Niacin activates the G protein estrogen receptor (GPER)-mediated signalling

Nicotinic acid, also known as niacin, is the water soluble vitamin B3 used for decades for the treatment of dyslipidemic diseases. Its action is mainly mediated by the G protein-coupled receptor (GPR) 109A; however, certain regulatory effects on lipid levels occur in a GPR109A-independent manner. The amide form of nicotinic acid, named nicotinamide, acts as a vitamin although neither activates the GPR109A nor exhibits the pharmacological properties of nicotinic acid. In the present study, we demonstrate for the first time that nicotinic acid and nicotinamide bind to and activate the GPER-mediated signalling in breast cancer cells and cancer-associated fibroblasts (CAFs). In particular, we show that both molecules are able to promote the up-regulation of well established GPER target genes through the EGFR/ERK transduction pathway. As a biological counterpart, nicotinic acid and nicotinamide induce proliferative and migratory effects in breast cancer cells and CAFs in a GPER-dependent fashion. Moreover, nicotinic acid prevents the up-regulation of ICAM-1 triggered by the pro-inflammatory cytokine TNF-α and stimulates the formation of endothelial tubes through GPER in HUVECs. Together, our findings concerning the agonist activity for GPER displayed by both nicotinic acid and nicotinamide broaden the mechanisms involved in the biological action of these molecules and further support the potential of a ligand to induce different responses mediated in a promiscuous manner by distinct GPCRs.     

Discovery of pyrazolopyrimidines as the first class of allosteric agonists for the high affinity nicotinic acid receptor GPR109A

Pyrazolopyrimidines were discovered as the first class of allosteric agonists for the high affinity nicotinic acid receptor GPR109A. In addition to its intrinsic activity, compound 9n significantly enhances nicotinic acid binding to the receptor, thereby potentiating the functional efficacy of nicotinic acid.     

Nicotinic acid receptor agonists differentially activate downstream effectors

Nicotinic acid remains the most effective therapeutic agent for the treatment and prevention of atherosclerosis resulting from low high density lipoprotein cholesterol. The therapeutic actions of nicotinic acid are mediated by GPR109A, a Gi protein-coupled receptor, expressed primarily on adipocytes, Langerhans cells, and macrophage. Unfortunately, a severe, cutaneous flushing side effect limits its use and patient compliance. The mechanism of high density lipoprotein elevation is not clearly established but assumed to be influenced by an inhibition of lipolysis in the adipose. The flushing side effect appears to be mediated by the release of prostaglandin D2 from Langerhans cells in the skin. We hypothesized that the signal transduction pathways mediating the anti-lipolytic and prostaglandin D2/flushing pathways are distinct and that agonists may be identified that are capable of selectively eliciting the therapeutic, anti-lipolytic pathway while avoiding the activation of the parallel flush-inducing pathway. We have identified a number of GPR109A pyrazole agonists that are capable of fully inhibiting lipolysis in vitro and in vivo and not only fail to elicit a flushing response but can antagonize the ability of nicotinic acid to elicit a flush response in vivo. In contrast to flushing agonists, exposure of cells expressing GPR109A to the non-flushing agonists fails to induce internalization of the receptor or to activate ERK 1/2 mitogen-activated protein kinase phosphorylation.     

Elucidation of signaling and functional activities of an orphan GPCR, GPR81

GPR81 is an orphan G protein-coupled receptor (GPCR) that has a high degree of homology to the nicotinic acid receptor GPR109A. GPR81 expression is highly enriched and specific in adipocytes. However, the function and signaling properties of GPR81 are unknown because of the lack of natural or synthetic ligands. Using chimeric G proteins that convert Gi-coupled receptors to Gq-mediated inositol phosphate (IP) accumulation, we show that GPR81 can constitutively increase IP accumulation in HEK293 cells and suggest that GPR81 couples to the Gi signaling pathway. We also constructed a chimeric receptor that expresses the extracellular domains of cysteinyl leukotriene 2 receptor (CysLT2R) and the intracellular domains of GPR81. We show that the CysLT2R ligand, leukotriene D(4) (LTD4), is able to activate this chimeric receptor through activation of the Gi pathway. In addition, LTD4 is able to inhibit lipolysis in adipocytes expressing this chimeric receptor. These results suggest that GPR81 couples to the Gi signaling pathway and that activation of the receptor may regulate adipocyte function and metabolism. Hence, targeting GPR81 may lead to the development of a novel and effective therapy for dyslipidemia and a better side effect profile than nicotinic acid.     

