Chronic administration of hydrolysed pine nut oil to mice improves insulin sensitivity and glucose tolerance and increases energy expenditure via a free fatty acid receptor 4-dependent mechanism.

A healthy diet is at the forefront of measures to prevent type 2 diabetes. Certain vegetable and fish oils, such as pine nut oil (PNO), have been demonstrated to ameliorate the adverse metabolic effects of a high-fat diet. The present study investigates the involvement of the free fatty acid receptors 1 (FFAR1) and 4 (FFAR4) in the chronic activity of hydrolysed PNO (hPNO) on high-fat diet-induced obesity and insulin resistance. Male C57BL/6J wild-type, FFAR1 knockout (-/-) and FFAR4-/- mice were placed on 60 % high-fat diet for 3 months. Mice were then dosed hPNO for 24 d, during which time body composition, energy intake and expenditure, glucose tolerance and fasting plasma insulin, leptin and adiponectin were measured. hPNO improved glucose tolerance and decreased plasma insulin in the wild-type and FFAR1-/- mice, but not the FFAR4-/- mice. hPNO also decreased high-fat diet-induced body weight gain and fat mass, whilst increasing energy expenditure and plasma adiponectin. None of these effects on energy balance were statistically significant in FFAR4-/- mice, but it was not shown that they were significantly less than in wild-type mice. In conclusion, chronic hPNO supplementation reduces the metabolically detrimental effects of high-fat diet on obesity and insulin resistance in a manner that is dependent on the presence of FFAR4.

FFAR4 activation inhibits lung adenocarcinoma via blocking respiratory chain complex assembly associated mitochondrial metabolism.

Despite notable advancements in the investigation and management of lung adenocarcinoma (LUAD), the mortality rate for individuals afflicted with LUAD remains elevated, and attaining an accurate prognosis is challenging. LUAD exhibits intricate genetic and environmental components, and it is plausible that free fatty acid receptors (FFARs) may bridge the genetic and dietary aspects. The objective of this study is to ascertain whether a correlation exists between FFAR4, which functions as the primary receptor for dietary fatty acids, and various characteristics of LUAD, while also delving into the potential underlying mechanism. The findings of this study indicate a decrease in FFAR4 expression in LUAD, with a positive correlation (P < 0.01) between FFAR4 levels and overall patient survival (OS). Receiver operating characteristic (ROC) curve analysis demonstrated a significant diagnostic value [area under the curve (AUC) of 0.933] associated with FFAR4 expression. Functional investigations revealed that the FFAR4-specific agonist (TUG891) effectively suppressed cell proliferation and induced cell cycle arrest. Furthermore, FFAR4 activation resulted in significant metabolic shifts, including a decrease in oxygen consumption rate (OCR) and an increase in extracellular acidification rate (ECAR) in A549 cells. In detail, the activation of FFAR4 has been observed to impact the assembly process of the mitochondrial respiratory chain complex and the malate-aspartate shuttle process, resulting in a decrease in the transition of NAD+ to NADH and the inhibition of LUAD. These discoveries reveal a previously unrecognized function of FFAR4 in the negative regulation of mitochondrial metabolism and the inhibition of LUAD, indicating its potential as a promising therapeutic target for the treatment and diagnosis of LUAD.

Eicosapentaenoic Acid Rescues Cav1.2-L-Type Ca2+ Channel Decline Caused by Saturated Fatty Acids via Both Free Fatty Acid Receptor 4-Dependent and -Independent Pathways in Cardiomyocytes.

