Fatty Acid Receptor GPR120 Research Articles

3D-Printed Bioactive Scaffold Loaded with GW9508 Promotes Critical-Size Bone Defect Repair by Regulating Intracellular Metabolism.

The process of bone regeneration is complicated, and it is still a major clinical challenge to regenerate critical-size bone defects caused by severe trauma, infection, and tumor resection. Intracellular metabolism has been found to play an important role in the cell fate decision of skeletal progenitor cells. GW9508, a potent agonist of the free fatty acid receptors GPR40 and GPR120, appears to have a dual effect of inhibiting osteoclastogenesis and promoting osteogenesis by regulating intracellular metabolism. Hence, in this study, GW9508 was loaded on a scaffold based on biomimetic construction principles to facilitate the bone regeneration process. Through 3D printing and ion crosslinking, hybrid inorganic-organic implantation scaffolds were obtained after integrating 3D-printed β-TCP/CaSiO3 scaffolds with a Col/Alg/HA hydrogel. The 3D-printed β-TCP/CaSiO3 scaffolds had an interconnected porous structure that simulated the porous structure and mineral microenvironment of bone, and the hydrogel network shared similar physicochemical properties with the extracellular matrix. The final osteogenic complex was obtained after GW9508 was loaded into the hybrid inorganic-organic scaffold. To investigate the biological effects of the obtained osteogenic complex, in vitro studies and a rat cranial critical-size bone defect model were utilized. Metabolomics analysis was conducted to explore the preliminary mechanism. The results showed that 50 μM GW9508 facilitated osteogenic differentiation by upregulating osteogenic genes, including Alp, Runx2, Osterix, and Spp1 in vitro. The GW9508-loaded osteogenic complex enhanced osteogenic protein secretion and facilitated new bone formation in vivo. Finally, the results from metabolomics analysis suggested that GW9508 promoted stem cell differentiation and bone formation through multiple intracellular metabolism pathways, including purine and pyrimidine metabolism, amino acid metabolism, glutathione metabolism, and taurine and hypotaurine metabolism. This study provides a new approach to address the challenge of critical-size bone defects.

AMPKα2 controls the anti-atherosclerotic effects of fish oils by modulating the SUMOylation of GPR120

Consuming fish oils (FO) is linked to reduced risk of cardiovascular disease in certain populations. However, FO failed to exhibit therapeutic effects in some patients with cardiovascular disease. This study aimed to determine the possible reasons for the inconsistent effects of FO. AMP-activated protein kinase (AMPK) α2 is an important energy metabolic sensor, which was reported to involve in FO mediated regulation of lipid and glucose metabolism. In an in vivo study, FO administration significantly reduced the aortic lesions and inflammation in the Ldlr−/− mouse model of atherosclerosis, but not in Ldlr−/−/Prkaa2−/−and Ldlr−/−/Prkaa2−/−Sm22Cre mice. Mechanistically, inactivation of AMPKα2 increased the SUMOylation of the fatty acid receptor GPR120 to block FO-induced internalization and binding to β-arrestin. In contrast, activation of AMPKα2 can phosphorylate the C-MYC at Serine 67 to inhibit its trans-localization into the nuclei and transcription of SUMO-conjugating E2 enzyme UBC9 and SUMO2/3 in vascular smooth muscle cells (VSMCs), which result in GPR120 SUMOylation. In human arteries, AMPKα2 levels were inversely correlated with UBC9 expression. In a cohort of patients with atherosclerosis, FO concentrations did not correlate with atherosclerotic severity, however, in a subgroup analysis a negative correlation between FO concentrations and atherosclerotic severity was found in patients with higher AMPKα2 levels. These data indicate that AMPKα2 is required for the anti-inflammatory and anti-atherosclerotic effects of FO.

Effect of Obesity on the Expression of Nutrient Receptors and Satiety Hormones in the Human Colon.

