Free fatty acids inhibit serum deprivation-induced apoptosis through GPR120 in a murine enteroendocrine cell line STC-1

Free fatty acids (FFAs) provide an important energy source and also act as signaling molecules. FFAs are known to exert a variety of physiological responses via their G protein-coupled receptors (GPCRs), such as the GPR40 family. Recently, we identified a novel FFA receptor, GPR120, that promotes secretion of glucagon-like peptide-1 (Hirasawa, A., Tsumaya, K., Awaji, T., Katsuma, S., Adachi, T., Yamada, M., Sugimoto, Y., Miyazaki, S., and Tsujimoto, G. (2005) Nat. Med. 11, 90-94). Here we showed that FFAs inhibit serum deprivation-induced apoptosis of murine enteroendocrine STC-1 cells, which express two types of GPCRs, GPR120 and GPR40, for unsaturated long chain FFA. We first found that linolenic acid potently activated ERK and Akt/protein kinase B (Akt) in STC-1 cells. ERK kinase inhibitors significantly reduced the anti-apoptotic effects of linolenic acid. Inhibitors for phosphatidylinositol 3-kinase (PI3K), a major target of which is Akt, significantly reduced the anti-apoptotic effects. Transfection of STC-1 cells with the dominant-negative form of Akt also inhibited the anti-apoptotic effect. These results suggested that the activation of ERK and PI3K-Akt pathways is required for FFA-induced anti-apoptotic effects on STC-1 cells. Transient transfection of STC-1 cells with GPR120 cDNA, but not GPR40 cDNA, enhanced inhibition of caspase-3 activation. RNA interference experiments showed that reduced expression of GPR120, but not GPR40, resulted in reduced ERK activation and reduced effects of FFAs on caspase-3 inhibition. Collectively, these results demonstrated that FFAs promote the activation of ERK and PI3K-Akt pathways mainly via GPR120, leading to the anti-apoptotic effect of STC-1 cells.     

G蛋白偶联受体介导游离脂肪酸的信号通路及生理功能

游离脂肪酸（free fatty acid,FFA）是动物一种重要能量来源,同时它还是一种重要的信号分子,其生理功能和作用机制长期以来倍受关注. 最近研究表明,细胞膜存在FFA的特定孤儿型G蛋白偶联膜受体家族.中长链游离脂肪酸是GPR40和GPR120的配基,而短链游离脂肪酸则是GPR41和GPR43的配基. 该受体家族可以介导游离脂肪酸,通过ERK、PI3K-Akt和MAPK信号通路,在维持机体内的葡萄糖稳态、脂肪形成、白细胞功能和细胞增殖等生理过程中发挥重要作用. 本文就游离脂肪酸G蛋白偶联受体的结构、分布、配体选择性、下游信号通路,及其介导FFA生理功能的最新研究进展进行简要综述.     

Medium-chain fatty acids as ligands for orphan G protein-coupled receptor GPR84

Free fatty acids (FFAs) play important physiological roles in many tissues as an energy source and as signaling molecules in various cellular processes. Elevated levels of circulating FFAs are associated with obesity, dyslipidemia, and diabetes. Here we show that GPR84, a previously orphan G protein-coupled receptor, functions as a receptor for medium-chain FFAs with carbon chain lengths of 9-14. Medium-chain FFAs elicit calcium mobilization, inhibit 3',5'-cyclic AMP production, and stimulate [35S]guanosine 5'-O-(3-thiotriphosphate) binding in a GPR84-dependent manner. The activation of GPR84 by medium-chain FFAs couples primarily to a pertussis toxin-sensitive G(i/o) pathway. In addition, we show that GPR84 is selectively expressed in leukocytes and markedly induced in monocytes/macrophages upon activation by lipopolysaccharide. Furthermore, we demonstrate that medium-chain FFAs amplify lipopolysaccharide-stimulated production of the proinflammatory cytokine interleukin-12 p40 through GPR84. Our results indicate a role for GPR84 in directly linking fatty acid metabolism to immunological regulation.     

