GPR109A Research Articles

A review of the mechanisms of action of dimethylfumarate in the treatment of psoriasis.

Fumaric acid esters (FAEs) such as dimethylfumarate (DMF) are used for the treatment of adults with moderate-to-severe psoriasis. The mode of action of FAEs is complex. Here, we provide a comprehensive review of the literature to describe the molecular mechanisms by which DMF and its active metabolite monomethylfumarate (MMF) exert their anti-inflammatory and immune modulatory effects. MMF can bind to the hydroxy-carboxylic acid receptor 2 (HCA2) on the cell surface and both DMF and MMF react with intracellular glutathione following cell penetration. DMF and to some extent also MMF modulate the activity of certain cellular signalling proteins such as the nuclear factor (erythroid-derived 2)-like 2 (Nrf2), nuclear factor kappa B (Nf-κB) and the cellular concentration of cyclic adenosine monophosphate. Some studies show that DMF can also affect the hypoxia-inducible factor 1-alpha (HIF-1α). These actions seem to be responsible for i) the downregulation of inflammatory cytokines and ii) an overall shift from a proinflammatory Th1/Th17 response to an anti-inflammatory/regulatory Th2 response. Both steps are necessary for the amelioration of psoriatic inflammation, although additional mechanisms have been proposed. There is a growing body of evidence to support the notion that DMF/MMF may also exert effects on granulocytes and non-immune cell lineages including keratinocytes and endothelial cells. A better understanding of the multiple molecular mechanisms involved in the cellular action of FAEs will help to adapt and further improve the use of such small molecules for the treatment of psoriasis and other chronic inflammatory diseases.

1317 Nicotinic acid suppresses sebaceous lipid synthesis of human sebocytes via activating hydroxycarboxylic acid receptor 2 (HCA2)

1317 Nicotinic acid suppresses sebaceous lipid synthesis of human sebocytes via activating hydroxycarboxylic acid receptor 2 (HCA2)

Comprehensive investigation of cytokine- and immune-related gene variants in HBV-associated hepatocellular carcinoma patients.

Host genotype may be closely related to the different outcomes of Hepatitis B virus (HBV) infection. To identify the association of variants and HBV infection, we comprehensively investigated the cytokine- and immune-related gene mutations in patients with HBV associated hepatocellular carcinoma (HBV-HCC). Fifty-three HBV-HCC patients, 53 self-healing cases (SH) with HBV infection history and 53 healthy controls (HCs) were recruited, the whole exon region of 404 genes were sequenced at >900× depth. Comprehensive variants and gene levels were compared between HCC and HC, and HCC and SH. Thirty-nine variants (adjusted P<0.0001, Fisher’s exact test) and 11 genes (adjusted P<0.0001, optimal unified approach for rare variant association test (SKAT-O) gene level test) were strongly associated with HBV-HCC. Thirty-four variants were from eight human leukocyte antigen (HLA) genes that were previously reported to be associated with HBV-HCC. The novelties of our study are: five variants (rs579876, rs579877, rs368692979, NM_145007:c.*131_*130delTG, NM_139165:exon5:c.623-2->TT) from three genes (REAT1E, NOD-like receptor (NLR) protein 11 (NLRP11), hydroxy-carboxylic acid receptor 2 (HCAR2)) were found strongly associated with HBV-HCC. We found 39 different variants in 11 genes that were significantly related to HBV-HCC. Five of them were new findings. Our data implied that chronic hepatitis B patients who carry these variants are at a high risk of developing HCC.

Dual action by fumaric acid esters synergistically reduces adhesion to human endothelium

Objective: Dimethyl fumarate (DMF) is prescribed against relapsing-remitting multiple sclerosis (MS). Here, we investigated the effects of DMF and monomethyl fumarate (MMF), its metabolite in vivo, at the (inflamed) blood–brain barrier (BBB). Methods: Effects of fumaric acid esters were analyzed using primary human brain–derived microvascular endothelial cells (HBMECs) in combination with peripheral blood mononuclear cells (PBMCs) derived from DMF-treated MS patients. Results: MMF-binding to brain endothelium cells leads to activation of nuclear factor (erythroid-derived 2)–related factor 2 (Nrf2)–induced downregulation of vascular cell adhesion molecule 1 (VCAM-1). This might be mediated via the G-protein-coupled receptor (GPCR) hydroxycarboxylic acid receptor 2 (HCA2), a known molecular target of MMF, as we could demonstrate its expression and regulation on HBMECs. DMF treatment in vivo led to a strongly reduced expression of VCAM-1’s ligand very late antigen 4 (VLA-4) by selectively reducing integrin high-expressing memory T cells of MS patients, potentially due to inhibition of their maturation by reduced trans-localization of NFκB. Conclusion: DMF-mediated VCAM-1 downregulation on the endothelial side and reduction in T cells with a migratory phenotype on the lymphocyte side result in a synergistic reduction in T-cell adhesion to activated endothelium and, therefore, to reduced BBB transmigration in the setting of MS.

