Selective transport of long-chain fatty acids by FAT/CD36 in skeletal muscle of broilers

Fatty acid translocase (FAT/CD36) is a membrane receptor that facilitates long-chain fatty acid uptake. To investigate its role in the regulation of long-chain fatty acid composition in muscle tissue, we studied and compared FAT/CD36 gene expression in muscle tissues of commercial broiler chickens and Chinese local Silky fowls. The results from gas chromatography–mass spectrometry analysis of muscle samples demonstrated that Chinese local Silky fowls had significantly higher (P < 0.05) proportions of linoleic acid (LA) and palmitic acid, lower proportions (P < 0.05) of arachidonic acid (AA) and oleic acid than the commercial broiler chickens. The mRNA expression levels of fatty acid (FA) transporters (FA transport protein-1, membrane FA-binding protein, FAT/CD36 and caveolin-1) in the m. ipsilateral pectoralis and biceps femoris were analyzed by Q-PCR, and FAT/CD36 expression levels showed significant differences between these types of chickens (P < 0.01). Interestingly, the levels of FAT/CD36 expression are positively correlated with LA content (r = 0.567, P < 0.01) but negatively correlated with palmitic acid content (r = −0.568, P < 0.01). Further experiments in the stably transfected Chinese hamster oocytes cells with chicken FAT/CD36 cDNA demonstrated that overexpression of FAT/CD36 improves total FA uptake with a significant increase in the proportion of LA and AA, and a decreased proportion of palmitic acid. These results suggest that chicken FAT/CD36 may selectively transport LA and AA, which may lead to the higher LA deposition in muscle tissue.     

GPR120 induces regulatory dendritic cells by inhibiting HK2-dependent glycolysis to alleviate fulminant hepatic failure

Fulminant hepatic failure (FHF) is a potentially fatal liver disease that is associated with intrahepatic infiltration of inflammatory cells. As the receptor of polyunsaturated long chain fatty acids, GPR120 can regulate cell differentiation, proliferation, metabolism, and immune response. However, whether GPR120 is involved in FHF remains unknown. Using Propionibacterium acnes (P. acnes)-primed, LPS-induced FHF in mice, we found that interference with GPR120 activity using pharmacological agonist attenuated the severity of the liver injury and mortality of FHF in mice, while a lack of GPR120 exacerbated the disease. GPR120 activation potently alleviated FHF and led to decreased T helper (Th) 1 cell response and expansion of regulatory T cells (Tregs). Interestingly, GPR120 agonist didn’t directly target T cells, but dramatically induced a distinct population of CD11c⁺MHC II^lowCD80^lowCD86^low regulatory DCs in the livers of FHF mice. GPR120 was found to restrict HIF-1α-dependent glycolysis. The augmented HIF-1α stabilization caused by GPR120 antagonism or deletion could be attenuated by the inhibition of ERK or by the activation of AMPK. Through the analysis of the clinical FHF, we further confirmed the activation of GPR120 was negatively associated with the severity in patients. Our findings indicated that GPR120 activation has therapeutic potential in FHF. Strategies to target GPR120 using agonists or free fatty acids (FFAs) may represent a novel approach to FHF treatment.Subject terms: Inflammation, Hepatitis     

Alteration of the Glucagon Axis in GPR120 (FFAR4) Knockout Mice: A ROLE FOR GPR120 IN GLUCAGON SECRETION

