Pharmacology of Free Fatty Acid Receptors and Their Allosteric Modulators

The physiological function of free fatty acids (FFAs) has long been regarded as indirect in terms of their activities as educts and products in metabolic pathways. The observation that FFAs can also act as signaling molecules at FFA receptors (FFARs), a family of G protein-coupled receptors (GPCRs), has changed the understanding of the interplay of metabolites and host responses. Free fatty acids of different chain lengths and saturation statuses activate FFARs as endogenous agonists via binding at the orthosteric receptor site. After FFAR deorphanization, researchers from the pharmaceutical industry as well as academia have identified several ligands targeting allosteric sites of FFARs with the aim of developing drugs to treat various diseases such as metabolic, (auto)inflammatory, infectious, endocrinological, cardiovascular, and renal disorders. GPCRs are the largest group of transmembrane proteins and constitute the most successful drug targets in medical history. To leverage the rich biology of this target class, the drug industry seeks alternative approaches to address GPCR signaling. Allosteric GPCR ligands are recognized as attractive modalities because of their auspicious pharmacological profiles compared to orthosteric ligands. While the majority of marketed GPCR drugs interact exclusively with the orthosteric binding site, allosteric mechanisms in GPCR biology stay medically underexploited, with only several allosteric ligands currently approved. This review summarizes the current knowledge on the biology of FFAR1 (GPR40), FFAR2 (GPR43), FFAR3 (GPR41), FFAR4 (GPR120), and GPR84, including structural aspects of FFAR1, and discusses the molecular pharmacology of FFAR allosteric ligands as well as the opportunities and challenges in research from the perspective of drug discovery.     

Regulator of G-Protein Signaling-4 Attenuates Cardiac Adverse Remodeling and Neuronal Norepinephrine Release-Promoting Free Fatty Acid Receptor FFAR3 Signaling

Propionic acid is a cell nutrient but also a stimulus for cellular signaling. Free fatty acid receptor (FFAR)-3, also known as GPR41, is a Gi/o protein-coupled receptor (GPCR) that mediates some of the propionate’s actions in cells, such as inflammation, fibrosis, and increased firing/norepinephrine release from peripheral sympathetic neurons. The regulator of G-protein Signaling (RGS)-4 inactivates (terminates) both Gi/o- and Gq-protein signaling and, in the heart, protects against atrial fibrillation via calcium signaling attenuation. RGS4 activity is stimulated by β-adrenergic receptors (ARs) via protein kinase A (PKA)-dependent phosphorylation. Herein, we examined whether RGS4 modulates cardiac FFAR3 signaling/function. We report that RGS4 is essential for dampening of FFAR3 signaling in H9c2 cardiomyocytes, since siRNA-mediated RGS4 depletion significantly enhanced propionate-dependent cAMP lowering, Gi/o activation, p38 MAPK activation, pro-inflammatory interleukin (IL)-1β and IL-6 production, and pro-fibrotic transforming growth factor (TGF)-β synthesis. Additionally, catecholamine pretreatment blocked propionic acid/FFAR3 signaling via PKA-dependent activation of RGS4 in H9c2 cardiomyocytes. Finally, RGS4 opposes FFAR3-dependent norepinephrine release from sympathetic-like neurons (differentiated Neuro-2a cells) co-cultured with H9c2 cardiomyocytes, thereby preserving the functional βAR number of the cardiomyocytes. In conclusion, RGS4 appears essential for propionate/FFAR3 signaling attenuation in both cardiomyocytes and sympathetic neurons, leading to cardioprotection against inflammation/adverse remodeling and to sympatholysis, respectively.     