Background: Receptors located on enteroendocrine cells (EECs) of the colon can detect nutrients in the lumen. These receptors regulate appetite through a variety of mechanisms, including hormonal and neuronal signals. We assessed the effect of obesity on the expression of these G-protein coupled receptors (GPCRs) and hormones at both mRNA and protein level. Methods: qPCR and immunohistochemistry were used to examine colonic tissue from cohorts of patients from the Netherlands (proximal and sigmoid tissue) and the United Kingdom (tissue from across the colon) and patients were grouped by body mass index (BMI) value (BMI < 25 and BMI ≥ 25). Results: The mRNA expression of the hormones/signaling molecules serotonin, glucagon, peptide YY (PYY), CCK and somatostatin were not significantly different between BMI groups. GPR40 mRNA expression was significantly increased in sigmoid colon samples in the BMI ≥ 25 group, but not proximal colon. GPR41, GPR109a, GPR43, GPR120, GPRC6A, and CaSR mRNA expression were unaltered between low and high BMI. At the protein level, serotonin and PYY containing cell numbers were similar in high and low BMI groups. Enterochromaffin cells (EC) showed high degree of co-expression with amino acid sensing receptor, CaSR while co-expression with PYY containing L-cells was limited, regardless of BMI. Conclusions: While expression of medium/long chain fatty acid receptor GPR40 was increased in the sigmoid colon of the high BMI group, expression of other nutrient sensing GPCRs, and expression profiles of EECs involved in peripheral mechanisms of appetite regulation were unchanged. Collectively, these data suggest that in human colonic tissue, EEC and nutrient-sensing receptor expression profiles are not affected despite changes to BMI.

Gene expression of nutrient-sensing molecules in I cells of CCK reporter male mice.

Cholecystokinin (CCK) is secreted from enteroendocrine I cells in response to fat, carbohydrate, and protein ingestion. Gene expression of nutrient-sensing molecules in I cells remains unclear, primarily due to the difficulty in distinguishing I cells from intestinal epithelial cells in vivo. In this study, we generated CCK reporter male mice in which the red fluorescence protein tdTomato (Tomato) is produced by activation of the native murine Cck promoter. Fluorescence microscopy revealed the presence of Tomato-positive cells in upper small intestine (SI), lower SI, and colon. Flow cytometer analysis revealed that Tomato-positive cells among epithelial cells of upper SI, lower SI, and colon occurred at the rate of 0.95, 0.54, and 0.06%, respectively. In upper SI and lower SI, expression levels of Cck mRNA were higher in Tomato-positive cells than those in Tomato-negative cells. The fatty acid receptors Gpr120, Gpr40, and Gpr43 and the oleoylethanolamide receptor Gpr119 were highly expressed in Tomato-positive cells isolated from SI, but were not found in Tomato-positive cells from colon. The glucose and fructose transporters Sglt1, Glut2, and Glut5 were expressed in both Tomato-positive cells and -negative cells, but these expression levels tended to be decreased in Tomato-positive cells from upper SI to colon. The peptide transporter Pept1 and receptor Gpr93 were expressed in both Tomato-positive cells and -negative cells, whereas Casr was expressed only in Tomato-positive cells isolated from SI. Thus, this transgenic mouse reveals that I cell number and gene expression in I cells vary according to region in the gastrointestinal tract.

The GPR120 Agonist TUG-891 Inhibits the Motility and Phagocytosis of Mouse Alveolar Macrophages

Movement and phagocytosis characterize the fundamental actions of macrophages. Although it is known that the free fatty acid receptor GPR120 is expressed in macrophages and regulates cytokine expression to exert anti-inflammatory activities, the effects of GPR120 activation on the motility and phagocytosis of macrophages are not clear. In this study, mouse alveolar macrophages (AM) were stimulated with the GPR120 agonist TUG-891, and the changes in cell motility, intracellular Ca2+ concentration ([Ca2+]i), and the ability of phagocytosis were measured. Mouse AM in controls exhibited active movement in vitro, and TUG-891 significantly restrained AM movement. Meanwhile, TUG-891 stimulated a quick increase in [Ca2+]i in AM, which was blocked separately by the Gq protein inhibitor YM-254890, the phospholipase C (PLC) inhibitor U73122, or depletion of endoplasmic reticulum (ER) Ca2+ store by thapsigargin. The inhibition of AM movement by TUG-891 was eliminated by YM-254890, U73122, thapsigargin, and chelation of cytosolic Ca2+ by BAPTA. Moreover, TUG-891 inhibited AM phagocytosis of fluorescent microspheres, which was also blocked by YM-254890, U73122, thapsigargin, and BAPTA. In conclusion, GPR120 activation in mouse AM increases [Ca2+]i but inhibits the motility and phagocytosis via Gq protein/PLC-mediated Ca2+ release from ER Ca2+ store.

