells

IntroductionCysteine rich angiogenic factor (CCN1/Cyr61) and connective tissue growth factor (CCN2/CTGF) are members of the CCN family of matricellular proteins. These secreted proteins interact with extracellular matrix proteins and integrins to modulate cell signaling. CCN proteins have been implicated in survival and other mitogenic responses in various cancers. CCN1 and CCN2 are the two early inducible genes in this family. Lysophosphatidic acid (LPA) and sphingosine‐1‐phosphate (S1P) are lipid growth factors known to contribute to tumor progression in prostate cancer. In previous work, our group showed that LPA stimulates proliferation and migration in prostate cancer cells via the G protein‐coupled receptor LPAR1. Free fatty acid receptor 4 (FFAR4) agonists, inhibit LPA‐induced responses in prostate cancer cells. We also demonstrated that CCN1 expression is induced by LPA in human prostate cancer cells. However, the roles of CCN1 and CCN2 in cancer cell signaling have not been fully elucidated.HypothesisWe hypothesize that CCN proteins contribute to LPA‐ and S1P‐induced adhesion and mitogenic signaling in prostate cancer cells.Methods and ResultsPC‐3 human prostate cancer cells were used in the current study. For all experiments, cells were serum starved for 24 hours and then treated with and without 10 µM LPA or S1P. Immunoblotting showed that CCN1 and CCN2 were induced by LPA after 2‐5 hours in PC‐3 cells. The response was inhibited by the LPA receptor antagonist Ki1625. TUG‐891, an FFAR4 agonist, inhibited LPA‐induced CCN1 expression in PC‐3 cells. S1P also induced CCN1 expression in PC‐3 cells. In related experiments, whole cell extracts, and extracellular matrix were subjected to immunoblotting for CCN1, and immunofluorescence microscopy was used to localize the induced CCN1. CCN1 protein levels in extracellular matrix were increased 2‐5 hours after LPA treatment, as compared to untreated controls, and CCN1 was detected outside the cells by confocal microscopy. In cell adhesion assays, LPA‐treated PC‐3 cells exhibited an increase in adhesion to fibronectin‐coated 96‐well plates that was detected by 2 hours. Erk MAPK activation in response to LPA was biphasic, with the later phase occurring after CCN1/2 induction.ConclusionThe results of this study suggest that CCN proteins may play roles in LPA‐ and S1P‐induced signaling in prostate cancer cells and are thus potential therapeutic targets.

Free Fatty Acid Receptor 4 (FFA4) Activation Ameliorates Imiquimod-Induced Psoriasis in Mice

Dietary supplementation with n-3 polyunsaturated fatty acids (n-3 PUFA) has been used as an adjunct therapy for psoriasis due to its anti-inflammatory properties. Free fatty acid receptor 4 (FFA4 or GPR120) is a receptor-sensing n-3 PUFA. In the present study, we examined whether FFA4 acted as a therapeutic target for n-3 PUFA in psoriasis therapy. Experimentally, psoriasis-like skin lesions were induced by treatment with imiquimod for 6 consecutive days. A selective FFA4 agonist, Compound A (30 mg/kg), was used in FFA4 WT and FFA4 KO mice. Imiquimod-induced psoriasis-like skin lesions, which present as erythematous papules and plaques with silver scaling, as well as markedly elevated IL-17/IL-23 cytokine levels in skin tissues, were significantly suppressed by Compound A in FFA4 WT mice, but not in FFA4 KO mice. Enlarged lymph nodes and spleens, as well as imiquimod-induced, elevated IL-17/IL-23 cytokine levels, were also strongly suppressed by Compound A in FFA4 WT mice, but not in FFA4 KO mice. Imiquimod-induced increases in the CD4+IL-17A+ T cell population in lymph nodes and spleens were suppressed by Compound A treatment in FFA4 WT mice; however, this was not seen in FFA4 KO mice. Furthermore, compound A suppressed the differentiation of CD4+ naïve T cells from splenocytes into TH17 cells in an FFA4-dependent manner. In conclusion, we demonstrated that the activation of FFA4 ameliorates imiquimod-induced psoriasis, and the suppression of the differentiation of TH17 cells may partly contribute to its efficacy. Therefore, we suggest that FFA4 could be a therapeutic target for psoriasis therapy.