The Anti-Atherosclerotic Action of FFAR4 Agonist TUG-891 in ApoE–Knockout Mice Is Associated with Increased Macrophage Polarization towards M2 Phenotype

Background: Over the past few years, a better understanding of the biology of G-protein coupled receptors (GPRs) has led to the identification of several receptors as novel targets for free fatty acids (FFAs). FFAR4 has received special attention in the context of chronic inflammatory diseases, including atherosclerosis, obesity and NAFLD, through to its anti-inflammatory effect. Methods: The present study investigates the influence of prolonged treatment with TUG-891-FFAR4 agonist on the development of atherosclerosis plaque in apoE-knockout mice, using morphometric and molecular methods. Results: TUG-891 administration has led to the reduction of atherosclerotic plaque size and necrotic cores in an apoE-knockout mice model. TUG-891-treated mice were administered subcutaneously at a dose of 20 mg/kg three times a week for 4 months. The FFAR4 agonist reduced the content of pro-inflammatory M1-like macrophages content in atherosclerotic plaques, as evidenced by immunohistochemical phenotyping and molecular methods. In atherosclerotic plaque, the population of smooth muscle cells increased as evidenced by α-SMA staining. We observed changes in G-CSF and eotaxin markers in the plasma of mice; changes in the levels of these markers in the blood may be related to macrophage differentiation. Importantly, we observed a significant increase in M2-like macrophage cells in atherosclerotic plaque and peritoneum. Conclusions: Prolonged administration of TUG-891 resulted in significant amelioration of atherogenesis, providing evidence that the strategy based on macrophage phenotype switching toward an M2-like activation state via stimulation of FFAR4 receptor holds promise for a new approach in the prevention or treatment of atherosclerosis.     

Gut Microbiota and Short Chain Fatty Acids: Implications in Glucose Homeostasis

Gut microbiota encompasses a wide variety of commensal microorganisms consisting of trillions of bacteria, fungi, and viruses. This microbial population coexists in symbiosis with the host, and related metabolites have profound effects on human health. In this respect, gut microbiota plays a pivotal role in the regulation of metabolic, endocrine, and immune functions. Bacterial metabolites include the short chain fatty acids (SCFAs) acetate (C2), propionate (C3), and butyrate (C4), which are the most abundant SCFAs in the human body and the most abundant anions in the colon. SCFAs are made from fermentation of dietary fiber and resistant starch in the gut. They modulate several metabolic pathways and are involved in obesity, insulin resistance, and type 2 diabetes. Thus, diet might influence gut microbiota composition and activity, SCFAs production, and metabolic effects. In this narrative review, we discuss the relevant research focusing on the relationship between gut microbiota, SCFAs, and glucose metabolism.     

The Role of Short-Chain Fatty Acids in the Interplay between a Very Low-Calorie Ketogenic Diet and the Infant Gut Microbiota and Its Therapeutic Implications for Reducing Asthma

Gut microbiota is well known as playing a critical role in inflammation and asthma development. The very low-calorie ketogenic diet (VLCKD) is suggested to affect gut microbiota; however, the effects of VLCKD during pregnancy and lactation on the infant gut microbiota are unclear. The VLCKD appears to be more effective than caloric/energy restriction diets for the treatment of several diseases, such as obesity and diabetes. However, whether adherence to VLCKD affects the infant gut microbiota and the protective effects thereof on asthma remains uncertain. The exact mechanisms underlying this process, and in particular the potential role of short chain fatty acids (SCFAs), are still to be unravelled. Thus, the aim of this review is to identify the potential role of SCFAs that underlie the effects of VLCKD during pregnancy and lactation on the infant gut microbiota, and explore whether it incurs significant implications for reducing asthma.     

The Metabolic Role of Gut Microbiota in the Development of Nonalcoholic Fatty Liver Disease and Cardiovascular Disease

The prevalence of metabolic disorders, such as type 2 diabetes (T2D), obesity, and non-alcoholic fatty liver disease (NAFLD), which are common risk factors for cardiovascular disease (CVD), has dramatically increased worldwide over the last decades. Although dietary habit is the main etiologic factor, there is an imperfect correlation between dietary habits and the development of metabolic disease. Recently, research has focused on the role of the microbiome in the development of these disorders. Indeed, gut microbiota is implicated in many metabolic functions and an altered gut microbiota is reported in metabolic disorders. Here we provide evidence linking gut microbiota and metabolic diseases, focusing on the pathogenetic mechanisms underlying this association.     

