Free Fatty Acid Receptor Research Articles

Research progress on free fatty acid receptors and bronchial asthma

Research progress on free fatty acid receptors and bronchial asthma

Modeling of culture conditions by culture system, glucose and propionic acid and their impact on metabolic profile in IPEC-J2.

The microbiological environment and their corresponding secreted metabolite spectrum are an essential modulator of the enterocyte function, effecting the whole organism. Intestinal porcine jejunal epithelial cell line (IPEC-J2) is an established in vitro model for differentiation of enterocytes in different cell culture models. An improved oxygen supply seems to be the main reason for differentiation in an air-liquid-interface culture, but this has not yet been conclusively clarified. In this context, the nutrition of the cell and its influence on the metabolism is also of crucial importance. The interest in short-chain fatty acids (SCFAs) has grown steadily in recent years due to their clinical relevance in certain diseases such as multiple sclerosis and other inflammatory diseases, but not much is known of FFAR2 and FFAR3 (free fatty acid receptor 2 and 3) in pigs. We want to address the questions: 1. about the distribution of FFAR2 and FFAR3 in vivo and in vitro in sus scrofa 2. whether there is an influence of propionic acid, glucose content and cultivation on metabolism of enterocytes? The morphological analysis of FFAR2 and FFAR3 in vivo was investigated through immunostaining of frozen sections of the porcine gut segments jejunum, ileum and colon. Both receptors are expressed along the gut and were found in the smooth muscle cells of the tunica muscularis and lamina muscularis mucosae. Furthermore, a high expression of FFAR2 and a low expression of FFAR3 in the enteric nerve system was also observed in jejunum, ileum and colon of sus scrofa. In addition, FFAR2 and FFAR3 within the vessels was investigated. FFAR3 showed a strong expression on endothelial cells of veins and lymphatic vessels but was not detectable on arteries. Furthermore, we demonstrate for the first time, FFAR2 and FFAR3 in IPEC-J2 cells on RNA- and protein level, as well as with confocal microscopy. In addition, ENO1 and NDUFA4 were investigated on RNA-level in IPEC-J2 cells as 2 important genes, which play an essential role in metabolism. Here, NDUFA4 is detected in the model animal sus scrofa as well as in the porcine cell line IPEC-J2. A potential impact of propionic acid and/or glucose and/or cultivation method on the metabolism of the cells was tested with the Seahorse analyzer. Here, a significant higher ECAR was observed in the SMC than in the OCR. In summary, we were able to show that the cultivation system appears to have a greater influence than the medium composition or nutrition of the cells. However, this can be modulated by incubation time or combination of different SCFAs.

In silico determination of fenthion, permethrin, and carbaryl as FFAR2 inhibitors: Type 2 diabetes mellitus pathomechanism study

Background: In the last few decades, many studies have shown that pesticides have a close relationship with increasing blood glucose levels and the incidence of diabetes. Some examples of pesticides include fenthion, permethrin, and carbaryl. Recently, free fatty acid receptor 2 (FFAR2) was identified as having a critical function in preventing insulin resistance. Activation of FFAR2 will reduce fat accumulation and induce glucagon-like peptide 1 (GLP-1) secretion, which plays an important role in regulating type 2 diabetes mellitus (T2DM) prevention.Objective: This study aims to determine a comparison of the binding ability between fenthion, permethrin, and carbaryl to FFAR2 protein for predicting the mechanism of pesticide toxicity to T2DM through an in silico study.Methods: This is an exploratory bioinformatic study. The protein structure was FFAR2 receptor (UniProt: O15552), while the ligand was fenthion (PubChem CID: 3346), permethrin (PubChem CID: 40326), and carbaryl (PubChem CID: 6129). This molecular docking was conducted in October 2022 using Asus X202XE with Intel® Core™ i3-3217U CPU equipped with BIOVIA Discovery Studio, AutoDockTools, and AutoDock Vina. Results: The binding affinity values generated after docking between fenthion, permethrin, and carbaryl with FFAR2 indicate that the binding affinity comparison is permethrin < carbaryl < fenthion. This explains that permethrin could form a stronger bond with FFAR2 protein than other pesticides. However, the visualisation results of the form of bond interactions show that permethrin does not bind to the active site of FFAR2, so it could not be called an inhibitor. This is different from fenthion and carbaryl, which could bind to several amino acid residues on the active site of FFAR2 and have the potential to become inhibitors.Conclusion: Carbaryl is a pesticide with the strongest FFAR2 inhibitor. Carbaryl could cause type 2 DM through its inhibitory pathway to FFAR2.