In Silico Searching for Alternative Lead Compounds to Treat Type 2 Diabetes through a QSAR and Molecular Dynamics Study.

Free fatty acid receptor 1 (FFA1) stimulates insulin secretion in pancreatic β-cells. An advantage of therapies that target FFA1 is their reduced risk of hypoglycemia relative to common type 2 diabetes treatments. In this work, quantitative structure–activity relationship (QSAR) approach was used to construct models to identify possible FFA1 agonists by applying four different machine-learning algorithms. The best model (M2) meets the Tropsha’s test requirements and has the statistics parameters R2 = 0.843, Q2CV = 0.785, and Q2ext = 0.855. Also, coverage of 100% of the test set based on the applicability domain analysis was obtained. Furthermore, a deep analysis based on the ADME predictions, molecular docking, and molecular dynamics simulations was performed. The lipophilicity and the residue interactions were used as relevant criteria for selecting a candidate from the screening of the DiaNat and DrugBank databases. Finally, the FDA-approved drugs bilastine, bromfenac, and fenofibric acid are suggested as potential and lead FFA1 agonists.

Ursodesoxycholic acid is an FFA4 agonist and reduces hepatic steatosis via FFA4 signaling

Ursodeoxycholic acid (UDCA) is a safe bile acid effective in reducing hepatic steatosis in non-alcoholic fatty liver disease (NAFLD). However, the mechanism of action linked to this effect is poorly defined. In the present study, we identified that UDCA acted as a free fatty acid receptor 4 (FFA4) agonist with EC50 of 10.4 ± 0.7 μM, and its activity was determined by dynamic mass redistribution, fluorometric imaging plate reader, inositol monophosphate and bioluminescence resonance energy transfer assays. Moreover, UDCA showed FFA4 selectivity over eleven other G protein-coupled receptors. Real-Time PCR and immunocytochemistry analyses showed that FFA4 was abundantly expressed in human hepatocytes HuH-7 cells. In an in vitro model of NAFLD induced by oleic acid (OA), UDCA downregulated lipid accumulation in HuH-7 cells and suppressed sterol-regulatory element binding protein-1c (SREBP-1c) mRNA expression. This suppression of SREBP-1c was restored when FFA4 expression was knocked down in siRNA assay. In a mouse model of hepatic steatosis, db/db mice were exposed to a high-fat diet (HFD), and treatment of UDCA or docosahexaenoic acid (DHA, an endogenous FFA4 agonist) effectively prevented body weight gain and hepatic fat deposition and reduced triglyceride (TG) levels in serum and liver. This study not only identified a new skeleton of FFA4 agonists, but also demonstrated that FFA4 signal was accounting for the protective effects of UDCA in the NAFLD treatment.

Design, synthesis, and biological evaluation of novel FFA1 partial agonists bearing oxime ether scaffold**

AbstractThe free fatty acid receptor 1 (FFA1) is a promising anti‐diabetic target, and many FFA1 agonists including TAK‐875 and AMG‐837 are reached in clinical studies. However, the excessive lipophilicity of AMG‐837 (ClogP=6.81) might be a potential downside attributed to the clinical failure of AMG‐837. In this study, we introduced the oxime ether moiety to replace the middle benzene of AMG‐837 to reduce the lipophilicity. After comprehensive structure‐activity relationship study, the optimal compound 7 was identified as a partial agonist with appropriate lipophilicity (EC50=37.6 nM, Efficacy=71 %, ClogP=4.73). Moreover, compound 7 exhibited significantly glucose‐lowering effects in a dose‐dependent manner, and the glucose‐lowering effect was equivalent to that of TAK‐875 at the dose of 20 mg/kg. In conclusion, this study provided a new series partial agonists bearing oxime ether scaffold, which is worthy for further exploration based on its excellent pharmacological activity and physicochemical property.

