Recombinant Human Free fatty acid receptor 4 (FFAR4)

What is the genomic characterization of human FFAR4?

Free Fatty Acid Receptor 4 (FFAR4), also known as G-protein coupled receptor 120 (GPR120), is encoded by the FFAR4 gene located on the long arm of chromosome 10 at position 23.33 (10q23.33). It belongs to the rhodopsin-like GPR family within the broader G protein-coupled receptors, which in humans are encoded by more than 800 different genes . FFAR4 is part of a small family of structurally and functionally related GPRs that include FFAR1 (GPR40), FFAR2 (GPR43), and FFAR3 (GPR41), all of which bind and are activated by specific fatty acids .

How does FFAR4 expression change in metabolic disorders?

Research has demonstrated significant downregulation of FFAR4 expression in metabolic syndrome (MetS). Studies in both mouse models and human subjects show decreased FFAR4 expression in hippocampal tissue of MetS models (HFD and db/db mice) with concurrent cognitive impairment . Analysis of human peripheral blood leukocytes revealed significantly decreased FFAR4 mRNA expression in MetS patients with cognitive impairment compared to those without cognitive dysfunction . These findings suggest FFAR4 expression levels could potentially serve as a biomarker, as ROC curve analysis indicated diagnostic potential for identifying MetS patients with cognitive impairment .

What experimental methods are recommended for measuring FFAR4 expression?

For experimental quantification of FFAR4 expression, researchers should consider tissue-specific approaches. In animal models, direct measurement from hippocampal tissue via qPCR has proven effective . For clinical studies, peripheral blood leukocytes offer an accessible tissue source that shows correlation with central nervous system expression patterns . When isolating microglia for FFAR4 expression analysis, researchers should confirm high knockdown or overexpression efficiency through validated qPCR protocols as demonstrated in recent studies using Cx3cr1-CreER conditional systems .

What are the primary physiological functions of FFAR4?

FFAR4 plays multifaceted roles in regulating numerous bodily functions. Studies primarily conducted on human and rodent cultured cells and in animal models suggest that FFAR4 regulates food preferences, food consumption, taste perception, body weight maintenance, blood glucose levels, inflammatory responses, atherosclerosis progression, and bone remodeling . Recent research has also identified crucial roles in cognitive function and anxiety regulation, particularly in the context of metabolic syndrome, suggesting a broader neurological significance than previously recognized .

How does microglial FFAR4 influence cognitive function?

Microglial FFAR4 has emerged as a key regulator of cognitive function, particularly in metabolic syndrome conditions. Conditional knockout studies using microglial-specific FFAR4 deletion (crossing Ffar4 flox/flox mice with Cx3cr1-CreER transgenic mice) demonstrated exacerbated high-fat diet (HFD)-induced cognitive impairment and anxiety in behavioral tests including Morris water maze, open-field test, and elevated plus maze . Conversely, microglial-specific FFAR4 overexpression improved cognitive performance and reduced anxiety behaviors in HFD-fed mice . These findings establish a direct mechanistic link between microglial FFAR4 expression and neurobehavioral outcomes in metabolic syndrome.

What signaling mechanisms underlie FFAR4 action in microglia?

FFAR4 in microglia functions primarily through regulation of type I interferon signaling pathways. Mechanistic studies have revealed that microglial FFAR4 deficiency leads to significantly increased IFN-β mRNA expression and protein levels . This activation occurs through the NF-κB pathway, resulting in increased phosphorylation of JAK1 and STAT1 in the hippocampus of mice lacking microglial FFAR4 . Experimental inhibition of this pathway using fludarabine (a type I IFN signaling inhibitor) improved cognitive function and reduced anxiety in FFAR4-deficient mice, confirming the causal relationship between FFAR4, IFN signaling, and neurobehavioral outcomes .

What are the recommended methods for generating FFAR4 knockout models?

For studying FFAR4 function, researchers have successfully employed both conventional global knockout and conditional tissue-specific approaches. Global FFAR4 knockout models provide valuable insights into systemic effects but may not distinguish between tissue-specific functions . For more refined analysis, conditional knockout models using Cre-lox systems have proven effective. The most successful approach for microglial-specific deletion involves crossing Ffar4 flox/flox mice with Cx3cr1-CreER transgenic mice and subsequent tamoxifen induction . Importantly, when studying peripheral effects, researchers should allow for 4 weeks after tamoxifen treatment to ensure replacement of peripheral monocytes before commencing metabolic challenge protocols .

How can researchers effectively generate FFAR4 overexpression models?

For conditional overexpression studies, researchers have successfully created microglial FFAR4 overexpression models by crossing Ffar4 cag/cag mouse strains with Cx3cr1-CreER transgenic mice . This approach allows for tamoxifen-inducible overexpression specifically in microglia. Expression verification through qPCR is essential to confirm overexpression efficiency. These models have demonstrated significant protection against HFD-induced metabolic disturbances, including reduced body weight, fasting blood glucose, plasma triglycerides, and low-density lipoproteins compared to littermate controls .

What behavioral tests best assess FFAR4-mediated cognitive effects?

To comprehensively evaluate FFAR4's impact on cognitive function and anxiety behaviors, researchers should employ a battery of validated tests. The Morris water maze (MWM) provides robust assessment of spatial learning and memory, with key metrics being escape latency during training and platform crossing frequency during probe trials . For anxiety evaluation, the open-field test (measuring center distance and center zone entries), elevated plus maze (time spent in open arms and entries into open arms), and light-dark box tests have all demonstrated sensitivity to FFAR4-mediated effects . Using this multimodal testing approach allows detection of subtle behavioral phenotypes across cognitive and affective domains.

How does FFAR4 interact with inflammatory signaling pathways?