GPR40 activation initiates store-operated Ca2+ entry and potentiates insulin secretion via the IP3R1/STIM1/Orai1 pathway in pancreatic \u03b2-cells

The long-chain fatty acid receptor GPR40 plays an important role in potentiation of glucose-induced insulin secretion (GIIS) from pancreatic β-cells. Previous studies demonstrated that GPR40 activation enhances Ca2+ release from the endoplasmic reticulum (ER) by activating inositol 1,4,5-triphosphate (IP3) receptors. However, it remains unknown how ER Ca2+ release via the IP3 receptor is linked to GIIS potentiation. Recently, stromal interaction molecule (STIM) 1 was identified as a key regulator of store-operated Ca2+ entry (SOCE), but little is known about its contribution in GPR40 signaling. We show that GPR40-mediated potentiation of GIIS is abolished by knockdown of IP3 receptor 1 (IP3R1), STIM1 or Ca2+-channel Orai1 in insulin-secreting MIN6 cells. STIM1 and Orai1 knockdown significantly impaired SOCE and the increase of intracellular Ca2+ by the GPR40 agonist, fasiglifam. Furthermore, β-cell-specific STIM1 knockout mice showed impaired fasiglifam-mediated GIIS potentiation not only in isolated islets but also in vivo. These results indicate that the IP3R1/STIM1/Orai1 pathway plays an important role in GPR40-mediated SOCE initiation and GIIS potentiation in pancreatic β-cells.

Protective effects of GPR120 agonist-programmed macrophages on renal interstitial fibrosis in unilateral ureteral obstruction (UUO) rats

Macrophages in the kidney play different roles in renal interstitial fibrosis (RIF) depending on their phenotypes. M2 phenotype macrophages are believed to protect the kidney against RIF. Free fatty acid receptor GPR120 is expressed in macrophages, and its activation induces macrophage transition to M2 phenotype. In this study, the effects of GPR120 agonist-programmed macrophages on RIF were investigated. The peritoneal macrophages collected from rats were incubated with GPR120 agonist TUG891 in vitro for 24 h, and then they were transplanted autologously to the kidney with ureteral obstruction by intrarenal injection for 7 days on the same day following unilateral ureteral obstruction (UUO) operation. RIF was identified by Masson trichrome histological staining, and the expression of RIF-related proteins was analyzed by immunohistochemistry and western blot. It was observed that TUG891-programmed macrophages up-regulated the expression of CD206 and arginase-1 while the expression of interleukin-6 and tumor necrosis factor-α were down-regulated. RIF in rats was significantly increased following UUO, which was markedly alleviated by TUG891-programmed macrophages but not untreated macrophages. TUG891-programmed macrophages inhibited the abnormal expression of TGF-β1 and SMAD2. The abnormal expression of epithelial-mesenchymal transition (EMT)-related proteins including vimentin, α-SMA and β-catenin was also significantly decreased in rats with transplantation of TUG891-programmed macrophages as compared to UUO rats. This study suggests that autologous administration of peritoneal macrophages programmed in vitro by GPR120 agonist to kidney has a protective effect against RIF following UUO.