Fumarate decreases edema volume and improves functional outcome after experimental stroke

BackgroundOxidative stress and inflammation exacerbate tissue damage in the brain after ischemic stroke. Dimethyl-fumarate (DMF) and its metabolite monomethyl-fumarate (MMF) are known to stimulate anti-oxidant pathways and modulate inflammatory responses. Considering these dual effects of fumarates, we examined the effect of MMF treatment after ischemic stroke in mice. MethodsPermanent middle cerebral artery occlusion (pMCAO) was performed using adult, male C57BL/6 mice. Thirty minutes after pMCAO, 20mg/kg MMF was administered intravenously. Outcomes were evaluated 6, 24 and 48h after pMCAO. First, we examined whether a bolus of MMF was capable of changing expression of kelch-like erythroid cell-derived protein with CNC homology-associated protein 1 (Keap1) and nuclear factor erythroid 2-related factor (Nrf)2 in the infarcted brain. Next, we studied the effect of MMF on functional recovery. To explore mechanisms potentially influencing functional changes, we examined infarct volumes, edema formation, the expression of heat shock protein (Hsp)72, hydroxycarboxylic acid receptor 2 (Hcar2), and inducible nitric oxide synthase (iNOS) in the infarcted brain using real-time PCR and Western blotting. Concentrations of a panel of pro- and anti-inflammatory cytokines (IFNγ, IL-1β, IL-2, IL-4, IL-5, IL-6, IL-10, IL-12p70, TNF) were examined in both the infarcted brain tissue and plasma samples 6, 24 and 48h after pMCAO using multiplex electrochemoluminiscence analysis. ResultsAdministration of MMF increased the protein level of Nrf2 6h after pMCAO, and improved functional outcome at 24 and 48h after pMCAO. MMF treatment did not influence infarct size, however reduced edema volume at both 24 and 48h after pMCAO. MMF treatment resulted in increased Hsp72 expression in the brain 6h after pMCAO. Hcar2 mRNA levels increased significantly 24h after pMCAO, but were not different between saline- and MMF-treated mice. MMF treatment also increased the level of the anti-inflammatory cytokine IL-10 in the brain and plasma 6h after pMCAO, and additionally reduced the level of the pro-inflammatory cytokine IL-12p70 in the brain at 24 and 48h after pMCAO. ConclusionsA single intravenous bolus of MMF improved sensory-motor function after ischemic stroke, reduced edema formation, and increased the levels of the neuroprotective protein Hsp72 in the brain. The early increase in IL-10 and reduction in IL-12p70 in the brain combined with changes in systemic cytokine levels may also contribute to the functional recovery after pMCAO.

Dimethyl fumarate mediates Nrf2-dependent mitochondrial biogenesis in mice and humans.

The induction of mitochondrial biogenesis could potentially alleviate mitochondrial and muscle disease. We show here that dimethyl fumarate (DMF) dose-dependently induces mitochondrial biogenesis and function dosed to cells in vitro, and also dosed in vivo to mice and humans. The induction of mitochondrial gene expression is more dependent on DMF's target Nrf2 than hydroxycarboxylic acid receptor 2 (HCAR2). Thus, DMF induces mitochondrial biogenesis primarily through its action on Nrf2, and is the first drug demonstrated to increase mitochondrial biogenesis with in vivo human dosing. This is the first demonstration that mitochondrial biogenesis is deficient in Multiple Sclerosis patients, which could have implications for MS pathophysiology and therapy. The observation that DMF stimulates mitochondrial biogenesis, gene expression and function suggests that it could be considered for mitochondrial disease therapy and/or therapy in muscle disease in which mitochondrial function is important.