A MECHANISM OF ACTION OF HUMAN-ORIGIN PROBIOTIC COCKTAIL IN ATTENUATING ALZHEIMER’S DISEASE PATHOLOGY

Abstract Alzheimer’s disease (AD) is a detrimental public health problem affecting millions of older adults in the USA alone, with no or limited treatment options. Emerging evidence indicates the pivotal role of gut microbiota in AD pathology, but mechanisms remain largely unknown. Also, microbiome modulators like probiotics are proven beneficial in preventing AD with little understanding of their mechanisms. Therefore, we investigated how a microbiota metabolite-short-chain free fatty acid receptor-2 (FFAR2) signaling in the intestine affects AD pathology in brain using intestinal FFAR2 knock-out APP/PS-1 mice; as well as determined if a human-origin-probiotic cocktail exhibits its actions via FFAR2 signaling. We measured changes in the microbiome, permeability in gut and blood-brain barriers (BBB), neuroinflammation, AD pathology, and cognitive behaviors. Results showed, the mice lacking FFAR2 in their intestine exacerbated AD pathology by developing abnormal changes in gut microbiome and intestinal permeability, which in turn promotes permeability in BBB and neuroinflammation. It’s also promoting microglia activation linked with increased Aβ accumulation and cognitive decline. It’s interesting to note that probiotic cocktail feeding reversed these abnormalities in AD mouse models. Further, this probiotic cocktail showed its mechanism in both FFAR2-dependent and independent manner. Overall, our findings indicate that the microbiome sensing mechanism FFAR2 signaling participates in regulating the gut-brain in AD pathology, and a human-origin probiotic cocktail improves the abnormal changes in the microbiome-intestinal & BBB permeability-neuroinflammation axis to ameliorate AD pathology. Thus, FFAR2 agonism and probiotic therapy can be viable options to prevent/ delay the risk of AD progression in older adults.

SUPPRESSION OF MICROBIOME SENSING—FREE FATTY ACID RECEPTOR 2 (FFAR2) SIGNALING EXHIBITS EARLY AGING

Abstract Abnormalities in gut microbiome contribute to age-related diseases, causal to unhealthy aging, and early mortality, but mechanisms still remain largely unknown. Our recent research shows that old microbiota lacks the capacity to produce beneficial metabolites like short-chain fatty acids (SCFAs), which in turn promote gut permeability and systemic inflammation. SCFAs activates free fatty acid receptor 2 and 3 (FFAR2/3) signaling, at least in part, to exhibit their benefits on host cells; however, the role of these signaling in aging process is not well understood. We discovered that in addition to gut the brains of old mice show lower expression of FFAR2. Further, FFAR2 deletion in the intestine shows a shorter life span compared to wild-type mice. They also show a higher cognitive decline at early age. Similarly, intestine specific FFAR2 KO in APP/PS1 (Alzheimer’s disease [AD] model) background shows higher progression of AD including amyloid beta (A-beta) and tau accumulation, linked with higher blood brain barrier (BBB) permeability and neuroinflammation. These changes are associated with increased gut permeability and systemic inflammaging. Interestingly, a newly discovered natural FFAR2 agonist- Fenchol reduces these abnormalities in gut-brain and prevented the progression of cognitive decline and AD pathology. These results show that microbiome sensing FFAR2 signaling deficiency develops an early aging phenotype in mice, while its agonism using natural compound can delay/ prevent the progression of early aging.

Antidiabetic efficacy of sea fish oil in ameliorating hyperglycaemia by enhancing FFAR1, GLP-1 and inhibiting DPP-4 signalling in the pancreatic tissues of high lipid diet and streptozotocin-induced type 2 diabetic rats