Oleic acid increases the transcriptional activity of FoxO1 by promoting its nuclear translocation and β-catenin binding in pancreatic β-cells

In the setting of metabolic overload, chronic elevations of free fatty acids in blood and tissues are associated with pancreatic β-cell lipotoxicity and failure. Ultimately, obesity combined with insulin resistance increases the dysfunctional demand of β-cells and contributes to the development of type 2 diabetes. Forkhead box O1 (FoxO1) is a potent transcriptional regulator of pancreatic β-cell function and tolerance to lipid stress. The present study examined the effects of stearoyl-CoA desaturase 1 (SCD1)-metabolized precursors and products, notably oleic acid, on the compensatory capacity of β-cells and their relationship with regulation of the FoxO1 and Wnt pathways. The trioleate-induced compromise of insulin sensitivity blunted the compensatory response of pancreatic β-cells in primary rat islets. These events were associated with increases in the nuclear accumulation and transcriptional activity of FoxO1. Such effects were also observed in INS-1E cells that were subjected to oleate treatment. The overexpression of human SCD1 that was accompanied by endogenously generated oleic acid also led to an increase in the nuclear abundance of FoxO1. The mechanism of the oleate-mediated subcellular localization of FoxO1 was independent of the fatty acid receptor GPR40. Instead, the mechanism involved diversion of the active β-catenin pool from an interaction with transcription factor 7-like 2 toward FoxO1-mediated transcription in β-cells. Our findings identify a unique role for oleic acid in the compensatory response of pancreatic β-cells and emphasize the importance of FoxO1 in β-cell failure in obesity-induced insulin resistance.

Activation of the Omega-3 Fatty Acid Receptor GPR120 Protects against Focal Cerebral Ischemic Injury by Preventing Inflammation and Apoptosis in Mice.

G protein-coupled receptor 120 (GPR120) has been shown to negatively regulate inflammation and apoptosis, but its role in cerebral ischemic injury remains unclear. Using an in vivo model of middle cerebral artery occlusion (MCAO) and an in vitro model of oxygen-glucose deprivation (OGD), we investigated the potential role and molecular mechanisms of GPR120 in focal cerebral ischemic injury. Increased GPR120 expression was observed in microglia and neurons following MCAO-induced ischemia in wild type C57BL/6 mice. Treatment with docosahexaenoic acid (DHA) inhibited OGD-induced inflammatory response in primary microglia and murine microglial BV2 cells, whereas silencing of GPR120 strongly exacerbated the inflammation induced by OGD and abolished the anti-inflammatory effects of DHA. Mechanistically, DHA inhibited OGD-induced inflammation through GPR120 interacting with β-arrestin2. In addition to its anti-inflammatory function, GPR120 also played a role in apoptosis as its knockdown impaired the antiapoptotic effect of DHA in OGD-induced rat pheochromocytoma (PC12) cells. Finally, using MCAO mouse model, we demonstrated that GPR120 activation protected against focal cerebral ischemic injury by preventing inflammation and apoptosis. Our study indicated that pharmacological targeting of GPR120 may provide a novel approach for the treatment of patients with ischemic stroke.

Upregulation of free fatty acid receptors in periodontal tissues of patients with metabolic syndrome and periodontitis

Metabolic syndrome (MetS) exacerbates periodontitis. Since saturated fatty acid (SFA) is increased in MetS and enhances lipopolysaccharide (LPS)-induced proinflammatory cytokine expression in macrophages, it has been considered to play a role in MetS-exacerbated periodontitis. However, it remains unknown how fatty acid receptors, which mediate the interaction of cells with SFA and uptake of SFA, are expressed and regulated in the periodontal tissue. In this study, we tested our hypothesis that the periodontal expression of fatty acid receptors GPR40 and CD36 is increased in patients with both MetS and periodontitis. We also determined the effect of SFA and LPS on GPR40 and CD36 expression in vitro. Periodontal tissue specimens were collected from 11 participants without MetS and periodontitis, 12 participants with MetS, 11 participants with periodontitis, and 14 participants with both MetS and periodontitis after surgeries. The tissues were processed, and GPR40 and CD36 were detected by immunohistochemistry. Furthermore, cultured macrophages and gingival fibroblasts were treated with LPS, palmitate, a major SFA, or LPS plus palmitate and the expression of GPR40 and CD36 was then quantified. Analysis of clinical data showed that age, smoker, gender, and race/ethnicity were not significantly different among 4 groups. Immunohistochemistry showed that GPR40 and CD36 were expressed by epithelial cells, fibroblasts, and immune cells. Quantitative data showed that GPR40 expression is increased in patients with periodontitis, MetS, or both periodontitis and MetS while CD36 expression is increased only in patients with both periodontitis and MetS. The in vitro studies showed that the expression of GPR40 and CD36 in macrophages and fibroblasts was upregulated by the combination of LPS and palmitate. Periodontal expression of GPR40 and CD36 was upregulated in patients with both MetS and periodontitis, and GPR40 and CD36 in macrophages and fibroblasts were upregulated in vitro by the combination of LPS and palmitate, suggesting that GPR40 and CD36 may be involved in MetS-exacerbated periodontitis.