Free fatty acids regulate gut incretin glucagon-like peptide-1 secretion through GPR120

Diabetes, a disease in which the body does not produce or use insulin properly, is a serious global health problem. Gut polypeptides secreted in response to food intake, such as glucagon-like peptide-1 (GLP-1), are potent incretin hormones that enhance the glucose-dependent secretion of insulin from pancreatic beta cells. Free fatty acids (FFAs) provide an important energy source and also act as signaling molecules in various cellular processes, including the secretion of gut incretin peptides. Here we show that a G-protein-coupled receptor, GPR120, which is abundantly expressed in intestine, functions as a receptor for unsaturated long-chain FFAs. Furthermore, we show that the stimulation of GPR120 by FFAs promotes the secretion of GLP-1 in vitro and in vivo, and increases circulating insulin. Because GLP-1 is the most potent insulinotropic incretin, our results indicate that GPR120-mediated GLP-1 secretion induced by dietary FFAs is important in the treatment of diabetes.     

GPR120的研究进展

游离脂肪酸作为组织能量来源以及介导各种细胞进程的信号分子,其生理功能长期以来受到广泛关注。外周游离脂肪酸水平的升高与肥胖、脂代谢紊乱以及糖尿病紧密相关。GPR120作为一新的长链脂肪酸受体,参与调节体内一系列的代谢过程,如激素分泌、细胞增殖及脂质生成等。作为肥胖、糖尿病的潜在治疗靶标,值得更深入的研究。     

Receptor-specific regulation of ERK1/2 activation by members of the “free fatty acid receptor” family

Context: The “free fatty acid receptors” (FFARs) GPR40, GPR41, and GPR43 regulate various physiological homeostases, and are all linked to activation of extracellular signal-regulated kinases (ERK)1/2.

Objective: Investigation of coupling of FFARs to two other mitogen-activated protein kinases (MAPKs) sometimes regulated by G protein-coupled receptors (GPCRs), c-Jun N-terminal kinase (JNK) and p38MAPK, and characterization of signaling proteins involved in the regulation of FFAR-mediated ERK1/2 activation.

Methods: FFARs were recombinantly expressed, cells challenged with the respective agonist, and MAPK activation quantitatively determined using an AlphaScreen SureFire assay. Inhibitors for signaling proteins were utilized to characterize ERK1/2 pathways.

Results: Propionate-stimulated GPR41 strongly coupled to ERK1/2 activation, while the coupling of linoleic acid-activated GPR40 and acetate-activated GPR43 was weaker. JNK and p38MAPK were weakly activated by FFARs. All three receptors activated ERK1/2 fully or partially via G_i/o and Rac. PI3K was relevant for GPR40- and GPR41-mediated ERK1/2 activation, and Src was essential for GPR40- and GPR43-induced activation. Raf-1 was not involved in the GPR43-triggered activation.

Conclusion: The results demonstrate a novel role of Rac in GPCR-mediated ERK1/2 signaling, and that GPCRs belonging to the same family can regulate ERK1/2 activation by different receptor-specific mechanisms.     

Free fatty acid receptor 3 is a key target of short chain fatty acid: What is the impact on the sympathetic nervous system?

Nervous system (NS) activity participates in metabolic homeostasis by detecting peripheral signal molecules derived from food intake and energy balance. High quality diets are thought to include fiber-rich foods like whole grain rice, breads, cereals, and grains. Several studies have associated high consumption of fiber-enriched diets with a reduced risk of diabetes, obesity, and gastrointestinal disorders. In the lower intestine, anaerobic fermentation of soluble fibers by microbiota produces short chain fatty acids (SCFAs), key energy molecules that have a recent identified leading role in the intestinal gluconeogenesis, promoting beneficial effects on glucose tolerance and insulin resistance¹. SCFAs are also signaling molecules that bind to specific G-protein coupled receptors (GPCRs) named Free Fatty Acid Receptor 3 (FFA3, GPR41) and 2 (FFA2, GPR43). However, how SCFAs impact NS activity through their GPCRs is poorly understood. Recently, studies have demonstrated the presence of FFA2 and FFA3 in the sympathetic NS of rat, mouse and human^{2, 3}. Two studies have showed that FFA3 activation by SCFAs increases firing and norepinephrine (NE) release from sympathetic neurons^{3, 4}. However, the recent study from the Ikeda Laboratory² revealed that activation of FFA3 by SCFAs impairs N-type calcium channel (NTCC) activity, which contradicts the idea of FFA3 activation leading to increased action potential evoked NE release. Here we will discuss the scope of the latter study and the putative physiological role of SCFAs and FFAs in the sympathetic NS.     