Eicosapentaenoic acid and docosahexaenoic acid are n-3 fatty acids that are highly available in sea-fishes. Many studies have revealed that n-3 fatty acids play antidiabetic activity. In this study antidiabetic activity of the three sea fishes oils, ‘volavetki’ (Panna microdon, Bleeker 1849), ‘ruli’ (Coilia dussumieri, Valenciennes 1848), and ‘tapra’ (Opisthopterus tardoore, Cuvier 1829), as well as three fresh-water fishes oils, ‘bata’ (Labeo bata, Hamilton 1822), ‘folui’ (Opisthopterus tardoore, Cuvier 1829), and ‘mourala’ (Amblypharyngodon mola, Hamilton 1822) evaluated high lipid diet (HLD) and STZ-induced type 2 diabetes mellitus (T2DM) rats. The GC-MS analysis revealed high n-3 fatty acid content in volavetki oil. Supplementation of sea-fish oils and antidiabetic effect was monitored by fasting plasma blood glucose and HbA1c. Also, insulin, c-peptide, glucagon-like peptide-1, dipeptidyl peptidase-4, free fatty acid receptor 1, glucokinase, glucose-6 phosphatase, superoxide dismutase, glutathione peroxidase, catalase, malondialdehyde, plasma total cholesterol, low density lipoprotein, triglycerides, high density lipoprotein, c-reactive protein, total protein, alkaline phosphatase, and histopathology of pancreatic tissues were evaluated. Out of six fish oils, oral supplementation with volavetki oil resulted in significant reduction of plasma blood glucose, HbA1c of T2DM rat. All other parameters in T2DM rats were returned to near normally by treatment of volavetki oil. These findings strongly indicate that volavetki oil possess antidiabetic activity.

Phosphorylation bar-coding of free fatty acid receptor 2 is generated in a tissue-specific manner.

Free fatty acid receptor 2 (FFAR2) is activated by short-chain fatty acids and expressed widely, including in white adipocytes and various immune and enteroendocrine cells. Using both wild-type human FFAR2 and a designer receptor exclusively activated by designer drug (DREADD) variant we explored the activation and phosphorylation profile of the receptor, both in heterologous cell lines and in tissues from transgenic knock-in mouse lines expressing either human FFAR2 or the FFAR2-DREADD. FFAR2 phospho-site-specific antisera targeting either pSer296/pSer297 or pThr306/pThr310 provided sensitive biomarkers of both constitutive and agonist-mediated phosphorylation as well as an effective means to visualise agonist-activated receptors in situ. In white adipose tissue, phosphorylation of residues Ser296/Ser297 was enhanced upon agonist activation whilst Thr306/Thr310 did not become phosphorylated. By contrast, in immune cells from Peyer's patches Thr306/Thr310 become phosphorylated in a strictly agonist-dependent fashion whilst in enteroendocrine cells of the colon both Ser296/Ser297 and Thr306/Thr310 were poorly phosphorylated. The concept of phosphorylation bar-coding has centred to date on the potential for different agonists to promote distinct receptor phosphorylation patterns. Here, we demonstrate that this occurs for the same agonist-receptor pairing in different patho-physiologically relevant target tissues. This may underpin why a single G protein-coupled receptor can generate different functional outcomes in a tissue-specific manner.

Phosphorylation bar-coding of free fatty acid receptor 2 is generated in a tissue-specific manner

Free fatty acid receptor 2 (FFAR2) is activated by short-chain fatty acids and expressed widely, including in white adipocytes and various immune and enteroendocrine cells. Using both wild-type human FFAR2 and a designer receptor exclusively activated by designer drug (DREADD) variant we explored the activation and phosphorylation profile of the receptor, both in heterologous cell lines and in tissues from transgenic knock-in mouse lines expressing either human FFAR2 or the FFAR2-DREADD. FFAR2 phospho-site-specific antisera targeting either pSer296/pSer297 or pThr306/pThr310 provided sensitive biomarkers of both constitutive and agonist-mediated phosphorylation as well as an effective means to visualise agonist-activated receptors in situ. In white adipose tissue, phosphorylation of residues Ser296/Ser297 was enhanced upon agonist activation whilst Thr306/Thr310 did not become phosphorylated. By contrast, in immune cells from Peyer’s patches Thr306/Thr310 become phosphorylated in a strictly agonist-dependent fashion whilst in enteroendocrine cells of the colon both Ser296/Ser297 and Thr306/Thr310 were poorly phosphorylated. The concept of phosphorylation bar-coding has centred to date on the potential for different agonists to promote distinct receptor phosphorylation patterns. Here, we demonstrate that this occurs for the same agonist-receptor pairing in different patho-physiologically relevant target tissues. This may underpin why a single G protein-coupled receptor can generate different functional outcomes in a tissue-specific manner.