Interaction of palmitate and LPS regulates cytokine expression and apoptosis through sphingolipids in human retinal microvascular endothelial cells

Studies have implicated saturated fatty acid (SFA) and lipopolysaccharide (LPS) in diabetic retinopathy. Since type 2 diabetes is associated with increases in both SFA and LPS in circulation, we investigated how SFA interacts with LPS to regulate proinflammatory cytokine expression and apoptosis in human retinal microvascular endothelial cells (HRMVECs) and the underlying mechanisms. HRMVECs were challenged with palmitate, a major SFA, LPS or palmitate plus LPS and the expression of proinflammatory cytokines were quantified using real-time PCR and enzyme-linked immunosorbent assay. The interaction between palmitate and LPS on inflammatory signaling and sphingolipid metabolism was demonstrated by immunoblotting and lipidomic analysis, respectively. The effect of palmitate and LPS on apoptosis was also studied by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) and histone-associated DNA fragment assays. Results showed that palmitate robustly stimulated the expression of proinflammatory cytokines including interleukin (IL)-6 and IL-1β, and the combination of palmitate and LPS further upregulated the proinflammatory cytokines by cooperatively stimulating inflammatory signaling pathways. Results also showed that while palmitate stimulated ceramide (CER) production via CER de novo synthesis and sphingomyelin (SM) hydrolysis, addition of LPS further increased CER de novo synthesis, but not SM hydrolysis. The involvement of sphingolipids in the cooperative stimulation by palmitate and LPS on cytokine expression was indicated by the findings that the inhibitor of CER de novo synthesis or SM hydrolysis attenuated the stimulation of IL-6 expression by palmitate and LPS. In addition, our study showed that fatty acid receptors GPR40 and CD36 were involved in the IL-6 upregulation by palmitate and LPS. Furthermore, palmitate induced apoptosis via CER production, but addition of LPS did not further increase apoptosis. Taken together, this study showed that palmitate interacted with LPS to upregulate cytokine expression via free fatty acid receptor-mediated inflammatory signaling and sphingolipid metabolism in HRMVECs. In contrast, the interaction between palmitate and LPS did not further increase apoptosis.

Role of wnt5a in Metabolic Inflammation in Humans.

Common nutrition-associated diseases like obesity and type 2 diabetes are linked to chronic low-grade inflammation. The secreted glycopeptide wingless-type mouse mammary tumor virus integration site family member 5a (wnt5a) has been implicated in metabolic inflammation in rodent models, suggesting a potential treatment target. Data on the role of wnt5a in human physiology have yielded conflicting results. Serum concentrations of wnt5a were measured in a cross-sectional cohort of 896 people to gain deeper insights into wnt5a physiology. Serum concentrations of wnt5a were measured by ELISA and related to several phenotyping and genotyping data. In vitro experiments were performed in THP-1 macrophages to examine potential molecular mechanisms. Wnt5a levels were significantly positively correlated to IL-6 and triglyceride levels. In subjects with diabetes, wnt5a levels were elevated and significantly correlated with fasting plasma glucose concentrations. Although wnt5a levels were not influenced by common single-nucleotide polymorphisms in the human wnt5a gene, environmental factors significantly altered wnt5a concentrations, as follows: (1) wnt5a levels were reduced in subjects with high nutritional load of the long-chain eicosatetraenoic acid independent of the total caloric intake and overall composition of the macronutrients, and (2) wnt5a levels were lower in humans with a high gut microbiome α diversity. In vitro experiments revealed that stimulation of the IL-6 receptor or the long-chain fatty acid receptor GPR40 directly affected wnt5a expression in human macrophages. Our data suggest that wnt5a is important in linking inflammation to metabolism. The nutrition and the microbiome might be interesting targets to prevent and/or treat wnt5a-mediated metabolic inflammation.