GPR40 (FFAR1) and GPR120 (FFAR4) are G-protein-coupled receptors (GPCRs) that are activated by long chain fatty acids (LCFAs). GPR40 is expressed at high levels in islets and mediates the ability of LCFAs to potentiate glucose-stimulated insulin secretion (GSIS). GPR120 is expressed at high levels in colon, adipose, and pituitary, and at more modest levels in pancreatic islets. The role of GPR120 in islets has not been explored extensively. Here, we confirm that saturated (e.g. palmitic acid) and unsaturated (e.g. docosahexaenoic acid (DHA)) LCFAs engage GPR120 and demonstrate that palmitate- and DHA-potentiated glucagon secretion are greatly reduced in isolated GPR120 KO islets. Remarkably, LCFA potentiated glucagon secretion is similarly reduced in GPR40 KO islets. Compensatory changes in mRNA expression of GPR120 in GPR40 KO islets, and vice versa, do not explain that LCFA potentiated glucagon secretion seemingly involves both receptors. LCFA-potentiated GSIS remains intact in GPR120 KO islets. Consistent with previous reports, GPR120 KO mice are hyperglycemic and glucose intolerant; however, our KO mice display evidence of a hyperactive counter-regulatory response rather than insulin resistance during insulin tolerance tests. An arginine stimulation test and a glucagon challenge confirmed both increases in glucagon secretion and liver glucagon sensitivity in GPR120 KO mice relative to WT mice. Our findings demonstrate that GPR120 is a nutrient sensor that is activated endogenously by both saturated and unsaturated long chain fatty acids and that an altered glucagon axis likely contributes to the impaired glucose homeostasis observed in GPR120 KO mice.     

Phenotypic characterization of GPR120-expressing cells in the interstitial tissue of pancreas

GPR120 functions as a plasma membrane receptor for unsaturated long-chain free fatty acids and involves in GLP-1 secretion, adipogenesis and the control of energy balance. Pancreas is the key organ in fuel and energy metabolism. Here GPR120 expression in human and rat pancreas was observed by RT-PCR, and the distribution and phenotypes of GPR120-positive cells in human and rat pancreas were shown by immunohistochemical staining. GPR120 mRNA expression was found in human and rat pancreas. GPR120-positive cells were scattered mainly in the interstitial tissues of human and rat pancreas, and they were not co-localized with nestin, vimentin, alpha-SMA and glucagon, respectively. However, GPR120 was distributed on the cells positively stained by CD68, the specific marker of macrophages, and on the cells positive stained by CD34 and CD117, the markers of interstitial cells. In conclusion, this study demonstrates the expression of GPR120 in pancreas and shows the distribution of GPR120 in human and rat pancreas.     

Role of free fatty acid receptors in the regulation of energy metabolism

Free fatty acids (FFAs) are energy-generating nutrients that act as signaling molecules in various cellular processes. Several orphan G protein-coupled receptors (GPCRs) that act as FFA receptors (FFARs) have been identified and play important physiological roles in various diseases. FFA ligands are obtained from food sources and metabolites produced during digestion and lipase degradation of triglyceride stores. FFARs can be grouped according to ligand profiles, depending on the length of carbon chains of the FFAs. Medium- and long-chain FFAs activate FFA1/GPR40 and FFA4/GPR120. Short-chain FFAs activate FFA2/GPR43 and FFA3/GPR41. However, only medium-chain FFAs, and not long-chain FFAs, activate GPR84 receptor. A number of pharmacological and physiological studies have shown that these receptors are expressed in various tissues and are primarily involved in energy metabolism. Because an impairment of these processes is a part of the pathology of obesity and type 2 diabetes, FFARs are considered as key therapeutic targets. Here, we reviewed recently published studies on the physiological functions of these receptors, primarily focusing on energy homeostasis.     

CD36, but not GPR120, is required for efficient fatty acid utilization during endurance exercise

Fatty acids (FA) are an important energy source during exercise. In addition to its role as an energy supply for skeletal muscle, FA may activate signaling pathways that regulate gene expression. FA translocase/cluster of differentiation 36 (CD36) and G protein-coupled receptor GPR120 are long-chain FA receptors. In this study, we investigated the impact of CD36 or GPR120 deletion on energy metabolism during exercise. CD36 has been reported to facilitate cellular transport and oxidation of FA during endurance exercise. We show that CD36 deletion decreased exogenous FA oxidation during exercise, using a combination of ¹³C-labeled FA oxidation measurement and indirect calorimetry. In contrast, GPR120 deletion had no observable effect on energy metabolism during exercise. Our results further substantiate that CD36-mediated FA transport plays an essential role in efficient FA oxidation during exercise.     