Ketone Bodies as Anti-Seizure Agents

There is growing evidence that ketone bodies (KB)-derived from fatty acid oxidation and produced during fasting or consumption of high-fat diets-can exert broad neuroprotective effects. With respect to epilepsy, KB (such as β-hydroxybutyrate or BHB, acetoacetate and acetone) have been shown to block acutely induced and spontaneous recurrent seizures in various animal models. Although the mechanisms underlying the anti-seizure effects of KB have not been fully elucidated, recent experimental studies have invoked ketone-mediated effects on both inhibitory (e.g., GABAergic, purinergic and ATP-sensitive potassium channels) and excitatory (e.g., vesicular glutamate transporters) neurotransmission, as well as mitochondrial targets (e.g., respiratory chain and mitochondrial permeability transition). Moreover, BHB appears to exert both epigenetic (i.e., inhibition of histone deacetylases or HDACs) and anti-inflammatory (i.e., peripheral modulation of hydroxycarboxylic acid receptor and inhibition of the NOD-like receptor protein 3 or NRLP3 inflammasome) activity. While the latter two effects of BHB have yet to be directly linked to ictogenesis and/or epileptogenesis, parallel lines of evidence indicate that HDAC inhibition and a reduction in neuroinflammation alone or collectively can block seizure activity. Nevertheless, the notion that KB are themselves anti-seizure agents requires clinical validation, as prior studies have not revealed a clear correlation between blood ketone levels and seizure control. Notwithstanding this limitation, there is growing evidence that KB are more than just cellular fuels, and can exert profound biochemical, cellular and epigenetic changes favoring an overall attenuation in brain network excitability.

Stimulation of lactate receptor (HCAR1) affects cellular DNA repair capacity

Numerous G-protein coupled receptors have been reported to enhance cancer cell survival and resistance to clinically used chemotherapeutics. Recently, hydroxycarboxylic acid receptor 1 (HCAR1) was shown to drive lactate-dependent enhancement of cell survival and metastasis in pancreatic and breast cancers. Furthermore, our previous study confirmed the involvement of HCAR1 in lactate-related enhancement of DNA repair in cervical cancer cells. In the present study, we examined the possible mechanisms of HCAR1-mediated enhancement of DNA repair capacity. We observed that the HCAR1 agonist dihydroxybenzoic acid (DHBA) up-regulated BRCA1 (breast cancer type 1 susceptibility protein) and NBS1 (Nijmegen breakage syndrome 1) expression in HeLa cells. Moreover, HCAR1 silencing decreased mRNA and protein levels of BRCA1 by 30% and 20%, respectively. Immunocytochemical analyses of BRCA1, nibrin and DNA-PKcs indicated an increased accumulation of these proteins in cell nuclei after DHBA stimulation. Subsequently, these changes in the DNA repair protein levels translated into an enhanced DNA repair rate after doxorubicin treatment, as shown by γ-H2AX and comet assay experiments. In contrast, the down-regulation of HCAR1 decreased the efficiency of DNA repair. Finally, we observed the abrogation of DHBA-driven BRCA1 protein up-regulation and enhanced DNA repair following the preincubation of cells with the PKC inhibitor Gö6983. Taken together, our data indicate that lactate receptor/HCAR1 expression in cervical carcinoma cells may contribute to the modulation of cellular DNA repair mechanisms.

Role of Dietary Metabolites in Regulating the Host Immune Response in Gastrointestinal Disease.

The host immune response to gastrointestinal (GI) infections, hypersensitivity reactions, or GI cancers comprises numerous pathways that elicit responses on different host cells. Some of these include (1) the stimulation of mast cells via their IgE receptor, (2) the production of antibodies leading to antibody-mediated cytotoxic T/natural killer cell killing, (3) the activation of the complement pathway, and (4) the activation of the adaptive immune response via antigen-presenting cell, T cell, and B cell interactions. Within the plethora of these different responses, several host immune cells represent major key players such as those of myeloid lineage (including neutrophils, macrophages, myeloid-derived suppressor cells) or lymphoid lineage (including T and B cells). In this review, we focus on newly identified metabolites and metabolite receptors that are expressed by either myeloid or lymphoid lineages. Irrespective of their source, these metabolites can in certain instances elicit responses on a wide range of cell types. The myeloid-expressed metabolic enzymes and receptors which we will discuss in this review include arginase 2 (Arg2), indoleamine-2,3-dioxygenase 1 (IDO1), hydroxycarboxylic acid receptor 2 (Hcar2; also called GPR109A), and immunoresponsive gene 1 (Irg1). We will also review the role of the lymphoid-expressed metabolite receptor that binds to the sphingosine-1-phosphate (S1P) sphingolipid. Moreover, we will describe the synthesis and metabolism of retinoic acid, and its effect on T cell activation. The review will then discuss the function of these metabolites in the context of GI disease. The review provides evidence that metabolic pathways operate in a disease- and context-dependent manner—either independently or concomitantly—in the GI tract. Therefore, an integrated approach and combinatorial analyses are necessary to devise new therapeutic strategies that can synergistically improve prognoses.