Butyrate reduces adherent-invasive E. coli-evoked disruption of epithelial mitochondrial morphology and barrier function: involvement of free fatty acid receptor 3

ABSTRACT Gut bacteria provide benefits to the host and have been implicated in inflammatory bowel disease (IBD), where adherent-invasive E. coli (AIEC) pathobionts (e.g., strain LF82) are associated with Crohn’s disease. E. coli-LF82 causes fragmentation of the epithelial mitochondrial network, leading to increased epithelial permeability. We hypothesized that butyrate would limit the epithelial mitochondrial disruption caused by E. coli-LF82. Human colonic organoids and the T84 epithelial cell line infected with E. coli-LF82 (MOI = 100, 4 h) showed a significant increase in mitochondrial network fission that was reduced by butyrate (10 mM) co-treatment. Butyrate reduced the loss of mitochondrial membrane potential caused by E. coli-LF82 and increased expression of PGC-1 α mRNA, the master regulator of mitochondrial biogenesis. Metabolomics revealed that butyrate significantly altered E. coli-LF82 central carbon metabolism leading to diminished glucose uptake and increased succinate secretion. Correlating with preservation of mitochondrial network form/function, butyrate reduced E. coli-LF82 transcytosis across T84-cell monolayers. The use of the G-protein inhibitor, pertussis toxin, implicated GPCR signaling as critical to the effect of butyrate, and the free fatty acid receptor three (FFAR3, GPR41) agonist, AR420626, reproduced butyrate’s effect in terms of ameliorating the loss of barrier function and reducing the mitochondrial fragmentation observed in E. coli-LF82 infected T84-cells and organoids. These data indicate that butyrate helps maintain epithelial mitochondrial form/function when challenged by E. coli-LF82 and that this occurs, at least in part, via FFAR3. Thus, loss of butyrate-producing bacteria in IBD in the context of pathobionts would contribute to loss of epithelial mitochondrial and barrier functions that could evoke disease and/or exaggerate a low-grade inflammation.

Metabolites 13,14-Dihydro-15-keto-PGE2 Participates in Bifidobacterium animalis F1-7 to Alleviate Opioid-Induced Constipation by 5-HT Pathway.

People suffer from constipation caused by many factors, including constipation (Opioid-Induced Constipation, OIC) during analgesic treatment. Microorganisms may be a potent solution to this problem, but the mechanism is still unclear. Based on models in vivo and in vitro, the potential mechanism involving Bifidobacterium animalis F1-7 (B. animalis F1-7), screened in the previous studies, is explored through non-targeted metabonomics, electrophysiological experiment and molecular level docking. The results showed that B. animalis F1-7 effectively alleviates OIC and promotes the expression of chromogranin A (CGA) and 5-hydroxytryptamine (5-HT). The metabolite 13,14-dihydro-15-keto-PGE2 related to B. animalis F1-7 is found, which has a potential improvement effect on OIC at 20mg kg BW-1 in vivo. At 30ng mL-1 it effectively stimulates secretion of CGA/5-HT (408.95± 1.18ngmL-1 ) by PC-12 cells and changes the membrane potential potassium ion current without affecting the sodium ion current in vitro. It upregulates the target of free fatty acid receptor-4 protein(FFAR4/β-actin, 0.81± 0.02). The results demonstrate that metabolite 13,14-dihydro-15-keto-PGE2 participated in B. animalis F1-7 to alleviate OIC via the 5-HT pathway.

Intestinal Microbiota and Metabolomics Reveal the Role of Auricularia delicate in Regulating Colitis-Associated Colorectal Cancer

Background: The edible fungus Auricularia delicate (ADe) is commonly employed in traditional medicine for intestinal disorders; however, its inhibitory effect on colitis-associated colorectal cancer (CAC) and the underlying mechanisms remain unexplored. (2) Methods: The inhibitory effect of ADe on CAC was investigated using a mouse model induced by azoxymethane/dextran sulfate sodium. Results: ADe effectively suppressed the growth and number of intestinal tumors in mice. Intestinal microbiota analyses revealed that ADe treatment increased Akkermansia and Parabacteroides while it decreased Clostridium, Turicibacter, Oscillospira, and Desulfovibrio. ADe regulated the levels of 2′-deoxyridine, creatinine, 1-palmitoyl lysophosphatidylcholine, and choline in serum. Furthermore, the levels of these metabolites were associated with the abundance of Oscillospira and Paraacteroides. ADe up-regulated the free fatty acid receptor 2 and β-Arrestin 2, inhibited the nuclear factor kappa B (NF-κB) pathway, and significantly attenuated the levels of inflammatory cytokines, thereby mitigating the inflammatory in CAC mice. Conclusions: The protective effect of ADe in CAC mice is associated with the regulation of intestinal microbiota, which leads to the inhibition of NF-kB pathway and regulation of inflammation.