GPR109a agonists. Part 2: Pyrazole-acids as agonists of the human orphan G-protein coupled receptor GPR109a

5-Alkyl and aryl-pyrazole-acids have been identified as a new class of selective, small-molecule, agonists of the human orphan G-protein-coupled receptor GPR109a, a high affinity receptor for the HDL-raising drug nicotinic acid.     

GPR109a agonists. Part 1: 5-Alkyl and 5-aryl-pyrazole–tetrazoles as agonists of the human orphan G-protein coupled receptor GPR109a

5-Alkyl and aryl-pyrazole–tetrazoles have been identified as a new class of selective, small-molecule, agonists of the human G-protein-coupled receptor GPR109a, a high affinity receptor for the HDL-raising drug nicotinic acid.     

Internalization of the Human Nicotinic Acid Receptor GPR109A Is Regulated by Gi,GRK2, and Arrestin3

Nicotinic acid (niacin) has been widely used as a favorable lipid-lowering drug for several decades, and the orphan G protein-coupled receptor GPR109A has been identified to be a receptor for niacin. Mechanistic investigations have shown that as a G_i-coupled receptor, GPR109A inhibits adenylate cyclase activity upon niacin activation, thereby inhibiting free fatty acid liberation. However, the underlying molecular mechanisms that regulate signaling and internalization of GPR109A remain largely unknown. To further characterize GPR109A internalization, we made a construct to express GPR109A fused with enhanced green fluorescent protein (EGFP) at its carboxyl-terminal end. In stable GPR109A-EGFP-expressing HEK-293 cells, GPR109A-EGFP was mainly localized at the plasma membrane and was rapidly internalized in a dose- and time-dependent manner upon agonist stimulation. GPR109A internalization was completely blocked by hypertonic sucrose, indicating that GPR109A internalizes via the clathrin-coated pit pathway. Further investigation demonstrated that internalized GPR109A was recycled to the cell surface after the removal of agonist, and recycling of the internalized receptors was not blocked by treatment with acidotropic agents, NH₄Cl and monensin. Pertussis toxin pretreatment not only inhibited forskolin-induced cAMP accumulation and intracellular Ca²⁺ mobilization; it also significantly attenuated agonist-promoted GPR109A internalization. Moreover, RNA interference experiments showed that knockdown of GRK2 (G protein-coupled receptor kinase 2) and arrestin3 expression significantly impaired receptor internalization. Taken together, these results indicate that the agonist-induced internalization of GPR109A receptors is regulated by GRK2 and arrestin3 in a pertussis toxin-sensitive manner and that internalized receptor recycling is independent of endosomal acidification.     

Impedance measurement: a new method to detect ligand-biased receptor signaling

Ligand-biased receptor signaling has been proposed for several G-protein coupled receptors including the niacin receptor GPR109A. Coupling to the G_i/o pathway has been shown to be responsible for the well described triglyceride lowering effect of nicotinic acid in mice, while activation of the β-arrestin pathway has been suggested to be responsible for its peripheral vasodilatory effect that causes cutaneous flushing. Several ligands have been described to selectively induce triglyceride lowering without inducing flushing.Cellular impedance has been demonstrated to determine G-protein coupled receptors activation in a G-protein specific manner. Agonists, which induce triglyceride lowering, but not flushing show a profile in cellular impedance that is distinct from the one induced by niacin and those compounds that induce triglyceride lowering as well as flushing. The strength of the signal correlates with the activation of β-arrestin.     

Neutrophil apoptosis mediated by nicotinic acid receptors (GPR109A)

G protein-coupled receptor (GPR)109A (HM74A) is a G(i) protein-coupled receptor, which is activated by nicotinic acid (NA), a lipid-lowering drug. Here, we demonstrate that mature human neutrophils, but not eosinophils, express functional GPR109A receptors. The induction of the GPR109A gene appears to occur late in the terminal differentiation process of neutrophils, since a mixed population of immature bone marrow neutrophils did not demonstrate evidence for its expression. NA accelerated apoptosis in cultured neutrophils in a concentration-dependent manner, as assessed by phosphatidylserine redistribution, caspase-3 activation, and DNA fragmentation assays. The pro-apoptotic effect of NA was abolished by pertussis toxin, which was used to block G(i) proteins, suggesting a receptor-mediated mechanism. Activation of GPR109A by NA resulted in decreased levels of cyclic adenosine monophosphate (cAMP), most likely due to G(i)-mediated inhibition of adenylyl cyclase activity. NA-induced apoptosis was reversed by the addition of cell-permeable cAMP, pointing to the possibility that reduced cAMP levels promote apoptosis in neutrophils. Distal mechanism involved in this process may include the post-translational modification of members of the Bcl-2 family, such as dephosphorylation of pro-apoptotic Bad and antiapoptotic Mcl-1 proteins. Taken together, following maturation in the bone marrow, neutrophils express functional GPR109A receptors, which might be involved in the regulation of neutrophil numbers. Moreover, this study identified a new cellular target of NA and future drugs activating GPR109A receptors, the mature neutrophil.     