AMP010014A09 in Sus Scrofa Encodes an Analog of G Protein-Coupled Receptor 109A, Which Mediates the Anti-Inflammatory Effects of Beta-Hydroxybutyric Acid

Background: Hydroxy-carboxylic acid receptor 2 (HCA₂, also called GPR109A) belongs to the G protein-coupled receptor (GPCR) family and is found in humans, rats, mice, hamsters and guinea pigs, but there are almost no reports of this protein in other species. In this investigation, we speculated that AMP010014A09 (AMP+) is a homologue of GPR109A in swine. Methods: To test this hypothesis, the following experiments were designed: monocytes isolated from the peripheral blood of swine were treated with LPS after pretreating with or without β-hydroxybutyric acid (BHBA), and the levels of pro-inflammatory cytokines and inflammatory proteins were assessed. cAMP levels induced by Forskolin in swine testicular (ST) and IPEC-J2 cells were detected with or without BHBA treatment and following silencing or stable transfection of the AMP+ gene. Results: AMP+ in swine exhibited a high level of homology with HM74A in humans and PUMA-G in mice. BHBA inhibited the LPS-induced secretion of the pro-inflammatory cytokines TNF-α, IL-6 and IL-1β and the inflammatory protein COX-2 in monocytes of swine. BHBA suppressed the Forskolin-induced cAMP level increase in ST cells, but failed to inhibit the accumulation of cAMP after the AMP+ gene was silenced with shRNA by transfecting cells with the pGPU6-GFP-Neo-AMP+-sus-392 plasmid. BHBA had no effect on cAMP levels in IPEC-J2 cells, but significantly inhibited the increase in cAMP induced by Forskolin treatment following transfection of the AMP+ gene into IPEC-J2 cells by a lentivirus vector. Conclusion: Our results indicated that AMP+ encodes a G protein-coupled receptor in Sus scrofa that inhibits cAMP levels and mediates anti-inflammatory effects in swine monocytes.

The roles of Niacin in the treatment of diabetic retinopathy and its mechanism

Objective To investigate the protective effect of Niacin on blood-retina barrier (BRB) in diabetic rats and related mechanism. Methods The male Wistar rats (60) were divided into control (CON) group, diabetes (DM) group and Niacin-treated (NA) group, 20 rats in each group. Rats diabetes models were induced with streptozotocin injection. Niacin (40 mg/kg·d) was administrated orally everyday in Niacin-treated group until sacrificed after 3 months. Pathological outcomes, total cholesterol (TC) and high-density lipoprotein (HDL) were evaluated at month 3. Optical microscopy was used to observe the retinal structure. The integrity of BRB and the vascular permeability was quantified by analyzing albumin leakage using Evans blue (EB) method. The relative expressions of Claudin-5, Occludin, zonula occluden (ZO)-1 and GPR109A mRNA in rat retinas were detected by reverse transcription PCR (RT-PCR) and relative expression of GPR109A, tumor necrosis factor (TNF)-α and interleukin (IL)-6 by Western blot. Results Compared to CON group, the TC content was increased and HDL content was decreased in DM group (t=4.034, 5.831; P<0.05). Compared to DM group, the TC content was decreased and HDL content was increased in NA group (t=6.868, 3.369; P<0.05). The retinal structure of CON group was normal. Pathological changes were found in the DM group, such as tumescent nuclei and disorganized structures. The retinal structure of NA group was similar to the control group. Evans blue dye that the microvascular leakage in DM group was increased compared with CON group (t=24.712, P<0.05), while in NA group was decreased compared with DM group (t=16.414, P<0.05). The mRNA expression of Occludin, Claudin-5, ZO-1 in DM group were decreased compared with CON group (t=11.422, 12.638, 12.060; P<0.05), while in NA group were increased compared with DM group (t=5.278, 3.952, 8.030; P<0.05). The mRNA expression of GPR109A in NA group were increased compared with DM group (t=5.053, P<0.05). The protein expression of GPR109A, IL-6, TNF-αin DM group were increased compared with CON group (t=4.915, 11.106, 6.582; P<0.05). Compared to DM group, the protein expression of GPR109A was increased (t=5.806, P<0.05), while the protein expression of IL-6 and TNF-α were decreased (t=10.131, 5.017; P<0.05). Conclusion Niacin has the protective effect for BRB by up-regulating GPR109A expression which may suppress inflammation. Key words: Blood-retinal barrier/drug effects; Niacin/agonists; Animal experimentation

Lactate Stimulates IL-4 and IL-13 Production in Activated HuT-78 T Lymphocytes Through a Process That Involves Monocarboxylate Transporters and Protein Hyperacetylation.