Grifolic acid induces GH3 adenoma cell death by inhibiting ATP production through a GPR120-independent mechanism

BackgroundGrifolic acid is a derivative of grifolin, an antitumor natural compound, and it was reported as an agonist of free fatty acid receptor GPR120. Little is known about its antitumor effects and the involvement of GPR120.MethodsGH3 cells, the rat anterior pituitary adenoma cells, were cultured and the cell death was measured by MTT assay and Annexin V/PI staining. The mitochondrial membrane potential (MMP) of GH3 cells was measured by JC-1 staining. Cellular ATP levels and the intracellular NAD/NADH ratio were measured. GPR120 expression in GH3 cells was observed by RT-PCR and Western Blot, and siRNA was used to inhibit GPR120 expression in GH3 cells.ResultsGrifolic acid dose- and time-dependently induced the necrosis of GH3 cells. Grifolic acid significantly reduced the mitochondrial membrane potential (MMP) and decreased cellular ATP levels in GH3 cells. In contrast, the MMP of isolated mitochondria was not decreased by grifolic acid. The intracellular NAD/NADH ratio was significantly increased by grifolic acid. GPR120 is expressed in GH3 cells, but GPR120 agonists such as EPA, GW9508 and TUG891 did not affect the viability of GH3 cells. Moreover, GPR120 siRNA knockdown showed no significant influence on grifolic acid-induced GH3 cell death.ConclusionGrifolic acid induces GH3 cell death by decreasing MMP and inhibiting ATP production, which may be due to the inhibition of NADH production through a GPR120-independent mechanism.

Flaxseed oil rich in omega-3 protects aorta against inflammation and endoplasmic reticulum stress partially mediated by GPR120 receptor in obese, diabetic and dyslipidemic mice models

The “first hit” to atherogenesis is driven by toll-like receptor 4, endoplasmic reticulum stress and ultimately metabolic dysfunction. In this study, we hypothesized that a flaxseed oil-enriched diet (FS) abolishes these inflammatory signaling pathway and restore metabolic homeostasis by activating the fatty acid receptor GPR120 in aorta of obese mice. Glucose homeostasis was assessed by GTT and ITT; lipidomics was performed using a Hybrid Ion Trap-Orbitrap Mass Spectrometer; serum lipids were measured using colorimetric assays; GPR120 and infiltrating macrophages were analyzed by immunofluorescence; protein immunoprecipitation and gene expression were evaluated by Western blot and RT-PCR, respectively. There were no differences in body weight and food intake between the groups from both strains (Swiss and LDLr-KO mice). GTT and cholesterol levels were improved by FS in both mice models. Lipidomics showed an increase in ω3 (C18:3) content, meanwhile stearic acid (C18:0) was not detected in endothelial tissue in response to FS. Moreover, FS markedly decreased pro-inflammatory (IL-1β, TNF-α, pIκBα, pIKKβ) and unfolded protein response markers (ATF6 and GRP78) in aorta. In Swiss mice, GPR120 was partially involved in the ω3-mediated anti-inflammatory actions, disrupting TLR4 pathway, but not in LDLr-KO mice. Partial replacement of dietary saturated by unsaturated ω3 fatty acids contributes to inhibition of cardiovascular risk markers, pro-inflammatory cytokines and ER stress sensors and effectors in the aorta. However, downregulation of inflammation is not mediated by arterial GPR120 activation.

Expression of the Fatty Acid Receptors GPR84 and GPR120 and Cytodifferentiation of Epithelial Cells in the Gastric Mucosa of Mouse Pups in the Course of Dietary Transition.