Intestinal FFA2 promotes obesity by altering food intake in Western diet-fed mice.

Short-chain fatty acids (SCFAs) are key nutrients that play a diverse set of roles in physiological function, including regulating metabolic homeostasis. Generated through the fermentation of dietary fibers in the distal colon by the gut microbiome, SCFAs and their effects are partially mediated by their cognate receptors, including free fatty acid receptor 2 (FFA2). FFA2 is highly expressed in the intestinal epithelial cells, where its putative functions are controversial, with numerous in vivo studies relying on global knockout mouse models to characterize intestine-specific roles of the receptor. Here, we used the Villin-Cre mouse line to generate a novel, intestine-specific knockout mouse model for FFA2 (Vil-FFA2) to investigate receptor function within the intestine. Because dietary changes are known to affect the composition of the gut microbiome, and can thereby alter SCFA production, we performed an obesogenic challenge on male Vil-FFA2 mice and their littermate controls (FFA2-floxed, FFA2fl/fl) to identify physiological changes on a high-fat, high-sugar 'Western diet' (WD) compared to a low-fat control diet (CD). We found that the WD-fed Vil-FFA2 mice were transiently protected from the obesogenic effects of the WD and had lower fat mass and improved glucose homeostasis compared to the WD-fed FFA2fl/fl control group during the first half of the study. Additionally, major differences in respiratory exchange ratio and energy expenditure were observed in the WD-fed Vil-FFA2 mice, and food intake was found to be significantly reduced at multiple points in the study. Taken together, this study uncovers a novel role of intestinal FFA2 in mediating the development of obesity.

Hypothalamic free fatty acid receptor-1 regulates whole-body energy balance

ObjectiveFree fatty acid receptor-1 (FFAR1) is a medium- and long-chain fatty acid sensing G protein-coupled receptor that is highly expressed in the hypothalamus. Here, we investigated the central role of FFAR1 on energy balance. MethodsCentral FFAR1 agonism and virogenic knockdown were performed in mice. Energy balance studies, infrared thermographic analysis of brown adipose tissue (BAT) and molecular analysis of the hypothalamus, BAT, white adipose tissue (WAT) and liver were carried out. ResultsPharmacological stimulation of FFAR1, using central administration of its agonist TUG-905 in diet-induced obese mice, decreases body weight and is associated with increased energy expenditure, BAT thermogenesis and browning of subcutaneous WAT (sWAT), as well as reduced AMP-activated protein kinase (AMPK) levels, reduced inflammation, and decreased endoplasmic reticulum (ER) stress in the hypothalamus. As FFAR1 is expressed in distinct hypothalamic neuronal subpopulations, we used an AAV vector expressing a shRNA to specifically knockdown Ffar1 in proopiomelanocortin (POMC) neurons of the arcuate nucleus of the hypothalamus (ARC) of obese mice. Our data showed that knockdown of Ffar1 in POMC neurons promoted hyperphagia and body weight gain. In parallel, these mice developed hepatic insulin resistance and steatosis. ConclusionsFFAR1 emerges as a new hypothalamic nutrient sensor regulating whole body energy balance. Moreover, pharmacological activation of FFAR1 could provide a therapeutic advance in the management of obesity and its associated metabolic disorders.

Butyrate functions as a histone deacetylase inhibitor to protect pancreatic beta cells from IL-1β-induced dysfunction.