Free Fatty Acid Composition of Human and Rat Peripheral Nerve

Abstract: The free fatty acid (FFA) composition of peripheral nerve resembles that of erythrocytes but the composition of both is different from that of brain and other tissues. Approximately 75% of FFAs of nerve and erythrocytes are saturated and <5% are polyunsaturated whereas in brain and other tissues, 30-45% of FFAs are saturated and 25-50% are polyunsaturated. Approximately 10-15% of the total FFA of nerve have very long chain lengths [C₂₄, C₂₆, C₂₈, and C₃₀]. The presence of these very long-chain FFAs in endoneurium cannot be accounted for by the retention of erythrocytes or by lipid degradation. During Wallerian degeneration a significant increase of 18:1, associated with a decrease of saturated FFAs, was found in rat sciatic endoneurium, but normal values were approached when fiber regeneration was well under way. The FFA composition with chain length ≥C₂₆ were not, however, significantly altered with degeneration or repair of nerves. The metabolic significance of this striking difference between nerve and brain FFA composition is unknown but may reflect different functional properties.     

Free fatty acid receptor 3 is a key target of short chain fatty acid

Nervous system (NS) activity participates in metabolic homeostasis by detecting peripheral signal molecules derived from food intake and energy balance. High quality diets are thought to include fiber-rich foods like whole grain rice, breads, cereals, and grains. Several studies have associated high consumption of fiber-enriched diets with a reduced risk of diabetes, obesity, and gastrointestinal disorders. In the lower intestine, anaerobic fermentation of soluble fibers by microbiota produces short chain fatty acids (SCFAs), key energy molecules that have a recent identified leading role in the intestinal gluconeogenesis, promoting beneficial effects on glucose tolerance and insulin resistance¹. SCFAs are also signaling molecules that bind to specific G-protein coupled receptors (GPCRs) named Free Fatty Acid Receptor 3 (FFA3, GPR41) and 2 (FFA2, GPR43). However, how SCFAs impact NS activity through their GPCRs is poorly understood.

Recently, studies have demonstrated the presence of FFA2 and FFA3 in the sympathetic NS of rat, mouse and human^{2, 3}. Two studies have showed that FFA3 activation by SCFAs increases firing and norepinephrine (NE) release from sympathetic neurons^{3, 4}. However, the recent study from the Ikeda Laboratory² revealed that activation of FFA3 by SCFAs impairs N-type calcium channel (NTCC) activity, which contradicts the idea of FFA3 activation leading to increased action potential evoked NE release. Here we will discuss the scope of the latter study and the putative physiological role of SCFAs and FFAs in the sympathetic NS.     

Long-Chain Free Fatty Acid Profiling Analysis by Liquid Chromatography–Mass Spectrometry in Mouse Treated with Peroxisome Proliferator-Activated Receptor α Agonist

A change in the free fatty acid (FFA) profile reflects an alteration in the lipid metabolism of peripheral tissue. A high-throughput quantitative analysis method for individual FFAs therefore needs to be established. We report here an optimized LC-MS assay for a high-throughput and high-sensitivity analysis of the 10 major long-chain FFAs in mouse plasma and liver. This assay enables quantification of individual FFAs by using trace amounts of samples (2 µL of plasma and 10 mg of liver tissue). We apply this method to analyze the FFA profile of plasma and liver samples from an obese mouse model treated with bezafibrate, the peroxisome proliferator-activated receptor α (PPARα) agonist, and show a change in the FFA profile, particularly in the palmitoleic and oleic acid contents. This assay is useful for quantifying individual FFAs and helpful for monitoring the condition of lipid metabolism.     

The regulation of adipogenesis through GPR120

Recently, it has been found that long-chain fatty acids activate the G protein-coupled receptors (GPRs), GPR120 and GPR40. However, there have been no reports to date on the possible physiological roles of these GPRs in adipose tissue development and adipocyte differentiation. GPR120 mRNA was highly expressed in the four different adipose tissues, and the amount of mRNA was elevated in adipose tissues of mice fed a high fat diet. However, GPR40 mRNA was not detected in any of the adipose tissues. The expression of GPR120 mRNA was higher in adipocytes compared to stromal-vascular (S-V) cells. The level of GPR120 mRNA increased during adipocyte differentiation in 3T3-L1 cells. Similar results were observed in human adipose tissue, human preadipocytes, and cultured adipocytes. Moreover, use of a small interference RNA (siRNA) to down-regulate GPR120 expression resulted in inhibition of adipocyte differentiation. Our results suggest that GPR120 regulates adipogenic processes such as adipocyte development and differentiation.     