FFAR4 serves as a critical negative regulator of inflammatory pathways, particularly through NF-κB-mediated signaling. Mechanistic studies have demonstrated that FFAR4 deficiency leads to enhanced activation of NF-κB, which subsequently increases transcription of IFN-β . This activation triggers the JAK1/STAT1 signaling cascade, promoting neuroinflammation and associated behavioral abnormalities . Experimental evidence shows that microglial depletion and NF-κB inhibition partially reversed cognitive dysfunction and anxiety in microglia-specific FFAR4 knockout mice, confirming the causal relationship between these pathways and neurobehavioral outcomes .

What is the relationship between FFAR4 and type I interferon signaling?

Research has established that FFAR4 negatively regulates type I interferon signaling through NF-κB. In the absence of FFAR4, particularly in microglia, there is significant upregulation of IFN-β (but not IFN-α) at both mRNA and protein levels . This activation leads to increased phosphorylation of downstream mediators JAK1 and STAT1 . The functional significance of this pathway was confirmed through pharmacological intervention, as administration of fludarabine (a type I IFN signaling inhibitor) improved cognitive impairment and anxiety behavior in microglia-specific FFAR4 knockout mice exposed to high-fat diet .

How do genetic variants affect FFAR4 function?

Genetic variation in FFAR4 significantly impacts its function and metabolic outcomes. Most notably, the p.R270H variant of the FFAR4 gene is associated with loss of function, reduced protein activity, and increased risk of metabolic disorders . This genetic variation may explain inconsistent results observed in clinical trials of omega-3 PUFA supplementation, as FFAR4 is a primary receptor for these fatty acids . Future experimental designs should consider genotyping for FFAR4 variants, especially when translating findings to clinical applications, as genetic variations may significantly influence treatment efficacy and experimental outcomes .

What potential does FFAR4 hold as a therapeutic target?

FFAR4 represents a promising therapeutic target for multiple conditions, including excessive fatty food consumption, obesity, type 2 diabetes, pathological inflammatory reactions, atherosclerosis, cardiovascular disease, bone repair, osteoporosis, and certain cancers . Recent evidence has expanded this potential to include cognitive impairment and anxiety associated with metabolic syndrome . The therapeutic value of FFAR4 manipulation is supported by evidence that microglial FFAR4 overexpression improved metabolic parameters, including body weight, fasting blood glucose, plasma triglycerides, and LDL levels in HFD-fed mice, while simultaneously enhancing cognitive performance .

How might combination therapies targeting FFAR4 and NF-κB be developed?

Research suggests that combined targeting of FFAR4 activation and NF-κB inhibition may offer synergistic therapeutic benefits for neuroinflammatory conditions . While NF-κB inhibition alone has shown therapeutic promise, systemic blockade has significant toxicity concerns limiting clinical application . Experimental data indicates that combined treatment approaches targeting both pathways may be more effective than monotherapy . Researchers should consider experimental designs that pair docosahexaenoic acid (DHA, a FFAR4 ligand) with selective NF-κB inhibitors to evaluate potential synergistic effects on neuroinflammation and cognitive outcomes associated with metabolic syndrome .

What methodological considerations apply to FFAR4 agonist development?

Development of FFAR4-specific agonists faces several methodological challenges. While DHA (the main omega-3 PUFA in the brain) activates FFAR4 and shows promising effects in vitro, it lacks specificity for FFAR4 and has uncertain bioavailability when administered orally . This non-specificity may explain variable results in clinical trials. Researchers should focus on developing highly selective FFAR4 agonists with favorable pharmacokinetic profiles for CNS penetration. Additionally, genetic variation in FFAR4 (such as the p.R270H variant) should be considered during drug development, as these variants may affect ligand binding and downstream signaling efficacy .

How should researchers address inconsistent findings in FFAR4 studies?

Inconsistent findings in FFAR4 research may stem from several factors requiring methodological attention. First, genetic variants of FFAR4, particularly the p.R270H variant associated with loss of function, may contribute to variable experimental outcomes . Studies should include genotyping components when possible. Second, different fatty acids can bind to FFAR4 with varying affinities and may activate distinct downstream signaling pathways based on their structural properties (e.g., double bonds) . Researchers should carefully specify and control for the exact FFAR4 ligands used. Finally, tissue-specific FFAR4 expression patterns necessitate careful experimental design using appropriate conditional knockout/overexpression models rather than relying solely on global manipulation approaches .

What are promising directions for FFAR4 research in neurodegenerative contexts?

Future FFAR4 research should explore its potential role in neurodegenerative conditions beyond metabolic syndrome-associated cognitive impairment. While clinical trials have failed to demonstrate beneficial effects of omega-3 PUFA supplementation in moderate or severe Alzheimer's disease, there is evidence of potential benefit in mild AD or mild cognitive impairment . This suggests timing-dependent effects that warrant further investigation. Research should also examine how FFAR4 interacts with established neurodegenerative disease pathways, particularly those involving neuroinflammation. The connection between FFAR4 and GLP-1 regulation is especially promising, as GLP-1 agonists have shown a 53% reduced risk of dementia development in MetS patients in clinical trials .

How might FFAR4 research inform personalized medicine approaches?

The identification of genetic variants affecting FFAR4 function suggests important implications for personalized medicine. Particularly, the p.R270H variant of FFAR4 is associated with reduced protein activity and increased metabolic disorder risk . This genetic variation may explain why some individuals respond differently to omega-3 PUFA supplementation in clinical trials. Future research should stratify participants based on FFAR4 genotypes to determine if therapeutic responses to FFAR4 targeting or omega-3 supplementation vary by genetic background. This approach could lead to more tailored therapeutic strategies where interventions are matched to patients based on their FFAR4 genetic profile, potentially improving treatment efficacy for metabolic syndrome-related cognitive impairments .