The oral lipid sensor GPR120 is not indispensable for the orosensory detection of dietary lipids in mice

Implication of the long-chain fatty acid (LCFA) receptor GPR120, also termed free fatty acid receptor 4, in the taste-guided preference for lipids is a matter of debate. To further unravel the role of GPR120 in the “taste of fat”, the present study was conducted on GPR120-null mice and their wild-type littermates. Using a combination of morphological [i.e., immunohistochemical staining of circumvallate papillae (CVP)], behavioral (i.e., two-bottle preference tests, licking tests and conditioned taste aversion) and functional studies [i.e., calcium imaging in freshly isolated taste bud cells (TBCs)], we show that absence of GPR120 in the oral cavity was not associated with changes in i) gross anatomy of CVP, ii) LCFA-mediated increases in intracellular calcium levels ([Ca²⁺]_i), iii) preference for oily and LCFA solutions and iv) conditioned avoidance of LCFA solutions. In contrast, the rise in [Ca²⁺]_i triggered by grifolic acid, a specific GPR120 agonist, was dramatically curtailed when the GPR120 gene was lacking. Taken together, these data demonstrate that activation of lingual GPR120 and preference for fat are not connected, suggesting that GPR120 expressed in TBCs is not absolutely required for oral fat detection in mice     

Endothelial lipase mediates efficient lipolysis of triglyceride-rich lipoproteins

Triglyceride-rich lipoproteins (TRLs) are circulating reservoirs of fatty acids used as vital energy sources for peripheral tissues. Lipoprotein lipase (LPL) is a predominant enzyme mediating triglyceride (TG) lipolysis and TRL clearance to provide fatty acids to tissues in animals. Physiological and human genetic evidence support a primary role for LPL in hydrolyzing TRL TGs. We hypothesized that endothelial lipase (EL), another extracellular lipase that primarily hydrolyzes lipoprotein phospholipids may also contribute to TRL metabolism. To explore this, we studied the impact of genetic EL loss-of-function on TRL metabolism in humans and mice. Humans carrying a loss-of-function missense variant in LIPG, p.Asn396Ser (rs77960347), demonstrated elevated plasma TGs and elevated phospholipids in TRLs, among other lipoprotein classes. Mice with germline EL deficiency challenged with excess dietary TG through refeeding or a high-fat diet exhibited elevated TGs, delayed dietary TRL clearance, and impaired TRL TG lipolysis in vivo that was rescued by EL reconstitution in the liver. Lipidomic analyses of postprandial plasma from high-fat fed Lipg^-/- mice demonstrated accumulation of phospholipids and TGs harboring long-chain polyunsaturated fatty acids (PUFAs), known substrates for EL lipolysis. In vitro and in vivo, EL and LPL together promoted greater TG lipolysis than either extracellular lipase alone. Our data positions EL as a key collaborator of LPL to mediate efficient lipolysis of TRLs in humans and mice.     

Kidney triglyceride accumulation in the fasted mouse is dependent upon serum free fatty acids

Lipid accumulation is a pathological feature of every type of kidney injury. Despite this striking histological feature, physiological accumulation of lipids in the kidney is poorly understood. We studied whether the accumulation of lipids in the fasted kidney are derived from lipoproteins or NEFAs. With overnight fasting, kidneys accumulated triglyceride, but had reduced levels of ceramide and glycosphingolipid species. Fasting led to a nearly 5-fold increase in kidney uptake of plasma [¹⁴C]oleic acid. Increasing circulating NEFAs using a β adrenergic receptor agonist caused a 15-fold greater accumulation of lipid in the kidney, while mice with reduced NEFAs due to adipose tissue deficiency of adipose triglyceride lipase had reduced triglycerides. Cluster of differentiation (Cd)36 mRNA increased 2-fold, and angiopoietin-like 4 (Angptl4), an LPL inhibitor, increased 10-fold. Fasting-induced kidney lipid accumulation was not affected by inhibition of LPL with poloxamer 407 or by use of mice with induced genetic LPL deletion. Despite the increase in CD36 expression with fasting, genetic loss of CD36 did not alter fatty acid uptake or triglyceride accumulation. Our data demonstrate that fasting-induced triglyceride accumulation in the kidney correlates with the plasma concentrations of NEFAs, but is not due to uptake of lipoprotein lipids and does not involve the fatty acid transporter, CD36.     