Molecular modeling aided design of nicotinic acid receptor GPR109A agonists

A homology model of the nicotinic acid receptor GPR109A was constructed based on the X-ray crystal structure of bovine rhodopsin. An HTS hit was docked into the homology model. Characterization of the binding pocket by a grid-based surface calculation of the docking model suggested that a larger hydrophobic body plus a polar tail would improve interaction between the ligand and the receptor. The designed compounds were synthesized, and showed significantly improved binding affinity and activation of GPR109A.     

Deorphanization of GPR109B as a Receptor for the ��-Oxidation Intermediate 3-OH-octanoic Acid and Its Role in the Regulation of Lipolysis

The orphan G-protein-coupled receptor GPR109B is the result of a recent gene duplication of the nicotinic acid and ketone body receptor GPR109A being found in humans but not in rodents. Like GPR109A, GPR109B is predominantly expressed in adipocytes and is supposed to mediate antilipolytic effects. Here we show that GPR109B serves as a receptor for the β-oxidation intermediate 3-OH-octanoic acid, which has antilipolytic activity on human but not on murine adipocytes. GPR109B is coupled to G_i-type G-proteins and is activated by 2- and 3-OH-octanoic acid with EC₅₀ values of about 4 and 8 μm, respectively. Interestingly, 3-OH-octanoic acid plasma concentrations reach micromolar concentrations under conditions of increased β-oxidation rates, like in diabetic ketoacidosis or under a ketogenic diet. These data suggest that the ligand receptor pair 3-OH-octanoic acid/GPR109B mediates in humans a negative feedback regulation of adipocyte lipolysis to counteract prolipolytic influences under conditions of physiological or pathological increases in β-oxidation rates.Triacylglycerols stored in the white adipose tissue serve as the major energy reserve in higher eukaryotes (1). Although they are constantly turned over by lipolysis and re-esterification, their mobilization and storage are precisely balanced by various hormones and other factors depending on the nutritional state (2). The net rate of lipolysis is increased during fasting or periods of increased energy demand. Fatty acids generated via lipolysis undergo β-oxidation in the muscle and liver to serve directly as a source of energy or as a precursor for ketone bodies (3). The major intracellular regulator of lipolysis is cyclic AMP, which stimulates cAMP-dependent kinase to activate lipolytic enzymes (2, 4–6). This lipolytic pathway is induced, for example, via β-adrenergic receptors that couple to the G-protein G_s and thereby stimulate adenylyl cyclase (7, 8). To adjust lipolysis at the appropriate rate, the effects of prolipolytic stimuli are balanced by various antilipolytic influences. Besides insulin, which promotes the degradation of cAMP via activation of phosphodiesterase 3B (2, 5, 7), several antilipolytic stimuli decrease cAMP levels by activation of G_i-coupled receptors, which mediate an inhibition of adenylyl cyclase (5, 8). One of these receptors, GPR109A, has recently been shown to mediate the anti-lipolytic effects of high concentrations of the ketone body 3-OH-butyrate thereby providing a negative feedback mechanism during fasting (9, 10). GPR109A also binds nicotinic acid (11–13) and mediates the anti-lipolytic effects of this anti-dyslipidemic drug (12).GPR109B, a close relative of GPR109A, is the result of a recent gene duplication being present in humans but not in rodents and most other mammals (14). GPR109B differs from GPR109A in an extended C-terminal tail as well as in 16 amino acids (11, 13). Despite its high homology to GPR109A, GPR109B does not bind nicotinic acid or 3-OH-butyrate with reasonable affinity (10, 11, 13). Because GPR109A and GPR109B have very similar expression patterns (11, 13, 15) and are likely to have the same basic signaling properties, agonists of GPR109B are expected to have physiological and pharmacological effects comparable with those of the GPR109A agonist 3-OH-butyrate and nicotinic acid, respectively. Recently, several synthetic compounds as well as various aromatic d-amino acids have been shown to be selective agonists at GPR109B (16–18). However, endogenous physiological anti-lipolytic ligands of GPR109B are unknown.In this study we tested endogenous carboxylic acids for their ability to activate GPR109B. We found that the fatty acid β-oxidation intermediate 3-OH-octanoic acid is a highly specific agonist of GPR109B. 3-OH-octanoic acid has anti-lipolytic activity, and its plasma concentration in humans reflects the β-oxidation flux. Our data suggest that 3-OH-octanoic acid and GPR109B mediate a negative feedback regulation of adipocyte lipolysis.     