Free fatty acid receptor 1: a ray of hope in the therapy of type 2 diabetes mellitus

Free fatty acid receptor 1 (FFAR1) is a G-protein coupled receptor with prominent expression on pancreatic beta cells, bones, intestinal cells as well as the nerve cells. This receptor mediates a multitude of functions in the body including release of incretins, secretion of insulin as well as sensation of pain. Since FFAR1 causes secretion of insulin and regulates glucose metabolism, efforts were made to unfold its structure followed by discovering agonists for the receptor and the utilization of these agonists in the therapy of type 2 diabetes mellitus. Development of such functional FFAR1 agonists is a necessity because the currently available therapy for type 2 diabetes mellitus has numerous drawbacks, of which, the major one is hypoglycemia. Since the most prominent effect of the FFAR1 agonists is on glucose concentration in the body, so the major research is focused on treating type 2 diabetes mellitus, though the agonists could benefit other metabolic disorders and neurological disorders as well. The agonists developed so far had one major limitation, i.e., hepatotoxicity. Although, the only agonist that could reach phase 3 clinical trials was TAK-875 developed by Takeda Pharmaceuticals but it was also withdrawn due to toxic effects on the liver. Thus, there are numerous agonists for the varied binding sites of the receptor but no drug available yet. There does seem to be a ray of hope in the drugs that target FFAR1 but a lot more efforts towards drug discovery would result in the successful management of type 2 diabetes mellitus.

FFAR1/GPR40 Contributes to the Regulation of Striatal Monoamine Releases and Facilitation of Cocaine-Induced Locomotor Activity in Mice.

The free fatty acid receptor 1 (FFAR1) is suggested to function as a G protein-coupled receptor (GPR40) for medium-to-long-chain free fatty acids. Previous studies on the expression of FFAR1 revealed that the nigrostriatal region is one of the areas which express abundant FFAR1 mRNA/protein in the central nervous system (CNS). However, the role of FFAR1 in the CNS has been still largely unclarified. Here, we examined a possible functional role of FFAR1 in the control of extracellular concentrations of striatal monoamines and cocaine-induced locomotor activity. Microdialysis analysis revealed that the basal level of extracellular dopamine (DA) was significantly elevated, while the basal serotonin (5-HT) level tended to be reduced in the striatum of FFAR1 knockout (−/−) mice. Interestingly, local application of a FFAR1 agonist, GW9508, markedly augmented the striatal 5-HT release in FFAR1 wild-type (+/+) mice, whereas topical application of a FFAR1 antagonist, GW1100, significantly reduced the 5-HT release. However, the enhanced 5-HT release was completely lost in −/− mice. Although acute administration of cocaine enhanced the locomotor activity in both +/+ and −/− mice, the magnitude of the enhancement was significantly reduced in −/− mice. In addition, intraperitoneal injection of GW1100 significantly decreased the cocaine-induced locomotor enhancement. These results suggest that FFAR1 has a facilitatory role in striatal 5-HT release, and the evoked 5-HT release might contribute to enhance cocaine-induced locomotor activity.

Screening of a novel free fatty acid receptor 1 (FFAR1) agonist peptide by phage display and machine learning based-amino acid substitution

Free fatty acid receptor 1 (FFAR1 or GPR40) has attracted attention for the treatment of type 2 diabetes mellitus, and various small-molecule agonists have been developed. However, most FFAR1 agonists as well as endogenous ligands, such as linoleic acids, have high lipophilicity, and their high lipophilicity is related to off-target toxicity. Therefore, we need to focus on new ligand candidates with less toxicity. In this study, we screened peptides with FFAR1 agonist activity as new ligand candidates. First, we used phage display to identify peptides with high affinity to FFAR1. Next, the agonist activities of peptides determined by the phage display were evaluated by the TGF-α shedding assay. Finally, to improve the FFAR1 agonist activity of the peptide, we performed an inclusive single amino acid substitution and sequence analysis. Logistic regression (LR) analysis using 120 physiochemical properties was performed to predict peptides with high FFAR1 agonist activity. STTGTQY determined by phage display promoted glucose-stimulated insulin secretion in pancreatic MIN6 cells. Furthermore, STKGTF predicted by the LR analysis showed high insulin secretion at low concentrations compared to STTGTQY. The results of this study suggest that peptides could be new candidates as FFAR1 agonists.