家鸡G蛋白偶联受体85基因的结构、序列与组织表达分析

G蛋白偶联受体(GPCR)超家族是细胞膜上广泛存在的一类受体,是细胞跨膜信号转导的一类重要受体分子,参与许多生理过程调节。它们中仍有很多至今尚未找到内源性配体,这类受体被称为孤儿型受体。G蛋白偶联受体85(GPR85)是GPCR超家族中孤儿型受体的一员。目前,在非哺乳类脊椎动物中,针对GPR85的研究极少。本研究以家鸡Gallus gallus domesticus为模型,通过反转录PCR和RACE-PCR等方法从脑中克隆到GPR85基因的cDNA全长序列,揭示其基因结构,并用实时荧光定量PCR(qPCR)方法探究了该基因在家鸡各组织中的表达情况。结果显示:家鸡GPR85基因位于1号染色体上,由2个外显子组成,其编码区位于第2个外显子上,长为1 113 bp,可编码1个370个氨基酸的7次跨膜受体蛋白。家鸡GPR85与其他脊椎动物(人Homo sapiens、小鼠Mus musculus、大鼠Rattus norvegicus、热带爪蟾Xenopus tropicalis和斑马鱼Danio rerio)的GPR85具有高度的氨基酸序列一致性(>93%)。qPCR分析发现,GPR85基因mRNA在家鸡全脑、垂体、肾上腺、精巢中有较高表达,而在所检测的其他外周组织中表达极低。本研究首次揭示了家鸡GPR85基因的结构与表达特征,为后续探究GPR85基因在家鸡等非哺乳类中的生理功能奠定基础。     

Ghrelin is a signal to facilitate the utilization of fatty acids and save glucose by the liver,skeletal muscle,and adipose tissues in chicks

Ghrelin, classically known as a central appetite-stimulating hormone, has recently been recognized to play an important role in peripheral tissue energy metabolism. In chicken, contrary to mammal, ghrelin acts as an anorexia signal, increased by fasting and further elevated after refed. In the present study, the effect of ghrelin on glucose/lipid utilization by peripheral tissues was investigated. Injection of exogenous acyl ghrelin reduced plasma triglyceride and glucose levels of chickens at both fasting and fed status. In the in vitro cultured chicken primary hepatocytes, adipocytes, and myoblasts, ghrelin suppressed glucose uptake, stimulated fatty acids uptake and oxidation, and decreased TG content. In hepatocyte, ghrelin increased the activities of LPL and HL, and upregulated the expression levels of gene ACC, CPT1, and PPARα. Ghrelin treatment markedly increased the protein level of p-ACC, PPARγ, PGC1α, and CPT1 in hepatocytes, adipocytes and myoblasts. Inhibition of AMPK activity by Compound C had no influence on glucose uptake by hepatocyte, adipocyte, and myoblast, but further amplified the stimulated fatty acid uptake of adipocyte by ghrelin. The present result demonstrates that ghrelin facilitates the uptake and oxidation of fatty acid and cut down the utilization of glucose by the liver, muscle, and adipose tissues. The result suggests that ghrelin functions as a signal of fatty acid oxidation. The study provides a vital framework for understanding the intrinsic role of ghrelin as a crucial factor in the concerted regulation of metabolic substrate of hepatocytes, adipocytes, and myoblasts.     