Dietary intake of omega-3 polyunsaturated fatty acids (eicosapentaenoic acid, EPA) exerts antiarrhythmic effects, although the mechanisms are poorly understood. Here, we investigated the possible beneficial actions of EPA on saturated fatty acid-induced changes in the L-type Ca2+ channel in cardiomyocytes. Cardiomyocytes were cultured with an oleic acid/palmitic acid mixture (OAPA) in the presence or absence of EPA. Beating rate reduction in cardiomyocytes caused by OAPA were reversed by EPA. EPA also retrieved a reduction in Cav1.2 L-type Ca2+ current, mRNA, and protein caused by OAPA. Immunocytochemical analysis revealed a distinct downregulation of the Cav1.2 channel caused by OAPA with a concomitant decrease in the phosphorylated component of a transcription factor adenosine-3',5'-cyclic monophosphate (cAMP) response element binding protein (CREB) in the nucleus, which were rescued by EPA. A free fatty acid receptor 4 (FFAR4) agonist TUG-891 reversed expression of Cav1.2 and CREB mRNA caused by OAPA, whereas an FFAR4 antagonist AH-7614 abolished the effects of EPA. Excessive reactive oxygen species (ROS) accumulation caused by OAPA decreased Cav1.2 and CREB mRNA expressions, which was reversed by an ROS scavenger. Our data suggest that EPA rescues cellular Cav1.2-Ca2+ channel decline caused by OAPA lipotoxicity and oxidative stresses via both free fatty acid receptor 4-dependent and -independent pathways.

Central activation of the fatty acid sensor GPR120 suppresses microglia reactivity and alleviates sickness- and anxiety-like behaviors.

G protein-coupled receptor 120 (GPR120, Ffar4) is a sensor for long-chain fatty acids including omega-3 polyunsaturated fatty acids (n-3 PUFAs) known for beneficial effects on inflammation, metabolism, and mood. GPR120 mediates the anti-inflammatory and insulin-sensitizing effects of n-3 PUFAs in peripheral tissues. The aim of this study was to determine the impact of GPR120 stimulation on microglial reactivity, neuroinflammation and sickness- and anxiety-like behaviors by acute proinflammatory insults. We found GPR120 mRNA to be enriched in  both murine and human microglia, and in situ hybridization revealed GPR120 expression in microglia of the nucleus accumbens (NAc) in mice. In a manner similar to or exceeding n-3 PUFAs, GPR120 agonism (Compound A, CpdA) strongly attenuated lipopolysaccharide (LPS)-induced proinflammatory marker expression in primary mouse microglia, including tumor necrosis factor-α (TNF-α) and interleukin-1ß (IL-1ß), and inhibited nuclear factor-ĸB translocation to the nucleus. Central administration of CpdA to adult mice blunted LPS-induced hypolocomotion and anxiety-like behavior and reduced TNF-α, IL-1ß and IBA-1 (microglia marker) mRNA in the NAc, a brain region modulating anxiety and motivation and implicated in neuroinflammation-induced mood deficits. GPR120 agonist pre-treatment attenuated NAc microglia reactivity and alleviated sickness-like behaviors elicited by central injection TNF-α and IL-1ß. These findings suggest that microglial GPR120 contributes to neuroimmune regulation and behavioral changes in response to acute infection and elevated brain cytokines. GPR120 may participate in the protective action of n-3 PUFAs at the neural and behavioral level and offers potential as treatment target for neuroinflammatory conditions.

Positive interplay between FFAR4/GPR120, DPP-IV inhibition and GLP-1 in beta cell proliferation and glucose homeostasis in obese high fat fed mice.

G-protein coupled receptor-120 (GPR120; FFAR4) is a free fatty acid receptor, widely researched for its glucoregulatory and insulin release activities. This study aimed to investigate the metabolic advantage of FFAR4/GPR120 activation using combination therapy. C57BL/6 mice, fed a High Fat Diet (HFD) for 120 days to induce obesity-diabetes, were subsequently treated with a single daily oral dose of FFAR4/GPR120 agonist Compound A (CpdA) (0.1µmol/kg) alone or in combination with sitagliptin (50 mg/kg) for 21 days. After 21-days, glucose homeostasis, islet morphology, plasma hormones and lipids, tissue genes (qPCR) and protein expression (immunocytochemistry) were assessed. Oral administration of CpdA improved glucose tolerance (34% p<0.001) and increased circulating insulin (38% p<0.001). Addition of CpdA with the dipeptidyl peptidase-IV (DPP-IV) inhibitor, sitagliptin, further improved insulin release (44%) compared to sitagliptin alone and reduced fat mass (p<0.05). CpdA alone (50%) and in combination with sitagliptin (89%) induced marked reductions in LDL-cholesterol, with greater effects in combination (p<0.05). All treatment regimens restored pancreatic islet and beta-cell area and mass, complemented with significantly elevated beta-cell proliferation rates. A marked increase in circulating GLP-1 (53%) was observed, with further increases in combination (38%). With treatment, mice presented with increased Gcg (proglucagon) gene expression in the jejunum (130% increase) and ileum (120% increase), indicative of GLP-1 synthesis and secretion. These data highlight the therapeutic promise of FFAR4/GPR120 activation and the potential for combined benefit with incretin enhancing DPP-IV inhibitors in the regulation of beta cell proliferation and diabetes.