Synthesis and evaluation of 3-(4-(phenoxymethyl)phenyl)propanoic acid and N-phenylbenzenesulfonamide derivatives as FFA4 agonists

Free fatty acid receptor 4 (FFA4) has been recognized as an attractive target in metabolic diseases. To find potent and selective FFA4 agonist, 28 compounds of 3-(4-(phenoxymethyl)phenyl)propanoic acid and N-phenylbenzenesulfonamide derivatives were designed and synthesized, featuring OC and SO2-N linkage. For the OC linkage compounds, 1g showed the most potent FFA4 agonistic activity with a pEC50 of 5.81 ± 0.04 and exhibited at least 64-fold selectivity against FFA1. For SO2-N linkage agonists, 2m had a pEC50 of 5.66 ± 0.04 and displayed>46-fold selectivity against FFA1. Among these two series of compounds, 1g was the most potent agonist at FFA4 and the best selectivity against FFA1, demonstrated by docking simulation. Moreover, 1g showed receptor selectivity on other seven GPCRs. In anti-diabetic evaluation, 1g dose-dependently reduced blood glucose, which was better than a clinical phase III drug TAK875. This study provides guidance for FFA4 ligand design and drug optimization.

Recent Updates on Free Fatty Acid Receptor 1 (GPR-40) Agonists for the Treatment of Type 2 Diabetes Mellitus.

The global incidence of type 2 diabetes mellitus (T2DM) has enthused the development of new antidiabetic targets with low toxicity and long-term stability. In this respect, free fatty acid receptor 1 (FFAR1), which is also recognized as a G protein-coupled receptor 40 (GPR40), is a novel target for the treatment of T2DM. FFAR1/GPR40 has a high level of expression in β-cells of the pancreas, and the requirement of glucose for stimulating insulin release results in immense stimulation to utilise this target in the medication of T2DM. The data used for this review is based on the search of several scienctific databases as well as various patent databases. The main search terms used were free fatty acid receptor 1, FFAR1, FFAR1 agonists, diabetes mellitus, G protein-coupled receptor 40 (GPR40), GPR40 agonists, GPR40 ligands, type 2 diabetes mellitus and T2DM. The present review article gives a brief overview of FFAR1, its role in T2DM, recent developments in small molecule FFAR1 (GPR40) agonists reported till now, compounds of natural/plant origin, recent patents published in the last few years, mechanism of FFAR1 activation by the agonists, and clinical status of the FFAR1/GPR40 agonists. The agonists of FFAR1/GRP40 showed considerable potential for the therapeutic control of T2DM. Most of the small molecule FFAR1/GPR40 agonists developed were aryl alkanoic acid derivatives (such as phenylpropionic acids, phenylacetic acids, phenoxyacetic acids, and benzofuran acetic acid derivatives) and thiazolidinediones. Some natural/plant-derived compounds, including fatty acids, sesquiterpenes, phenolic compounds, anthocyanins, isoquinoline, and indole alkaloids, were also reported as potent FFAR1 agonists. The clinical investigations of the FFAR1 agonists demonstrated their probable role in the improvement of glucose control. Though, there are some problems still to be resolved in this field as some FFAR1 agonists terminated in the late phase of clinical studies due to "hepatotoxicity." Currently, PBI-4050 is under clinical investigation by Prometic. Further investigation of pharmacophore scaffolds for FFAR1 full agonists as well as multitargeted modulators and corresponding clinical investigations will be anticipated, which can open up new directions in this area.

HWL-088, a new and highly effective FFA1/PPARδ dual agonist, attenuates nonalcoholic steatohepatitis by regulating lipid metabolism, inflammation and fibrosis.