Mucosal cells of the gastrointestinal and female reproductive tract are constantly exposed to l- and d-lactate of bacterial origin. These compounds not only protect the host from pathogen colonization but also modulate the activity of mucosal immune cells, thereby playing an important role in inflammatory host responses. In this study, we demonstrated that exposure of anti-CD3/CD28 or phorbol 12-myristate 13-acetate (PMA)/ionomycin-activated HuT-78 T lymphocyte cells to 10-20 mM d-lactate significantly increased IL-4 and IL-13 production. Interestingly, the d-lactate isomer, exclusively produced locally by gut or cervicovaginal microbiota, was found to be more potent than the l-isomer. Interestingly, neither of the strong histone deacetylase inhibitors [structurally similar butyrate and suberoylanilide hydroxamic acid (SAHA)] was as effective in the stimulation of IL-13 production as d-lactate. Lactate transport through monocarboxylate transporters was required for lactate-enhanced IL-13 production in a manner that was not hydroxycarboxylic acid receptor 1-dependent. Furthermore, lactate treatment increased the acetylation of GATA-3, a critical regulator of Th1/Th2 differentiation and resulted in H3 and H4 histone hyperacetylation state, which is a characteristic feature of transcriptionally active chromatin. Both lactate isomers also enhanced IL4 and IL13 promoter-driven activity of reporter constructs in murine and human cells. Together, these findings demonstrate that a local millimolar concentration of l- or d-lactate may play an important role in the modulation of inflammation-mediated processes.

Expression of metabolic sensing receptors in adipose tissues of periparturient dairy cows with differing extent of negative energy balance

We recently showed that the mRNA expression of genes encoding for specific nutrient sensing receptors, namely the free fatty acid receptors (FFAR) 1, 2, 3, and the hydroxycarboxylic acid receptor (HCAR) 2, undergo characteristic changes during the transition from late pregnancy to lactation in certain adipose tissues (AT) of dairy cows. We hypothesised that divergent energy intake achieved by feeding diets with either high or low portions of concentrate (60% v. 30% concentrate on a dry matter basis) will alter the mRNA expression of FFAR 1, 2, 3, as well as HCAR2 in subcutaneous (SCAT) and retroperitoneal AT (RPAT) of dairy cows in the first 3 weeks postpartum (p.p.). For this purpose, 20 multiparous German Holstein cows were allocated to either the high concentrate ration (HC, n=10) or the low concentrate ration (LC, n=10) from day 1 to 21 p.p. Serum samples and biopsies of SCAT (tail head) and RPAT (above the peritoneum) were obtained at day −21, 1 and 21 relative to parturition. The mRNA abundances were measured by quantitative PCR. The concentrations of short-chain fatty acid (SCFA) in serum were measured by gas chromatography-flame ionisation detector. The FFAR1 and FFAR2 mRNA abundance in RPAT was higher at day −21 compared to day 1. At day 21 p.p. the FFAR2 mRNA abundance was 2.5-fold higher in RPAT of the LC animals compared to the HC cows. The FFAR3 mRNA abundance tended to lower values in SCAT of the LC group at day 21. The HCAR2 mRNA abundance was neither affected by time nor by feeding in both AT. On day 21 p.p. the HC group had 1.7-fold greater serum concentrations of propionic acid and lower concentrations of acetic acid (trend: 1.2-fold lower) compared with the LC group. Positive correlations between the mRNA abundance of HCAR2 and peroxisome proliferator-activated receptor γ-2 (PPARG2) indicate a link between HCAR2 and PPARG2 in both AT. We observed an inverse regulation of FFAR2 and FFAR3 expression over time and both receptors also showed an inverse mRNA abundance as induced by different portions of concentrate. Thus, indicating divergent nutrient sensing of both receptors in AT during the transition period. We propose that the different manifestation of negative EB in both groups at day 21 after parturition affect at least FFAR2 expression in RPAT.

Longitudinal changes in adipose tissue of dairy cows from late pregnancy to lactation. Part 1: The adipokines apelin and resistin and their relationship to receptors linked with lipolysis