Molecular identification of high and low affinity receptors for nicotinic acid

Nicotinic acid has been used clinically for over 40 years in the treatment of dyslipidemia producing a desirable normalization of a range of cardiovascular risk factors, including a marked elevation of high density lipoprotein and a reduction in mortality. The precise mechanism of action of nicotinic acid is unknown, although it is believed that activation of a G(i)-G protein-coupled receptor may contribute. Utilizing available information on the tissue distribution of nicotinic acid receptors, we identified candidate orphan receptors. The selected orphan receptors were screened for responses to nicotinic acid, in an assay for activation of G(i)-G proteins. Here we describe the identification of the G protein-coupled receptor HM74 as a low affinity receptor for nicotinic acid. We then describe the subsequent identification of HM74A in follow-up bioinformatics searches and demonstrate that it acts as a high affinity receptor for nicotinic acid and other compounds with related pharmacology. The discovery of HM74A as a molecular target for nicotinic acid may facilitate the discovery of superior drug molecules to treat dyslipidemia.     

Fluorinated pyrazole acids are agonists of the high affinity niacin receptor GPR109a

A series of 5-alkyl pyrazole-3-carboxylic acids were prepared and found to act as potent and selective agonists of the human GPCR, GPR109a, the high affinity nicotinic acid receptor. No activity was observed at the highly homologous low affinity niacin receptor, GPR109b. A further series of 4-fluoro-5-alkyl pyrazole-3-carboxylic acids were shown to display similar potency. One example from the series was shown to have improved properties in vivo compared to niacin.     

Inflammation stimulates niacin receptor (GPR109A/HCA2) expression in adipose tissue and macrophages

Many of the beneficial and adverse effects of niacin are mediated via a G protein receptor, G protein-coupled receptor 109A/hydroxycarboxylic acid 2 receptor (GPR109A/HCA2), which is highly expressed in adipose tissue and macrophages. Here we demonstrate that immune activation increases GPR109A/HCA2 expression. Lipopolysaccharide (LPS), TNF, and interleukin (IL) 1 increase GPR109A/HCA2 expression 3- to 5-fold in adipose tissue. LPS also increased GPR109A/HCA2 mRNA levels 5.6-fold in spleen, a tissue rich in macrophages. In peritoneal macrophages and RAW cells, LPS increased GPR109A/HCA2 mRNA levels 20- to 80-fold. Zymosan, lipoteichoic acid, and polyinosine-polycytidylic acid, other Toll-like receptor activators, and TNF and IL-1 also increased GPR109A/HCA2 in macrophages. Inhibition of the myeloid differentiation factor 88 or TIR-domain-containing adaptor protein inducing IFNβ pathways both resulted in partial inhibition of LPS stimulation of GPR109A/HCA2, suggesting that LPS signals an increase in GPR109A/HCA2 expression by both pathways. Additionally, inhibition of NF-κB reduced the ability of LPS to increase GPR109A/HCA2 expression by ∼50% suggesting that both NF-κB and non-NF-κB pathways mediate the LPS effect. Finally, preventing the LPS-induced increase in GPR109A/HCA2 resulted in an increase in TG accumulation and the expression of enzymes that catalyze TG synthesis. These studies demonstrate that inflammation stimulates GPR109A/HCA2 and there are multiple intracellular signaling pathways that mediate this effect. The increase in GPR109A/HCA2 that accompanies macrophage activation inhibits the TG accumulation stimulated by macrophage activation.     

Distinct Kinetic and Spatial Patterns of Protein Kinase C (PKC)- and Epidermal Growth Factor Receptor (EGFR)-dependent Activation of Extracellular Signal-regulated Kinases 1 and 2 by Human Nicotinic Acid Receptor GPR109A