Nonalcoholic fatty liver (NAFLD), a chronic progressive liver disease, is highly correlated with pathoglycemia, dyslipidemia and oxidative stress. The free fatty acid receptor 1 (FFA1) agonists have been reported to improve liver steatosis and fibrosis, and the peroxisome proliferator-activated receptor δ (PPARδ) plays a synergistic role with FFA1 in energy metabolism and fibrosis. HWL-088, a PPARδ/FFA1 dual agonist, exerts better glucose-lowering effects than the representative FFA1 agonist TAK-875. However, the ability of HWL-088 to protect NAFLD was unknown. This study aimed to discover a new strategy for the treatment of NAFLD. The methionine- and choline-deficient diet (MCD)-induced Nonalcoholic steatohepatitis (NASH) model was constructed to evaluate the effects of HWL-088. Administration of HWL-088 exerted multiple benefits on glucose control, lipid metabolism and fatty liver. Further mechanism research indicated that HWL-088 promotes lipid metabolism by decreasing lipogenesis and increasing lipolysis. Moreover, HWL-088 attenuates NASH by regulating the expression levels of genes related to inflammation, fibrosis and oxidative stress. These positive results indicated that PPARδ/FFA1 dual agonist HWL-088 might be a potential candidate to improve multiple pathogenesis of NASH.

ZLY032, the first-in-class dual FFA1/PPARδ agonist, improves glucolipid metabolism and alleviates hepatic fibrosis

The free fatty acid receptor 1 (FFA1) and peroxisome proliferator-activated receptor δ (PPARδ) are considered as anti-diabetic targets based on their role in improving insulin secretion and resistance. Based on their synergetic mechanisms, we have previously identified the first-in-class dual FFA1/PPARδ agonist ZLY032. After long-term treatment, ZLY032 significantly improved glucolipid metabolism and alleviated fatty liver in ob/ob mice and methionine choline-deficient diet-fed db/db mice, mainly by regulating triglyceride metabolism, fatty acid β-oxidation, lipid synthesis, inflammation, oxidative stress and mitochondrial function. Notably, ZLY032 exhibited greater advantages on lipid metabolism, insulin sensitivity and pancreatic β-cell function than TAK-875, the most advanced candidate of FFA1 agonists. Moreover, ZLY032 prevented CCl4-induced liver fibrosis by reducing the expressions of genes involved in inflammation and fibrosis development. These results suggest that the dual FFA1/PPARδ agonists such as ZLY032 may be useful for the treatment of metabolic disorders.

Free fatty acid receptor 4 activation protects against choroidal neovascularization in mice.

To examine whether free fatty acid receptor 4 (FFAR4) activation can protect against choroidal neovascularization (CNV), which is a common cause of blindness, and to elucidate the mechanism underlying the inhibition, we used the mouse model of laser-induced CNV to mimic angiogenic aspects of age-related macular degeneration (AMD). Laser-induced CNV was compared between groups treated with an FFAR4 agonist or vehicle, and between FFAR4 wild-type (Ffar4+/+) and knock out (Ffar4-/-) mice on a C57BL/6J/6N background. The ex vivo choroid-sprouting assay, including primary retinal pigment epithelium (RPE) and choroid, without retina was used to investigate whether FFAR4 affects choroidal angiogenesis. Western blotting for pNF-ĸB/NF-ĸB and qRT-PCR for Il-6, Il-1β, Tnf-α, Vegf, and Nf-ĸb were used to examine the influence of FFAR4 on inflammation, known to influence CNV. RPE isolated from Ffar4+/+ and Ffar4-/- mice were used to assess RPE contribution to inflammation. The FFAR4 agonist suppressed laser-induced CNV in C57BL/6J mice, and CNV increased in Ffar4-/- compared to Ffar4+/+ mice. We showed that the FFAR4 agonist acted through the FFAR4 receptor. The FFAR4 agonist suppressed mRNA expression of inflammation markers (Il-6, Il-1β) via the NF-ĸB pathway in the retina, choroid, RPE complex. The FFAR4 agonist suppressed neovascularization in the choroid-sprouting ex vivo assay and FFAR4 deficiency exacerbated sprouting. Inflammation markers were increased in primary RPE cells of Ffar4-/- mice compared with Ffar4+/+ RPE. In this mouse model, the FFAR4 agonist suppressed CNV, suggesting FFAR4 to be a new molecular target to reduce pathological angiogenesis in CNV.