The transition from pregnancy to lactation is characterized by major changes in glucose and adipose tissue metabolism. Anti- and prolipolytic pathways mediated via the hydroxycarboxylic acid receptors 1 (HCAR1) and 2 (HCAR2) and tumor necrosis factor-α receptor 1 (TNFR1), as well as the adipokines apelin and resistin, are likely involved in regulating these processes. This study aimed to determine the mRNA abundance of the aforementioned receptors in both subcutaneous and visceral adipose tissue, to characterize the adipokine concentrations in serum, and to test the effects of feeding diets with either high or low portions of concentrate and a concomitant niacin supplementation from late gestation to early lactation. Twenty pluriparous German Holstein cows were all kept on the same silage-based diet until d 42 antepartum, when they were allocated to 2 feeding groups: until d 1 antepartum, 10 animals each were assigned to either a high-concentrate (60:40 concentrate-to-roughage ratio) or a low-concentrate diet (30:70). Both groups were further subdivided into a control and a niacin group, the latter receiving 24g/d of nicotinic acid from d −42 until 24. From d 1 to 24 postpartum, the concentrate portion was increased from 30 to 50% for all cows. Biopsies of subcutaneous (SCAT) and retroperitoneal adipose tissue (RPAT) were taken at d −42, 1, 21, and 100 relative to parturition. Blood samples were drawn along with the biopsies and on d −14, 3, 7, 14, and 42. The concentrations of the adipokines apelin and resistin in serum were measured via ELISA. The mRNA of the 3 receptors in AT was quantified as well as the protein abundance of HCAR2 by Western blot. The feeding regimen did not affect the variables examined. The concentrations of apelin remained fairly constant during the observation period, whereas the resistin concentrations increased toward parturition and decreased to precalving levels within 1 wk after calving. The mRNA abundance of HCAR1, HCAR2, and TNFR1 changed in SCAT and RPAT during the considered time period. For the HCAR2 protein, time-dependent changes were restricted to SCAT. The mRNA abundance of all receptors was greater in RPAT than in SCAT. The tissue-specific correlations observed between the receptors point to a link between these factors and may indicate different regulatory roles in the respective tissues. This study provides insight into the complex metabolic adaptations during the transition period and supports a differential regulation of lipolysis among SCAT and RPAT in dairy cows.

The role of the C-terminus of the human hydroxycarboxylic acid receptors 2 and 3 in G protein activation using Gα-engineered yeast cells

In the present study we focused our attention on the family of hydroxycarboxylic acid (HCA) receptors, a GPCR family of three members, of which the HCA2 and HCA3 receptors share 95% high sequence identity but differ considerably in C-terminus length with HCA3 having the longest tail. The two receptors were expressed and analysed for their activation profile in Saccharomyces cerevisiae MMY yeast strains that have different G protein Gα subunits. The hHCA2 receptor was promiscuous in its G protein coupling preference. In the presence of nicotinic acid the hHCA2 receptor activated almost all G protein pathways except Gαq (MMY14). However, the Gα protein coupling profile of the hHCA3 receptor was less promiscuous, as the receptor only activated Gαi1 (MMY23) and Gαi3 (MMY24) pathways.We then constructed two mutant receptors by ‘swapping’ the short (HCA2) and long (HCA3) C-terminus. The differences in HCA2 and HCA3 receptor activation and G protein selectivity were not controlled, however, by their C-terminal tails, as we observed only minor differences between mutant and corresponding wild-type receptor. This study provides new insights into the G protein coupling profiles of the HCA receptors and the function of the receptor's C terminus, which may be extended to other GPCRs.

L- and D-lactate enhance DNA repair and modulate the resistance of cervical carcinoma cells to anticancer drugs via histone deacetylase inhibition and hydroxycarboxylic acid receptor 1 activation.

BackgroundThe consideration of lactate as an active metabolite is a newly emerging and attractive concept. Recently, lactate has been reported to regulate gene transcription via the inhibition of histone deacetylases (HDACs) and survival of cancer cells via hydroxycarboxylic acid receptor 1 (HCAR1). This study examined the role of L- and D-lactate in the DNA damage response in cervical cancer cells.MethodsThree cervical cancer cell lines were examined: HeLa, Ca Ski and C33A. The inhibitory activity of lactate on HDACs was analysed using Western blot and biochemical methods. The lactate-mediated stimulation of DNA repair and cellular resistance to neocarzinostatin, doxorubicin and cisplatin were studied using γ-H2AX, comet and clonogenic assays. HCAR1 and DNA repair gene expression was quantified by real-time PCR. DNA-PKcs activity and HCAR1 protein expression were evaluated via immunocytochemistry and Western blot, respectively. HCAR1 activation was investigated by measuring intracellular cAMP accumulation and Erk phosphorylation. HCAR1 expression was silenced using shRNA.ResultsL- and D-lactate inhibited HDACs, induced histone H3 and H4 hyperacetylation, and decreased chromatin compactness in HeLa cells. Treating cells with lactate increased LIG4, NBS1, and APTX expression by nearly 2-fold and enhanced DNA-PKcs activity. Based on γ-H2AX and comet assays, incubation of cells in lactate-containing medium increased the DNA repair rate. Furthermore, clonogenic assays demonstrated that lactate mediates cellular resistance to clinically used chemotherapeutics. Western blot and immunocytochemistry showed that all studied cell lines express HCAR1 on the cellular surface. Inhibiting HCAR1 function via pertussis toxin pretreatment partially abolished the effects of lactate on DNA repair. Down-regulating HCAR1 decreased the efficiency of DNA repair, abolished the cellular response to L-lactate and decreased the effect of D-lactate. Moreover, HCAR1 shRNA-expressing cells produced significantly lower mRNA levels of monocarboxylate transporter 4. Finally, the enhancement of DNA repair and cell survival by lactate was suppressed by pharmacologically inhibiting monocarboxylate transporters using the inhibitor α-cyano-4-hydroxycinnamic acid (α-CHCA).ConclusionsOur data indicate that L- and D-lactate present in the uterine cervix may participate in the modulation of cellular DNA damage repair processes and in the resistance of cervical carcinoma cells to anticancer therapy.Electronic supplementary materialThe online version of this article (doi:10.1186/s12964-015-0114-x) contains supplementary material, which is available to authorized users.