During weaning, the ingested food of mouse pups changes from exclusively milk to solid food. In contrast to the protein- and carbohydrate-rich solid food, high fat milk is characterized primarily by fatty acids of medium chain length particularly important for the suckling pups. Therefore, it seems conceivable that the stomach mucosa may be specialized for detecting these important nutrients during the suckling phase. Here, we analyzed the expression of the G protein coupled receptors GPR84 and GPR120 (FFAR4), which are considered to be receptors for medium and long chain fatty acids (LCFAs), respectively. We found that the mRNA levels for GPR84 and GPR120 were high during the suckling period and progressively decreased in the course of weaning. Visualization of the receptor-expressing cells in 2-week-old mice revealed a high number of labeled cells, which reside in the apical as well as in the basal region of the gastric glands. At the base of the gastric glands, all GPR84-immunoreactive cells and some of the GPR120-positive cells also expressed chromogranin A (CgA), suggesting that they are enteroendocrine cells. We demonstrate that the majority of the CgA/GPR84 cells are X/A-like ghrelin cells. The high degree of overlap between ghrelin and GPR84 decreased post-weaning, whereas the overlap between ghrelin and GPR120 increased. At the apical region of the glands the fatty acid receptors were mainly expressed in unique cell types. These contain lipid-filled vacuole- and vesicle-like structures and may have absorptive functions. We detected decreased immunoreactivity for GPR84 and no lipid droplets in surface cells post-weaning. In conclusion, expression of GPR84 in ghrelin cells as well as in surface cells suggests an important role of medium chain fatty acids (MCFAs) in the developing gastric mucosa of suckling mice.

Structural basis for the cooperative allosteric activation of the free fatty acid receptor GPR40.

Clinical studies indicate that partial agonists of the G-protein-coupled, free fatty acid receptor 1 GPR40 enhance glucose-dependent insulin secretion and represent a potential mechanism for the treatment of type 2 diabetes mellitus. Full allosteric agonists (AgoPAMs) of GPR40 bind to a site distinct from partial agonists and can provide additional efficacy. We report the 3.2-Å crystal structure of human GPR40 (hGPR40) in complex with both the partial agonist MK-8666 and an AgoPAM, which exposes a novel lipid-facing AgoPAM-binding pocket outside the transmembrane helical bundle. Comparison with an additional 2.2-Å structure of the hGPR40-MK-8666 binary complex reveals an induced-fit conformational coupling between the partial agonist and AgoPAM binding sites, involving rearrangements of the transmembrane helices 4 and 5 (TM4 and TM5) and transition of the intracellular loop 2 (ICL2) into a short helix. These conformational changes likely prime GPR40 to a more active-like state and explain the binding cooperativity between these ligands.

Discovery of Potent and Orally Bioavailable GPR40 Full Agonists Bearing Thiophen-2-ylpropanoic Acid Scaffold

The free fatty acid receptor GPR40 is predominantly expressed in pancreatic β-cells and enhances insulin secretion in a glucose dependent manner. Therefore, GPR40 agonists are possible novel insulin secretagogues with reduced or no risk of hypoglycemia for the treatment of type 2 diabetes mellitus (T2DM). Chemically and structurally diverse GPR40 agonists with high safety are pursued for the clinical development of GPR40-based pharmacotherapeutics. Herein we report our design and discovery of a new chemotype of GPR40 agonists free of the typical phenylpropanoic acid scaffold. The thiophen-2-ylpropanoic acid containing GPR40 modulators functioned as full agonists with high-efficacy response (Emax) and reduced lipophilicity. Significantly, the lead compound in this series, (R)-7k, exhibited more potent in vitro glucose-stimulated insulin secretion and in vivo glucose-lowering effects (10 mg/kg, po) than the GPR40 partial agonist TAK-875, which was once in phase III clinical trials, and high selectivity over the relevant receptors GPR120 and PPARγ.

Long-Chain Free Fatty Acid Receptor GPR120 Mediates Oil-Induced GIP Secretion Through CCK in Male Mice.