Butyrate, a gut microbial metabolite, has beneficial effects on glucose homeostasis and has become an attractive drug candidate for type 2 diabetes (T2D). Recently, we showed that butyrate protects pancreatic beta cells against cytokine-induced dysfunction. In this study, we explored the underlying mechanisms of butyrate action. Pancreatic mouse islets were exposed to a non-cytotoxic concentration of interleukin-1β (IL-1β) for 10 days to mimic low-grade inflammation in T2D. Similar to the effect of butyrate, an isoform-selective histone deacetylase 3 (HDAC3) inhibitor normalized IL-1β-reduced glucose-stimulated insulin secretion and insulin content. In contrast, free fatty acid receptor 2 and 3 (FFAR2/3) agonists failed to normalize IL-1β-induced beta cell dysfunction. Furthermore, butyrate inhibited HDAC activity and increased the acetylation of histone H3 and H4 by 3- and 10-fold, respectively. Genome-wide analysis of histone H3 lysine 27 acetylation (H3K27ac) revealed that butyrate mainly increased H3K27ac at promoter regions (74%), while H3K27ac peaks regulated by IL-1β were more equally distributed at promoters (38%), introns (23%) and intergenic regions (23%). Gene ontology analysis showed that butyrate increased IL-1β-reduced H3K27ac levels near several genes related to hormone secretion and reduced IL-1β-increased H3K27ac levels near genes associated with inflammatory responses. Butyrate alone increased H3K27ac near many genes related to MAPK signaling, hormone secretion, and differentiation, and decreased H3K27ac at genes involved in cell replication. Together, these results suggest that butyrate prevents IL-1β-induced pancreatic islet dysfunction by inhibition of HDACs resulting in changes in H3K27ac levels at genes relevant for beta cell function and inflammatory responses.

Characterization of expression response in post-prandial situation of food sensing system in rainbow trout (Oncorhynchus mykiss) fed a plant-based diet: Focus on free fatty acid receptors and their signaling pathways

Recently identified and characterized in rainbow trout (Oncorhynchus mikyss), the main gustatory receptors for fatty acid, known as free fatty acids receptor (FFAR) 1 and 2, may play a crucial role in nutrient detection and regulation of feeding behavior. However, our understanding of their post-prandial roles remains limited. This study investigates the impact of a single meal (acute effect) with an alternative plant-based diet (vegetal diet, V diet devoid of fish meal and fish oil) on the food detection mechanisms in trout. After 30 days of feeding with commercial-like diet (C diet made up of a mix of fish meal, fish oil and plant ingredients) followed 5 days of fasting, we conducted a post-prandial kinetic experiment after single meal with C or V diet. We assessed the expression pattern of ffar genes, their associated signaling pathways (calcium and indolamine pathways) in the tongue along with the expression pattern of appetite-regulating neuropeptides in the hypothalamus. Additionally, we evaluated central activity through brain activity markers in the telencephalon of trout at various post-meal time points, including 20 min, 2, 6, 10 and 24 h. Our results revealed that the diet nature (C vs V) influences the expression of ffar genes, while some paralogs of ffar2 (ffar2a2 and ffar2b1.2) were early up-regulated by V diet, ffar2b1.1 and ffar1 were latter up-regulated by C diet. Differential regulations in the tongues of V diet fish were also observed on calcium signaling and an early alteration in serotonin turnover. The expression increase of neuropeptide npya from 6 to 10 h after meal, could suggest a greater sensation of hunger in fish fed the C diet. Finally, our findings suggest that rainbow trout exhibit a keen ability to detect a novel feed after a single meal with V diet at the brain level, particularly in areas associated with decision-making and behavior control. Altogether, results not only show that the integrative feeding response appears to be dysregulated by V diet, but also highlight the noteworthy sensitivity of nutrient detection mechanisms in rainbow trout to new food sources. FFARs may play a role in finely discerning diets with varying levels of ω-3 LC-PUFAs in rainbow trout. This heightened detection sensitivity holds the potential to significantly influence the regulation of feeding behavior and food detection in farmed fish.

High levels of short-chain fatty acids secreted by Candida albicans hyphae induce neutrophil chemotaxis via free fatty acid receptor 2.