Nrf2 and beyond: deciphering the mode of action of fumarates in the inflamed central nervous system.

observed in degenerating cells with considerable differences in expression patterns depending on cell type. This detailed description of cellular expression patterns (and sources) of Nrf2 in different CNS regions of pathological brain specimens is relevant since it (re)emphasizes that oxidative stress seems to be an important pathomechanism of demyelination and neurodegeneration in MS. It further supports the view that therapeutic targeting of the Nrf2 pathway might indeed be a reasonable strategy in preventing oligodendrocyte degeneration. Notably, Nrf2 expression differed considerably between neural and glial cell types: immunoreactivity against Nrf2 was only rarely detectable in neurons. It might therefore be tempting to speculate that a potential protective role of DMF against oxidative stress might be more effective in active white matter lesions as compared to grey matter. This work using human materials provides important insight into “real” disease not obtainable using experimental models, while conclusions on DMF drug effects or even more general—on the role of Nrf2 in the dynamics of lesion evolution versus resolution—naturally remain difficult. In the second publication, Uccelli and co-workers report that MMF (the biochemical derivate of DMF after metabolization in vivo) can switch the functional phenotype of activated microglia from a pro-inflammatory type to an (indirect) anti-inflammatory (or “neuroprotective”) type [8]. Interestingly, this effect was mediated through activation of the hydroxycarboxylic acid receptor 2 (HCAR2), a G protein-coupled membrane receptor. This receptor has already been reported to be critically involved in therapeutic efficacy of DMF in preclinical EAE studies, its specific role, however, remained elusive at that stage [1]. In the present article, the authors demonstrate that LPS-activated microglia express HCAR2 and that binding of MMF to this receptor in vitro activates a downstream pathway mediated Dimethyl fumarate (DMF) is an oral drug based on recent phase III studies that has been approved by the US Food and Drug Administration and European Medicines Agency for the treatment of relapsing forms of multiple sclerosis (MS) [2, 4]. Its mode of action, however, still remains elusive. In experimental autoimmune encephalomyelitis (EAE), an animal model of MS, DMF demonstrated immunomodulatory as well as neuroprotective effects translating into disease amelioration and clinical improvement [7, 9]. DMF is rapidly metabolized to monomethyl fumarate (MMF), both of which covalently modify glutathione and KEAP-1, the inhibitor of the antioxidant transcription factor nuclear factor erythroid-derived 2–related factor 2 (Nrf2), and induce hemoxygenase-1 (HMOX1), NADP(H) quinoline oxidoreductase-1 (NQO1), and other genes [3, 7]. Whether the covalent modifications occur in vivo and eventually translate in attenuation of MS pathology is still unclear [1]. However, one key hypothesis for the therapeutic efficacy of DMF is its action through a Nrf2-dependent anti-oxidative pathway [5]. In the present issue of Acta Neuropathologica, two publications provide further evidence for Nrf2 as a relevant therapeutic target in MS: In the first article, Licht-Mayer and colleagues report positive immunoreactivity against Nrf2 in active MS lesions from formalin-fixed autopsy brain tissue [6]. Nrf2 was particularly localized to mature oligodendrocytes and most extensive expression was