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.     

Featured Article: Deficiency of G-protein coupled receptor 40, a lipid-activated receptor,heightens in?vitro- and in?vivo-induced murine osteoarthritis

Osteoarthritis (OA) is an age-related degenerative joint disease. To date, its management is focused on symptoms (pain and inflammation). Studies suggest that fatty acids can reduce the expression of inflammatory and catalytic mediators, and improve in vivo joint function. Free fatty acid receptors (FFARs) such as G-protein coupled receptor 40 (GPR40) are proposed as attractive therapeutic targets to counteract inflammation and cartilage degradation observed in OA. This study aims to elucidate the involvement of GPR40 in OA. In this study, we used an in vitro model of OA, and surgically induced OA by ligament transection and partial meniscectomy in wild-type and GPR40 deficient mice. OA phenotype was investigated in vivo by histology and genes expression. We demonstrate that IL-1β-treated GPR40^−/− chondrocytes secret more inflammatory mediators (nitric oxide, interleukin-6, prostaglandin E2) and active catabolic enzymes (metalloproteinase-2, -9 [MMP-2, MMP-9]), and show decreased anabolism (glycoaminoglycan) compared to GPR40^+/+ cells. In accordance with these results, we show that GPR40^−/− mice exhibit an aggravated OA-induced phenotype characterized by higher tidemark exposure, frequency of osteophyte formation and subchondral bone sclerosis. Altogether our results demonstrate that GPR40 deficiency leads to an extended OA phenotype, providing evidence that increasing GPR40 activity, by natural or synthetic ligands, could be a new strategy in the management of OA.     

Induction of GPR40 positively regulates cell motile and growth activities in breast cancer MCF-7 cells

Abstract

Free fatty acid (FFA) receptors belong to a member of G-protein-coupled receptors. GPCR 120 (GPR120) and GPR40 are identified as FFA receptors and activated via the binding of long- and medium-chain FFAs. The aim of this study was to assess the effects of GPR120 and GPR40 on cell motility and growth in breast cancer cells treated with tamoxifen (TAM). MCF-7 cells were continuously treated with TAM for approximately 6?months. The expression level of GPR40 gene was markedly higher in the long-term TAM treated (MCF-TAM) cells than in MCF-7 cells. In cell motility assay, MCF-TAM cells indicated the high cell motile activity, compared with MCF-7 cells. The cell motile activity of MCF-TAM cells was suppressed by a selective GPR40 antagonist, GW1100. To evaluate the effects of GPR40 on cell growth activity under estrogen-free conditions, cells were maintained in serum-free DMEM without phenol red for 2?days. In estrogen-free conditioned medium, the cell growth rate of MCF-TAM cells was significantly higher than that of MCF-7 cells. In addition, treatment of GW1100 reduced the cell growth rate of MCF-TAM cells. These results suggest that the cell motile and growth activities may be positively regulated through the induction of GPR40 by the long-term TAM treatment in MCF-7 cells.     

Structural basis for GPCR signaling by small polar versus large lipid metabolites—discovery of non-metabolite ligands

Key metabolites act through specific G protein-coupled receptors (GPCRs) as extracellular signals of fuel availability and metabolic stress. Here, we focus on the succinate receptor SUCNR1/GPR91 and the long chain fatty acid receptor FFAR1/GPR40, for which 3D structural information is available. Like other small polar acidic metabolites, succinate is excreted from the cell by transporter proteins to bind to an extracellular, solvent-exposed pocket in SUCNR1. Non-metabolite pharmacological tool compounds are currently being designed based on the structure of the SUCNR1 binding pocket. In FFAR1, differently signaling lipid mimetics bind in two distinct membrane-exposed sites corresponding to each of the lipid bilayer leaflets. Conceivably endogenous lipid ligands gain access to these sites by way of the membrane and probably occupy both sites under physiological circumstances. Design of polar agonists for a dynamic, solvent-exposed pocket in FFAR1 underlines the possibility of structure-based approaches for development of novel tool compounds even in lipid sensing metabolite GPCRs.     