Selective Enrichment of Omega-3 Fatty Acids in Oils by Phospholipase A1

Omega fatty acids are recognized as key nutrients for healthier ageing. Lipases are used to release ω-3 fatty acids from oils for preparing enriched ω-3 fatty acid supplements. However, use of lipases in enrichment of ω-3 fatty acids is limited due to their insufficient specificity for ω-3 fatty acids. In this study use of phospholipase A1 (PLA1), which possesses both sn-1 specific activity on phospholipids and lipase activity, was explored for hydrolysis of ω-3 fatty acids from anchovy oil. Substrate specificity of PLA1 from Thermomyces lenuginosus was initially tested with synthetic p-nitrophenyl esters along with a lipase from Bacillus subtilis (BSL), as a lipase control. Gas chromatographic characterization of the hydrolysate obtained upon treatment of anchovy oil with these enzymes indicated a selective retention of ω-3 fatty acids in the triglyceride fraction by PLA1 and not by BSL. ¹³C NMR spectroscopy based position analysis of fatty acids in enzyme treated and untreated samples indicated that PLA1 preferably retained ω-3 fatty acids in oil, while saturated fatty acids were hydrolysed irrespective of their position. Hydrolysis of structured triglyceride,1,3-dioleoyl-2-palmitoylglycerol, suggested that both the enzymes hydrolyse the fatty acids at both the positions. The observed discrimination against ω-3 fatty acids by PLA1 appears to be due to its fatty acid selectivity rather than positional specificity. These studies suggest that PLA1 could be used as a potential enzyme for selective concentrationof ω-3 fatty acids.     

Modulation of Antimicrobial Host Defense Peptide Gene Expression by Free Fatty Acids

Routine use of antibiotics at subtherapeutic levels in animal feed drives the emergence of antimicrobial resistance. Development of antibiotic-alternative approaches to disease control and prevention for food animals is imperatively needed. Previously, we showed that butyrate, a major species of short-chain fatty acids (SCFAs) fermented from undigested fiber by intestinal microflora, is a potent inducer of endogenous antimicrobial host defense peptide (HDP) genes in the chicken (PLoS One 2011, 6: e27225). In the present study, we further revealed that, in chicken HD11 macrophages and primary monocytes, induction of HDPs is largely in an inverse correlation with the aliphatic hydrocarbon chain length of free fatty acids, with SCFAs being the most potent, medium-chain fatty acids moderate and long-chain fatty acids marginal. Additionally, three SCFAs, namely acetate, propionate, and butyrate, exerted a strong synergy in augmenting HDP gene expression in chicken cells. Consistently, supplementation of chickens with a combination of three SCFAs in water resulted in a further reduction of Salmonella enteritidis in the cecum as compared to feeding of individual SCFAs. More importantly, free fatty acids enhanced HDP gene expression without triggering proinflammatory interleukin-1β production. Taken together, oral supplementation of SCFAs is capable of boosting host immunity and disease resistance, with potential for infectious disease control and prevention in animal agriculture without relying on antibiotics.     

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.     

G protein-coupled receptors in human fat taste perception

In contrast to carbohydrates and proteins, which are detected by specialized taste receptors in the forms of their respective building blocks, sugars, and L-amino acids, the third macronutrient, lipids, has until now not been associated with gustatory receptors. Instead, the recognition of fat stimuli was believed to rely mostly on textural, olfactory, and postingestive cues. During the recent years, however, research done mainly in rodent models revealed an additional gustatory component for the detection of long-chain fatty acids (LCFAs), the main taste-activating component of lipids. Concomitantly, a number of candidate fat taste receptors were proposed to be involved in rodent's gustatory fatty acid perception. Compared with rodent models, much less is known about human fat taste. In order to investigate the ability of the human gustatory system to respond to fat components, we performed sensory experiments with fatty acids of different chain lengths and derivatives thereof. We found that our panelists discriminated a "fatty" and an irritant "scratchy" taste component, with the "fatty" percept restricted to LCFAs. Using functional calcium-imaging experiments with the human orthologs of mouse candidate fat receptors belonging to the G protein-coupled receptor family, we correlated human sensory data with receptor properties characterized in vitro. We demonstrated that the pharmacological activation profile of human GPR40 and GPR120, 2 LCFA-specific receptors associated with gustatory fat perception in rodents, is inconsistent with the "scratchy" sensation of human subjects and more consistent with the percept described as "fatty." Expression analysis of GPR40 and GPR120 in human gustatory tissues revealed that, while the GPR40 gene is not expressed, GPR120 is detected in gustatory and nongustatory epithelia. On a cellular level, we found GPR120 mRNA and protein in taste buds as well as in the surrounding epithelial cells. We conclude that GPR120 may indeed participate in human gustatory fatty acid perception.     