Free fatty acid receptors GPR120 and GPR40 are involved in the secretion of gut hormones. GPR120 and GPR40 are expressed in enteroendocrine K cells, and their activation induces the secretion of the incretin glucose-dependent insulinotropic polypeptide (GIP). However, the role of these receptors in fat-induced GIP secretion in vivo and the associated mechanisms are unclear. In this study, we investigated corn oil-induced GIP secretion in GPR120-knockout (GPR120-/-) and GPR40-knockout (GPR40-/-) mice. Oil-induced GIP secretion was reduced by 50% and 80% in GPR120-/- and GPR40-/- mice, respectively, compared with wild-type mice. This was not associated with a significant difference in K-cell number or GIP content in K cells, nor messenger RNA levels of the lipid receptor GPR119, nor bile acid receptors TGR5 and farnesoid X receptor. GPR120-/- and GPR40-/- mice also exhibited substantially decreased levels of cholecystokinin (CCK), a hormone from I cells that promotes bile and pancreatic lipase secretion, and this decrease was associated with impaired gallbladder contraction. Notably, treatment with a CCK analog resulted in recovery of oil-induced GIP secretion in GPR120-/- mice but not in GPR40-/- mice. These results indicate that corn oil-induced GIP secretion from K cells involves both GPR120 and GPR40 signaling pathways, and GPR120-induced GIP secretion is indirectly mediated by CCK.

Expression of fatty acid sensing G-protein coupled receptors in peripartal Holstein cows

BackgroundG-protein coupled receptors (GPCR), also referred as Free Fatty Acid Receptors (FFAR), are widely studied within human medicine as drug targets for metabolic disorders. To combat metabolic disorders prevalent in dairy cows during the transition period, which co-occur with negative energy balance and changes to lipid and glucose metabolism, it may be helpful to identify locations and roles of FFAR and other members of the GPCR family in bovine tissues.ResultsQuantitative RT-PCR (qPCR) of subcutaneous adipose, liver, and PMNL samples during the transition period (-10, +7, and +20 or +30 d) were used for expression profiling of medium- (MCFA) and long-chain fatty acid (LCFA) receptors GPR120 and GPR40, MCFA receptor GPR84, and niacin receptor HCAR2/3. Adipose samples were obtained from cows with either high (HI; BCS ≥ 3.75) or low (LO; BCS ≤ 3.25) body condition score (BCS) to examine whether FFAR expression is correlated with this indicator of health and body reserves. Supplementation of rumen-protected methionine (MET), which may improve immune function and production postpartum, was also compared with unsupplemented control (CON) cows for liver and blood polymorphonuclear leukocytes (PMNL) samples. In adipose tissue, GPR84 and GPR120 were differentially expressed over time, while GPR40 was not expressed; in PMNL, GPR40 was differentially expressed over time and between MET vs. CON, GPR84 expression differed only between dietary groups, and GPR120 was not expressed; in liver, GPCR were either not expressed or barely detectable.ConclusionsThe data indicate that there is likely not a direct role in liver for the selected GPCR during the transition period, but they do play variable roles in adipose and PMN. In future, these receptors may prove useful targets and/or markers for peripartal metabolism and immunity.

Discovery of benzofuran propanoic acid GPR120 agonists: From uHTS hit to mechanism-based pharmacodynamic effects

The transformation of an aryloxybutanoic acid ultra high-throughput screening (uHTS) hit into a potent and selective series of G-protein coupled receptor 120 (GPR120) agonists is reported. uHTS hit 1 demonstrated an excellent rodent pharmacokinetic profile and selectivity over the related fatty acid receptor GPR40, but only modest GPR120 potency. Optimization of the “left-hand” aryl group led to compound 6, which demonstrated a GPR120 mechanism-based pharmacodynamic effect in a mouse oral glucose tolerance test (oGTT). Further optimization gave rise to the benzofuran propanoic acid series (exemplified by compound 37), which demonstrated acute mechanism-based pharmacodynamic effects. The combination of in vivo efficacy and attractive rodent pharmacodynamic profiles suggests compounds generated from this series may afford attractive candidates for the treatment of Type 2 diabetes.