Altered Gut Microbiota and Its Metabolites in Hypertension of Developmental Origins: Exploring Differences between Fructose and Antibiotics Exposure

Gut microbiota-derived metabolites, in particular short chain fatty acids (SCFAs) and their receptors, are linked to hypertension. Fructose and antibiotics are commonly used worldwide, and they have a negative impact on the gut microbiota. Our previous study revealed that maternal high-fructose (HF) diet-induced hypertension in adult offspring is relevant to altered gut microbiome and its metabolites. We, therefore, intended to examine whether minocycline administration during pregnancy and lactation may further affect blood pressure (BP) programmed by maternal HF intake via mediating gut microbiota and SCFAs. Pregnant Sprague-Dawley rats received a normal diet or diet containing 60% fructose throughout pregnancy and lactation periods. Additionally, pregnant dams received minocycline (50 mg/kg/day) via oral gavage or a vehicle during pregnancy and lactation periods. Four groups of male offspring were studied (n = 8 per group): normal diet (ND), high-fructose diet (HF), normal diet + minocycline (NDM), and HF + minocycline (HFM). Male offspring were killed at 12 weeks of age. We observed that the HF diet and minocycline administration, both individually and together, causes the elevation of BP in adult male offspring, while there is no synergistic effect between them. Four groups displayed distinct enterotypes. Minocycline treatment leads to an increase in the F/B ratio, but decreased abundance of genera Lactobacillus, Ruminococcus, and Odoribacter. Additionally, minocycline treatment decreases plasma acetic acid and butyric acid levels. Hypertension programmed by maternal HF diet plus minocycline exposure is related to the increased expression of several SCFA receptors. Moreover, minocycline- and HF-induced hypertension, individually or together, is associated with the aberrant activation of the renin–angiotensin system (RAS). Conclusively, our results provide a new insight into the support of gut microbiota and its metabolite SCAFs in the developmental programming of hypertension and cast new light on the role of RAS in this process, which will help prevent hypertension programmed by maternal high-fructose and antibiotic exposure.     

Activation of Free Fatty Acid Receptor 4 (FFA4) Ameliorates Ovalbumin-Induced Allergic Asthma by Suppressing Activation of Dendritic and Mast Cells in Mice

Epidemiological and clinical studies have suggested that intake of n-3 polyunsaturated fatty acids (PUFA) reduces the incidence of allergic airway diseases and improves pulmonary function in patients with allergic asthma. However, the pharmacological targets of PUFA have not been elucidated upon. We investigated whether free fatty acid receptor 4 (FFA4, also known as GPR120) is a molecular target for beneficial PUFA in asthma therapy. In an ovalbumin (OVA)-induced allergic asthma model, compound A (a selective agonist of FFA4) was administrated before OVA sensitization or OVA challenge in FFA4 wild-type (WT) and knock-out (KO) mice. Compound A treatment of RBL-2H3 cells suppressed mast cell degranulation in vitro in a concentration-dependent manner. Administration of compound A suppressed in vivo allergic characteristics in bronchoalveolar lavage fluid (BALF) and lungs, such as inflammatory cytokine levels and eosinophil accumulation in BALF, inflammation and mucin secretion in the lungs. Compound A-induced suppression was not only observed in mice treated with compound A before OVA challenge, but in mice treated before OVA sensitization as well, implying that compound A acts on mast cells as well as dendritic cells. Furthermore, this suppression by compound A was only observed in FFA4-WT mice and was absent in FFA4-KO mice, implying that compound A action is mediated through FFA4. Activation of FFA4 may be a therapeutic target of PUFA in allergic asthma by suppressing the activation of dendritic cells and mast cells, suggesting that highly potent specific agonists of FFA4 could be a novel therapy for allergic asthma.     