The Beneficial Effects of n-3 Polyunsaturated Fatty Acids on Diet Induced Obesity and Impaired Glucose Control Do Not Require Gpr120

GPR120 (Ffar4) has been postulated to represent an important receptor mediating the improved metabolic profile seen upon ingestion of a diet enriched in polyunsaturated fatty acids (PUFAs). GPR120 is highly expressed in the digestive system, adipose tissue, lung and macrophages and also present in the endocrine pancreas. A new Gpr120 deficient mouse model on pure C57bl/6N background was developed to investigate the importance of the receptor for long-term feeding with a diet enriched with fish oil. Male Gpr120 deficient mice were fed two different high fat diets (HFDs) for 18 weeks. The diets contained lipids that were mainly saturated (SAT) or mainly n-3 polyunsaturated fatty acids (PUFA). Body composition, as well as glucose, lipid and energy metabolism, was studied. As expected, wild type mice fed the PUFA HFD gained less body weight and had lower body fat mass, hepatic lipid levels, plasma cholesterol and insulin levels and better glucose tolerance as compared to those fed the SAT HFD. Gpr120 deficient mice showed a similar improvement on the PUFA HFD as was observed for wild type mice. If anything, the Gpr120 deficient mice responded better to the PUFA HFD as compared to wild type mice with respect to liver fat content, plasma glucose levels and islet morphology. Gpr120 deficient animals were found to have similar energy, glucose and lipid metabolism when fed HFD PUFA compared to wild type mice. Therefore, GPR120 appears to be dispensable for the improved metabolic profile associated with intake of a diet enriched in n-3 PUFA fatty acids.     

Fatty Acid Solubilizer from the Oral Disk of the Blowfly

Background

Blowflies are economic pests of the wool industry and potential vectors for epidemics. The establishment of a pesticide-free, environmentally friendly blowfly control strategy is necessary. Blowflies must feed on meat in order to initiate the cascade of events that are involved in reproduction including juvenile hormone synthesis, vitellogenesis, and mating. During feeding blowflies regurgitate salivary lipase, which may play a role in releasing fatty acids from triglycerides that are found in food. However, long-chain fatty acids show low solubility in aqueous solutions. In order to solubilize and ingest the released hydrophobic fatty acids, the blowflies must use a solubilizer.

Methodology

We applied native PAGE, Edman degradation, cDNA cloning, and RT-PCR to characterize a protein that accumulated in the oral disk of the black blowfly, Phormia regina. In situ hybridization was carried out to localize the expression at the cellular level. A fluorescence competitive binding assay was used to identify potential ligands of this protein.

Conclusion

A protein newly identified from P. regina (PregOBP56a) belonged to the classic odorant-binding protein (OBP) family. This gene was expressed in a cluster of cells that was localized between pseudotracheae on the oral disk, which are not accessory cells of the taste peg chemosensory sensilla that normally synthesize OBPs. At pH 7 and pH 6, PregOBP56a bound palmitic, stearic, oleic, and linoleic acids, that are mainly found in chicken meat. The binding affinity of PregOBP56a decreased at pH 5. We propose that PregOBP56a is a protein that solubilizes fatty acids during feeding and subsequently helps to deliver the fatty acids to the midgut where it may help in the process of reproduction. As such, PregOBP56a is a potential molecular target for controlling the blowfly.