Candida albicans belongs to our commensal mucosal flora and in immune-competent individuals in the absence of epithelial damage, this fungus is well tolerated and controlled by our immune defense. However, C. albicans is an opportunistic microorganism that can cause different forms of infections, ranging from superficial to life-threatening systemic infections. C. albicans is polymorphic and switches between different phenotypes (e.g. from yeast form to hyphal form). C. albicans hyphae are invasive and can grow into tissues to eventually reach circulation. During fungal infections, neutrophils in particular play a critical role for the defense, but how neutrophils are directed toward the invasive forms of fungi is less well understood. We set out to investigate possible neutrophil chemoattractants released by C. albicans into culture supernatants. We found that cell-free culture supernatants from the hyphal form of C. albicans induced both neutrophil chemotaxis and concomitant intracellular calcium transients. Size separation and hydrophobic sorting of supernatants indicated small hydrophilic factors as responsible for the activity. Further analysis showed that the culture supernatants contained high levels of short-chain fatty acids with higher levels from hyphae as compared to yeast. Short-chain fatty acids are known neutrophil chemoattractants acting via the neutrophil free fatty acid receptor 2. In line with this, the calcium signaling in neutrophils induced by hyphae culture supernatants was blocked by a free fatty acid receptor 2 antagonist and potently increased in the presence of a positive allosteric modulator. Our data imply that short-chain fatty acids may act as a recruitment signal whereby neutrophils can detect C. albicans hyphae.

Phosphodiesterase 4 activity uniquely regulates ciliary cAMP-dependent 3T3-L1 adipogenesis

Recent evidence indicates that the presence of a primary cilium (PC), and of selective cAMP signaling within this smallest of organelles, promotes adipogenic differentiation of 3T3-L1 preadipocytes incubated in media supplemented with either a natural (docosahexaenoic acid, DHA), or a synthetic (TUG-891), free fatty acid receptor 4 (FFAR4) agonist. Indeed, in this earlier work, activation of ciliary FFAR4 in 3T3-L1 cells was correlated with selective increases in PC cAMP and adipogenesis in these cells. However, this study was silent on the role of local PC cAMP phosphodiesterases (PDEs)-mediated events in regulating these adipogenic responses and on the identity of cAMP PDEs that could regulate the “pool” of ciliary cAMP accessed by FFAR4 agonists. In this context, we have identified the PDEs expressed by 3T3-L1 preadipocytes and showed that of these, only PDE4 inhibition promotes FFAR4-mediated adipogenesis. We propose that this work will identify more selective therapeutic targets through which to control adipogenesis, and perhaps the differentiation of other stem cells in which ciliary cAMP is critical.

A high-throughput structural dynamics approach for identification of potential agonists of FFAR4 for type 2 diabetes mellitus therapy

Diabetes mellitus is a metabolic disorder that persists as a global threat to the world. A G-protein coupled receptor (GPCR), free fatty acid receptor 4 (FFAR4), has emerged as a potential target for type 2 diabetes mellitus (T2DM) and obesity-related disorders. The current study has investigated the FFAR4, deploying 3-dimensional structure modeling, molecular docking, machine learning, and high-throughput virtual screening methods to unravel the receptor’s crucial and non-crucial binding site residues. We screened four lakh compounds and shortlisted them based on binding energy, stereochemical considerations, non-bonded interactions, and pharmacokinetic profiling. Out of the screened compounds, four compounds were selected for ligand-bound simulations. The molecular dynamic simulations were carried out for 1µs for native FFAR4 and 500 ns each for complexes of FFAR4 with compound 1, compound 2, compound 3, and compound 4. Our findings showed that in addition to reported binding site residues ARG99, ARG183, and VAL98 in known agonists like TUG-891, the amino acids ARG22, ARG24, THR23, TRP305, and GLU43 were also critical binding site residues. These amino acids impart stability to the FFAR4 complexes and contribute to the stronger binding affinity of the compounds. The study also indicated that aromatic residues like PHE211 are crucial for recognizing the active site’s pi-pi and C-C double bonds. Since FFAR4 is a membrane protein, the simulation studies give an insight into the mechanisms of the crucial protein-lipid and lipid-water interactions. The analysis of the molecular dynamics trajectories showed all four compounds as potential hit molecules that can be developed further into potential agonists for T2DM therapy. Amongst the four compounds, compound 4 showed relatively better binding affinity, stronger non-bonded interactions, and a stable complex. Communicated by Ramaswamy H. Sarma

Dapagliflozin downregulates G protein-coupled receptor Kinase-2 and upregulates regulator of g protein signaling-4 in adrenals to exert sympatholysis in heart failure