HWL-088, a new potent free fatty acid receptor 1 (FFAR1) agonist, improves glucolipid metabolism and acts additively with metformin in ob/ob diabetic mice.

The free fatty acid receptor 1 (FFAR1) plays an important role in glucose-stimulated insulin secretion making it an attractive anti-diabetic target. This study characterizes the pharmacological profile of HWL-088 (2-(2-fluoro-4-((2'-methyl-[1,1'- biphenyl]-3-yl)methoxy)phenoxy)acetic acid), a novel highly potent FFAR1 agonist in vitro and in vivo. Moreover, we investigated the long-term effects of HWL-088 alone and in combination with metformin in diabetic mice. In vitro effects of HWL-088 on FFAR1 and PPARα/γ/δ were studied in cell-based assays. Glucose-dependent insulinotropic effects were evaluated in MIN6 cell line and in rats. Long-term effects on glucose and lipid metabolism were investigated in ob/ob mice. HWL-088 is a highly potent FFAR1 agonist (EC50 = 18.9 nM) with moderate PPARδ activity (EC50 = 570.9 nM) and promotes glucose-dependent insulin secretion in vitro and in vivo. Long-term administration of HWL-088 exhibited better glucose control and plasma lipid profiles than those of another FFAR1 agonist, TAK-875, and synergistic improvements were observed when combined with metformin. Moreover, HWL-088 and combination therapy improved β-cell function by up-regulation of pancreas duodenum homeobox-1, reduced fat accumulation in adipose tissue and alleviated fatty liver in ob/ob mice. The effect of HWL-088 involves a reduction in hepatic lipogenesis and oxidative stress, increased lipoprotein lipolysis, glucose uptake, mitochondrial function and fatty acid β-oxidation. These data indicate that long-term treatment with HWL-088, a highly potent FFAR1 agonist, improves glucose and lipid metabolism and may be useful for the treatment of diabetes mellitus by mono-therapy or combination with metformin.

Regulation of cell survival through free fatty acid receptor 1 (FFA1) and FFA4 induced by endothelial cells in osteosarcoma cells

Free fatty acid receptor 1 (FFA1) and FFA4 belong to a family of free fatty acid (FFA) receptors. FFA1- and FFA4-mediated signaling regulates a variety of malignant properties in cancer cells. It is known that stromal cells in the tumor microenvironment promote tumor progression. In the present study, to assess the roles of FFA1 and FFA4 in cellular functions modulated by endothelial cells, highly migratory MG63-CR7(F2) cells were generated from osteosarcoma MG-63 cells, using endothelial F2 cell supernatants. Expression levels of FFAR1 and FFAR4 genes in MG63-CR7(F2) cells were significantly higher than those of MG-63 cells. In cell survival assay, cells were treated with cisplatin (CDDP) every 24 h for 2 days. The cell survival rate of MG-63 cells was significantly elevated by an FFA1 agonist TUG-770 as well as an FFA4 agonist TUG-891. Moreover, the cell survival rate of MG63-CR7(F2) cells was higher than that of MG-63 cells in the presence of TUG-770 or TUG-891, correlating with FFAR1 and FFAR4 expression levels. To validate the effects of FFA1 and FFA4 on cell survival to CDDP, FFA1 and FFA4 knockdown cell were generated from MG-63 cells. The cell survival rate of MG-63 cells was markedly inhibited by FFA1 or FFA4 knockdown. These results suggest that FFA1 and FFA4 may play an important role in the modulation of cellular functions by endothelial cells in osteosarcoma cells.