Role of Short-Chain Fatty Acids Produced by Gut Microbiota in Innate Lung Immunity and Pathogenesis of the Heterogeneous Course of Chronic Obstructive Pulmonary Disease

Chronic obstructive pulmonary disease (COPD) is a widespread socially significant disease. The development of COPD involves the innate immune system. Interestingly, the regulation of the innate lung immune system is related to the gut microbiota. This connection is due to the production by gut microorganisms of short-chain fatty acids (SCFAs) such as acetate, propionate, and butyrate. Nutritional disturbances and changes in the structure of the intestinal microbiota lead to a decrease in SCFAs production and their effect on pulmonary immunity. The presence of a metabolic and immune axis linking the lungs and gut plays an important role in the pathogenesis of COPD. In addition, the nature of nutrition and SCFAs may participate in the development of the clinically heterogeneous course of COPD.     

Effect of FFA of Crude Rice Bran Oil on the Properties of Diesel Blends

Crude rice bran oil (CRBO) with high free fatty acid (FFA) content is not suitable for eating purposes, however, it can be used as a fuel to partially replace or fully replace No.2 diesel. The main objective of the present work was to analyse the effect of FFA content of CRBO on the combustion properties such as viscosity, calorific value, volatility and aniline point. CRBO with different FFAs were collected and mixed with No.2 diesel to prepare CRBO-diesel blends. It was observed that the viscosity of the blends increased with increase in FFA while the calorific value decreased. Significant variations were observed in the distillation curve for the CRBO blends with different FFA. Aniline point of the blends was 10–15% lower than that of diesel and it is indirectly proportional to the FFA of CRBO in the blend. Experimental results showed that the combustion properties of CRBO are the function of the FFA in the oil. As a dilute blend with diesel, CRBO with high FFA content showed comparable combustion properties to that of diesel. The properties differed in magnitude by 10–15% when compared with diesel. From the present investigation it is concluded that in blended form, CRBO with high FFA can be a potential resource to utilize it as an alternative fuel for CI (compression ignition) engines.     

The Importance of Free Fatty Chain Length on the Lipid Organization in the Long Periodicity Phase

The skin’s barrier ability is an essential function for terrestrial survival, which is controlled by intercellular lipids within the stratum corneum (SC) layer. In this barrier, free fatty acids (FFAs) are an important lipid class. As seen in inflammatory skin diseases, when the lipid chain length is reduced, a reduction in the barrier’s performance is observed. In this study, we have investigated the contributing effects of various FFA chain lengths on the lamellar phase, lateral packing. The repeat distance of the lamellar phase increased with FFA chain length (C20–C28), while shorter FFAs (C16 to C18) had the opposite behaviour. While the lateral packing was affected, the orthorhombic to hexagonal to fluid phase transitions were not affected by the FFA chain length. Porcine SC lipid composition mimicking model was then used to investigate the proportional effect of shorter FFA C16, up to 50% content of the total FFA mixture. At this level, no difference in the overall lamellar phases and lateral packing was observed, while a significant increase in the water permeability was detected. Our results demonstrate a FFA C16 threshold that must be exceeded before the structure and barrier function of the long periodicity phase (LPP) is affected. These results are important to understand the lipid behaviour in this unique LPP structure as well as for the understanding, treatment, and development of inflammatory skin conditions.     

Synthesis and Pharmacological Properties of Silicon‐Containing GPR81 and GPR109A Agonists

The GPR81 and GPR109A receptors mediate antilipolytic effects and are potential drug targets for the treatment of metabolic disorders such as dyslipidemia and type 2 diabetes. There is still a need to identify potent GPR81 agonists as pharmacological tools. A high‐throughput screen identified an acylurea‐based GPR81 agonist lead series, with activities at the GPR109A receptor as well. To expand the chemical scope and to explore the pharmacological and pharmacokinetic consequences, a series of structurally related organosilicon compounds with a 6‐sila‐4,5,6,7‐tetrahydrobenzo[d]thiazole skeleton was synthesized and studied for their physicochemical properties [octanol/water distribution coefficient (pH 7.4), solubility in HBSS buffer (pH 7.4)], agonistic potency at rat GPR81 and GPR109A receptors, and intrinsic clearance in human liver microsomes and rat hepatocytes. The straightforward synthesis of these organosilicon compounds offered a valuable expansion of the chemical scope in the above‐mentioned GPR81 agonist lead series, provided potency and efficacy SAR, and yielded compounds with sub‐micromolar GPR81 potency. This work supports the value of including silicon chemistry into the toolbox of medicinal chemistry.     