G protein-coupled receptors for free fatty acids

Free fatty acids (FFAs) are not only an important direct source of energy but they also play key roles in regulating a range of physiological responses. Although it was long assumed that such effects of FFAs must occur following cellular uptake, and potentially via their conversion to fatty acyl-CoAs, it is now apparent that FFAs also function directly as agonists at a number of G protein-coupled receptors (GPCRs). Tissue distribution studies and, subsequently, siRNA knock-down experiments have indicated key roles for these GPCRs in glucose homeostasis, adipogenesis, white cell recruitment and potentially in a range of other processes. Considerable interest is thus now centred on the generation of potent and selective small molecule ligands, both as tool compounds to further unravel the biology and physiological role of this group of GPCRs and as starting points for possible therapeutic intervention in a range of areas, particularly those associated with 'metabolic syndrome'.     

Exploration of phenylpropanoic acids as agonists of the free fatty acid receptor 4 (FFA4): Identification of an orally efficacious FFA4 agonist

The long chain free fatty acid receptor 4 (FFA4/GPR120) has recently been recognized as lipid sensor playing important roles in nutrient sensing and inflammation and thus holds potential as a therapeutic target for type 2 diabetes and metabolic syndrome. To explore the effects of stimulating this receptor in animal models of metabolic disease, we initiated work to identify agonists with appropriate pharmacokinetic properties to support progression into in vivo studies. Extensive SAR studies of a series of phenylpropanoic acids led to the identification of compound 29, a FFA4 agonist which lowers plasma glucose in two preclinical models of type 2 diabetes.     

Adipose tissue metabolism -- an aspect we should not neglect?

Free fatty acids (FFAs) are the most metabolically important products of adipose tissue lipolysis. Experimentally creating high FFA concentrations can reproduce the metabolic abnormalities of obesity in lean, healthy persons and lowering FFA concentrations can improve the metabolic health of upper body obese individuals. FFA concentrations are determined by both the release of FFAs into the bloodstream and the clearance of FFAs from the bloodstream. Normal FFA release rates are different in men and women and total FFA release is closely linked to resting energy expenditure. Upper body subcutaneous fat, visceral fat, and leg fat depots contribute differently to the exposure of various tissues to FFAs. The implications of regional adipose tissue lipolysis to systemic FFA availability and the effect of different approaches to treatment of obesity are discussed.     

游离脂肪酸受体蛋白研究进展

游离脂肪酸不仅是人和动物体的一种重要能量来源,也是一种重要的信号分子。最近研究表明,游离脂肪酸受体蛋白在维持机体内的葡萄糖稳衡、脂肪形成、白细胞功能等生理过程中都有重要的作用,对于调控人或动物的营养代谢及疾病发生具有重要生理意义。     

Melittin exerts multiple effects on the release of free fatty acids from L1210 cells: lack of selective activation of phospholipase A2 by melittin

Melittin is known as a phospholipase A2 (PLA2) activator, but the selectivity of its effect on PLA2 is uncertain. We examined the selectivity of melittin effect on the release of free fatty acids (FFAs) from L1210 cells using various inhibitors. A systemic lipid analysis by HPLC and GLC revealed that melittin induced release of various FFAs including saturated, monounsaturated, and polyunsaturated FFAs. Various PLA2 inhibitors examined exerted only minimal effects on the melittin-induced arachidonic acid (AA) and palmitic acid (PAL) releases. Specific inhibitors of phosphatidylinositol-phospholipase C (U73122) and diacylglycerol lipase (RHC80267) exerted significant inhibitory effects on both AA and PAL releases. These results suggest that melittin-induced FFA release is most likely due to multiple participations of various types of lipases. Since BAPTA/AM, an intracellular Ca2+ chelator, did not influence the FFA release, the Ca2+ influxed by melittin appeared not to be a key factor for the FFA release. The mimicking of the melittin-induced FFA release by digitonin, a membrane-permeabilizing agent, implies that the membrane-perturbing action of melittin is likely the cause of the FFA release. Melittin also induced release of multiple FFAs from other cell lines including P388D1 and HL60. The rapid melittin-stimulated phospholipase D (PLD) observed in L1210 cells appeared not directly related to the steady release of FFA, as indicated by the fact that the PLD was not blocked by RHC80267. In view of melittin's multiple effects on the composition of cellular lipids, we conclude that melittin does neither exclusively release any single FFA nor selectively activate PLA2 in L1210 cells. The problem of using melittin as a PLA2 activator is discussed.