Abstract Dapagliflozin is a sodium-glucose co-transporter (SGLT)-2 inhibitor with beneficial cardiovascular effects independent of its anti-diabetic actions. Among these is sympatholysis, i.e., norepinephrine (NE) lowering which is beneficial in chronic human heart failure (HF). Adrenal G protein-coupled receptor kinase (GRK)-2 upregulation is a major driver of circulating catecholamine (CA) elevation and sympathetic nervous system (SNS) hyperactivity in HF due to dysregulation of adrenal sympatho-inhibitory α2-adrenergic receptors (ARs) [1]. On the other hand, we recently reported that signaling by neuronal free fatty acid receptor (FFAR)-3, which stimulates NE release from sympathetic neurons, is blocked by Regulator of G protein Signaling (RGS)-4 [2,3]. We hypothesized herein that dapagliflozin may lower SNS hyperactivity in the adrenal gland by improving α2AR, and antagonizing FFAR3 signaling in adrenal chromaffin cells. We used the rat pheochromocytoma PC12 cell line expressing human α2A-AR, as well as freshly isolated adrenal glands from rats treated with dapagliflozin in vivo. Dapagliflozin treatment for 7 consecutive days (20 mg/kg/d in drinking water) led to a significant reduction in blood circulating NE levels (217+67 pg/ml, n=6), compared to control, vehicle-treated rats (363+77 pg/ml, n=6, p<.05), suggesting reduced SNS activity. This was accompanied by reduced GRK2 and tyrosine hydroxylase (TH) mRNA and protein levels in dapagliflozin-treated rat adrenals vs. vehicle-treated animal-derived glands. In contrast, RGS4 mRNA levels were increased in dapagliflozin-treated adrenals. Adrenal α2AR density was higher in dapagliflozin- vs. vehicle-treated rats (51.3+7.3 vs. 26.1+8.1 fmol/mg of protein, respectively; n=12 glands from 6 animals per group, p<.05). These results (i.e., GRK2 & TH downregulation with RGS4 upregulation) were completely recapitulated in PC12 cells in culture, treated with 5 microM dapagliflozin (or vehicle) for 24 hours. Importantly, α2AR-dependent G protein-mediated signaling towards inhibition of CA secretion was markedly enhanced, whereas FFAR3-dependent phospholipase C/calcium signaling was reduced, at 24 hrs post-dapagliflozin treatment of PC12 cells. GRK2 overexpression partially reversed dapagliflozin`s effect on cholinergic-induced CA secretion. In conclusion, dapagliflozin exerts a sympatholytic action in the adrenal medulla via a) downregulation of TH, which reduces CA biosynthesis, and GRK2, which reduces α2AR desensitization; and b) upregulation of RGS4 to block FFAR3-stimulated CA secretion (Figure).Figure

Effect of pregnancy on the expression of nutrient-sensors and satiety hormones in mice

Small intestinal satiation pathways involve nutrient-induced stimulation of chemoreceptors leading to release of satiety hormones from intestinal enteroendocrine cells (ECCs). Whether adaptations in these pathways contribute to increased maternal food intake during pregnancy is unknown.To determine the expression of intestinal nutrient-sensors and satiety hormone transcripts and proteins across pregnancy in mice.Female C57BL/6J mice (10–12 weeks old) were randomized to mating and then tissue collection at early- (6.5 d), mid- (12.5 d) or late-pregnancy (17.5 d), or to an unmated age matched control group. Relative transcript expression of intestinal fatty acid, peptide and amino acid and carbohydrate chemoreceptors, as well as gut hormones was determined across pregnancy. The density of G-protein coupled receptor 93 (GPR93), free fatty acid receptor (FFAR) 4, cholecystokinin (CCK) and glucagon-like peptide1 (GLP-1) immunopositive cells was then compared between non-pregnant and late-pregnant mice.Duodenal GPR93 expression was lower in late pregnant than non-pregnant mice (P < 0.05). Ileal FFAR1 expression was higher at mid- than at early- or late-pregnancy. Ileal FFAR2 expression was higher at mid-pregnancy than in early pregnancy. Although FFAR4 expression was consistently lower in late-pregnant than non-pregnant mice (P < 0.001), the density of FFAR4 immunopositive cells was higher in the jejunum of late-pregnant than non-pregnant mice.A subset of protein and fatty acid chemoreceptor transcripts undergo region-specific change during murine pregnancy, which could augment hormone release and contribute to increased food intake. Further investigations are needed to determine the functional relevance of these changes.