Rapid colorimetric determination of free fatty acids

In 1964, a method was described for the determination of free fatty acids (FFAs) in vegetable oil. This paper describes an expansion of that work, improving the sensitivity and reproducibility of the method, as well as examination of solubilities of the copper soaps as a function of chain length and unsaturation. Involvement of the micellar structure was reviewed. Finally, a procedure is described that permits very rapid determination of FFA at the 2.0–14.0 μmol (0.5–4.0 mg oleic acid) level, and the results with several oils are given. Particular attention was given to evaluation of solvent systems which would extract the copper complexes. Presented in part at the AOCS Meeting in San Francisco, April 1969. Technical Paper No. 4036, Oregon Agricultural Experiment Station.     

Interactions between medium-chain and long-chain triacylglycerols in lipid and energy metabolism in growing chicks

The combined effects of dietary medium-chain triacylglycerols (MCT) and long-chain triacylglycerols (LCT) on lipid and energy utilization in chicks were investigated. Corn oil was used as the LCT source, and trioctanoin (8∶0) was used as the MCT source. The efficiency of dietary energy utilization (such as metabolizable energy values and fat and energy retention) decreased linearly as the level of MCT increased, but the efficiency of dietary protein utilization (protein retained per protein consumed) was not affected in a consistent manner. Fecal saturated and monounsaturated fatty acid composition was dependent on the dietary fatty acid composition, whereas fecal linoleic acid content was not. It is concluded that dietary MCT and LCT influence each other to some degree, with respect to protein and lipid metabolism in chicks. Moreover, in most cases, the nutritional characteristics of each triacylglycerol, including food efficiency and fat and energy retention, are independent of each other in growing chicks.     

The Role of Gut Microbiota and Gut–Brain Interplay in Selected Diseases of the Central Nervous System

The gut microbiome has attracted increasing attention from researchers in recent years. The microbiota can have a specific and complex cross-talk with the host, particularly with the central nervous system (CNS), creating the so-called “gut–brain axis”. Communication between the gut, intestinal microbiota, and the brain involves the secretion of various metabolites such as short-chain fatty acids (SCFAs), structural components of bacteria, and signaling molecules. Moreover, an imbalance in the gut microbiota composition modulates the immune system and function of tissue barriers such as the blood–brain barrier (BBB). Therefore, the aim of this literature review is to describe how the gut–brain interplay may contribute to the development of various neurological disorders, combining the fields of gastroenterology and neuroscience. We present recent findings concerning the effect of the altered microbiota on neurodegeneration and neuroinflammation, including Alzheimer’s and Parkinson’s diseases, as well as multiple sclerosis. Moreover, the impact of the pathological shift in the microbiome on selected neuropsychological disorders, i.e., major depressive disorders (MDD) and autism spectrum disorder (ASD), is also discussed. Future research on the effect of balanced gut microbiota composition on the gut–brain axis would help to identify new potential opportunities for therapeutic interventions in the presented diseases.     

Biodiesel Production using Lewis Catalysts under Ultrasonic Activation

Biodiesel can be produced by transesterification of oil triglycerides, as well as by esterification of fatty acids using feedstock materials rich in free fatty acids)FFAs). Utilization of such feedstocks can gain substantial economical profit and solve a problem of disposal of residual raw materials. To find an effective method of biodiesel production from FFA, we developed esterification of FFAs under ultrasonic activation using Lewis acids as catalysts. Homologous catalyst BF₃ and heterogeneous AlCl₃ were highly effective in reactions of model FFAs methylation under ultrasonic activation, when the reaction yield reached 100 %. Ethylation at the same conditions was less successful.