Characterization of the hydroxycarboxylic acid receptor 2 in cats

The hydroxycarboxylic acid receptor 2 (HCA2) belongs to a family of nutrient-sensing receptors that bind β-hydroxybutyrate, an alternative fuel source produced during a negative energy balance. The HCA2 receptor has not been identified or characterized in cats. Therefore, the following were the objectives of this study: (1) identify the feline HCA2 receptor protein sequence and compare against known human and rodent sequences, (2) determine tissue distribution and relative expression in lean, healthy cats, and (3) demonstrate in vitro functionality in feline adipose tissue. Tissues (n = 6) and primary adipocytes (n = 4) were collected from lean, healthy, female cats. The published genomic sequence for cats was used to design primers for polymerase chain reaction isolation of HCA2. Relative tissue distribution was evaluated using reverse transcriptase-polymerase chain reaction with RNA isolated from 9 different tissues (spleen, pancreas, lymph node, jejunum, kidney, liver, heart, and subcutaneous and abdominal adipose tissue). Receptor function was evaluated in primary feline adipocyte culture, and changes were compared with basal lipolysis. The in silico predicted feline HCA2 protein sequence exhibited 83.1% and 86.5% amino acid similarity to human and mouse sequences, respectively. The feline HCA2 receptor is predominantly expressed in adipose tissue and spleen. Exposure of feline adipocytes to niacin, a pharmacologic ligand of HCA2, inhibited lipolysis to a similar degree as insulin, a potent lipolytic inhibitor. In conclusion, the feline HCA2 receptor is similar to human and murine receptors in sequence, distribution, and functionality. By gaining a better understanding of the HCA2 receptor in cats, we will be able to better manage feline patients.

Fumarates modulate microglia activation through a novel HCAR2 signaling pathway and rescue synaptic dysregulation in inflamed CNS.

Dimethyl fumarate (DMF), recently approved as an oral immunomodulatory treatment for relapsing-remitting multiple sclerosis (MS), metabolizes to monomethyl fumarate (MMF) which crosses the blood–brain barrier and has demonstrated neuroprotective effects in experimental studies. We postulated that MMF exerts neuroprotective effects through modulation of microglia activation, a critical component of the neuroinflammatory cascade that occurs in neurodegenerative diseases such as MS. To ascertain our hypothesis and define the mechanistic pathways involved in the modulating effect of fumarates, we used real-time PCR and biochemical assays to assess changes in the molecular and functional phenotype of microglia, quantitative Western blotting to monitor activation of postulated pathway components, and ex vivo whole-cell patch clamp recording of excitatory post-synaptic currents in corticostriatal slices from mice with experimental autoimmune encephalomyelitis (EAE), a model for MS, to study synaptic transmission. We show that exposure to MMF switches the molecular and functional phenotype of activated microglia from classically activated, pro-inflammatory type to alternatively activated, neuroprotective one, through activation of the hydroxycarboxylic acid receptor 2 (HCAR2). We validate a downstream pathway mediated through the AMPK–Sirt1 axis resulting in deacetylation, and thereby inhibition, of NF-κB and, consequently, of secretion of pro-inflammatory molecules. We demonstrate through ex vivo monitoring of spontaneous glutamate-mediated excitatory post-synaptic currents of single neurons in corticostriatal slices from EAE mice that the neuroprotective effect of DMF was exerted on neurons at pre-synaptic terminals by modulating glutamate release. By exposing control slices to untreated and MMF-treated activated microglia, we confirm the modulating effect of MMF on microglia function and, thereby, its indirect neuroprotective effect at post-synaptic level. These findings, whereby DMF-induced activation of a new HCAR2-dependent pathway on microglia leads to the modulation of neuroinflammation and restores synaptic alterations occurring in EAE, represent a possible novel mechanism of action for DMF in MS.Electronic supplementary materialThe online version of this article (doi:10.1007/s00401-015-1422-3) contains supplementary material, which is available to authorized users.

Hydroxycarboxylic acid receptors are essential for breast cancer cells to control their lipid/fatty acid metabolism.

Cancer cells exhibit characteristic changes in their metabolism with efforts being made to address them therapeutically. However, targeting metabolic enzymes as such is a major challenge due to their essentiality for normal proliferating cells. The most successful pharmaceutical targets are G protein-coupled receptors (GPCRs), with more than 40% of all currently available drugs acting through them.We show that, a family of metabolite-sensing GPCRs, the Hydroxycarboxylic acid receptor family (HCAs), is crucial for breast cancer cells to control their metabolism and proliferation.We found HCA1 and HCA3 mRNA expression were significantly increased in breast cancer patient samples and detectable in primary human breast cancer patient cells. Furthermore, siRNA mediated knock-down of HCA3 induced considerable breast cancer cell death as did knock-down of HCA1, although to a lesser extent. Liquid Chromatography Mass Spectrometry based analyses of breast cancer cell medium revealed a role for HCA3 in controlling intracellular lipid/fatty acid metabolism. The presence of etomoxir or perhexiline, both inhibitors of fatty acid β-oxidation rescues breast cancer cells with knocked-down HCA3 from cell death.Our data encourages the development of drugs acting on cancer-specific metabolite-sensing GPCRs as novel anti-proliferative agents for cancer therapy.