Predicting FFAR4 agonists using structure-based machine learning approach based on molecular fingerprints.

Free Fatty Acid Receptor 4 (FFAR4), a G-protein-coupled receptor, is responsible for triggering intracellular signaling pathways that regulate various physiological processes. FFAR4 agonists are associated with enhancing insulin release and mitigating the atherogenic, obesogenic, pro-carcinogenic, and pro-diabetogenic effects, normally associated with the free fatty acids bound to FFAR4. In this research, molecular structure-based machine-learning techniques were employed to evaluate compounds as potential agonists for FFAR4. Molecular structures were encoded into bit arrays, serving as molecular fingerprints, which were subsequently analyzed using the Bayesian network algorithm to identify patterns for screening the data. The shortlisted hits obtained via machine learning protocols were further validated by Molecular Docking and via ADME and Toxicity predictions. The shortlisted compounds were then subjected to MD Simulations of the membrane-bound FFAR4-ligand complexes for 100 ns each. Molecular analyses, encompassing binding interactions, RMSD, RMSF, RoG, PCA, and FEL, were conducted to scrutinize the protein-ligand complexes at the inter-atomic level. The analyses revealed significant interactions of the shortlisted compounds with the crucial residues of FFAR4 previously documented. FFAR4 as part of the complexes demonstrated consistent RMSDs, ranging from 3.57 to 3.64, with minimal residue fluctuations 5.27 to 6.03 nm, suggesting stable complexes. The gyration values fluctuated between 22.8 to 23.5 nm, indicating structural compactness and orderliness across the studied systems. Additionally, distinct conformational motions were observed in each complex, with energy contours shifting to broader energy basins throughout the simulation, suggesting thermodynamically stable protein-ligand complexes. The two compounds CHEMBL2012662 and CHEMBL64616 are presented as potential FFAR4 agonists, based on these insights and in-depth analyses. Collectively, these findings advance our comprehension of FFAR4's functions and mechanisms, highlighting these compounds as potential FFAR4 agonists worthy of further exploration as innovative treatments for metabolic and immune-related conditions.

An atlas of GPCRs in dopamine neurons: Identification of the free fatty acid receptor 4 as a regulator of food and water intake.

Midbrain dopaminergic neurons (DANs) are subject to extensive metabotropic regulation, but the repertoire of G protein-coupled receptors (GPCRs) present in these neurons has not been mapped. Here, we isolate DANs from Dat-eGFP mice to generate a GPCR atlas by unbiased qPCR array expression analysis of 377 GPCRs. Combined with data mining of scRNA-seq databases, we identify multiple receptors in DAN subpopulations with 38 of these receptors representing the majority of transcripts. We identify 41 receptors expressed in midbrain DANs but not in non-DAN midbrain cells, including the free fatty acid receptor 4 (FFAR4). Functional expression of FFAR4 is validated by ex vivo Ca2+ imaging, and in vivo experiments support that FFAR4 negatively regulates food and water intake and bodyweight. In addition to providing a critical framework for understanding metabotropic DAN regulation, our data suggest fatty acid sensing by FFAR4 as a mechanism linking high-energy intake to the dopamine-reward pathway.