Nicotinic acid (niacin) has been widely used as a lipid-lowering drug for several decades, and recently, orphan G protein-coupled receptor GPR109A has been identified as a receptor for niacin. Mechanistic investigations have shown that, upon niacin activation, GPR109A couples to a G_i protein and inhibits adenylate cyclase activity, leading to inhibition of liberation of free fatty acid. However, the underlying molecular mechanisms for GPR109A signaling remain largely unknown. Using CHO-K1 cells stably expressing GPR109A and A431 cells, which are a human epidermoid cell line with high levels of endogenous expression of functional GPR109A receptors, we found that activation of extracellular signal-regulated kinases 1 and 2 (ERK1/2) by niacin was rapid, peaking at 5 min, and was significantly blocked by pertussis toxin. Furthermore, time course experiments with different kinase inhibitors demonstrated that GPR109A induced ERK1/2 activation via the matrix metalloproteinase/epidermal growth factor receptor transactivation pathway at both early and later time points (2–5 min); this pathway was distinct from the PKC pathway-mediated ERK1/2 phosphorylation that occurs at early time points (≤2 min) in response to niacin. Overexpression of Gβγ subunit scavengers βARK1-CT and the Gα subunit of transducin led to a significant reduction of ERK1/2 phosphorylation, suggesting a critical role for βγ subunits in GPR109A-activated ERK1/2 phosphorylation. Using arrestin-2/3-specific siRNA and an internalization-deficient GPR109A mutant, we found that arrestin-2 and arrestin-3 were not involved in GPR109A-mediated ERK1/2 activation. In conclusion, our findings demonstrate that upon binding to niacin GPR109A receptors initially activate G_i, leading to dissociation of the Gβγ subunit from activated G_i, and subsequently induce ERK1/2 activation via two distinct pathways, one PKC-dependent pathway occurring at a peak time of ≤2 min and the other matrix metalloproteinase-dependent growth factor receptor transactivation occurring at both early and later time points (2–5 min).     

Micromolar concentration of kynurenic acid in rat small intestine

Kynurenic acid is an antagonist of glutamate and alpha-7 nicotinic acetylcholine receptors and an agonist of the G: -protein-coupled receptor GPR35, which is predominantly expressed in immune and gastrointestinal tissues. In this study, we report that kynurenic acid is present in the lumen of rat small intestine in micromolar concentration sufficient to affect the GPR35 receptor. Moreover, we show that kynurenic acid can be produced by Escherichia coli. We suggest that kynurenic acid may modulate gastrointestinal function and integrity.     

PUMA-G and HM74 are receptors for nicotinic acid and mediate its anti-lipolytic effect

Nicotinic acid (niacin), a vitamin of the B complex, has been used for almost 50 years as a lipid-lowering drug. The pharmacological effect of nicotinic acid requires doses that are much higher than those provided by a normal diet. Its primary action is to decrease lipolysis in adipose tissue by inhibiting hormone-sensitive triglyceride lipase. This anti-lipolytic effect of nicotinic acid involves the inhibition of cyclic adenosine monophosphate (cAMP) accumulation in adipose tissue through a G(i)-protein-mediated inhibition of adenylyl cyclase. A G-protein-coupled receptor for nicotinic acid has been proposed in adipocytes. Here, we show that the orphan G-protein-coupled receptor, 'protein upregulated in macrophages by interferon-gamma' (mouse PUMA-G, human HM74), is highly expressed in adipose tissue and is a nicotinic acid receptor. Binding of nicotinic acid to PUMA-G or HM74 results in a G(i)-mediated decrease in cAMP levels. In mice lacking PUMA-G, the nicotinic acid-induced decrease in free fatty acid (FFA) and triglyceride plasma levels was abrogated, indicating that PUMA-G mediates the anti-lipolytic and lipid-lowering effects of nicotinic acid in vivo. The identification of the nicotinic acid receptor may be useful in the development of new drugs to treat dyslipidemia.     

Differential tissue and ligand-dependent signaling of GPR109A receptor: Implications for anti-atherosclerotic therapeutic potential

Until recently, the anti-atherosclerotic effects of niacin were attributed primarily to its lipid modification properties mediated by adipocyte G-protein coupled receptor GPR109A, though recent studies have raised significant doubts about this mechanism. In fact, in rodents it has recently been demonstrated that niacin inhibits progression of atherosclerosis through actions on immune cells, particularly via macrophage-expressed GPR109A, independent of lipid-modifying properties. Here, we studied GPR109A signal transduction in human Langerhans cells, macrophages and adipocytes. We find that the consequences of receptor activation are profoundly influenced by cellular context and that ligand-biased signaling significantly impacts functionally relevant signaling. In Langerhans cells, niacin initiates GPR109A-mediated signaling pathways (Erk1/2 and Ca^2 +) responsible for the release of vasodilatory prostanoids, while the synthetic GPR109A agonist MK-0354 fails to elicit any signaling, providing a mechanistic basis for the latter compound's inability to cause flushing. While GPR109A mediates inhibition of cAMP in adipocytes, in macrophages GPR109A signaling via G_βγ subunits results in paradoxical augmentation of intracellular cAMP levels. Also, in macrophages niacin and GPR109A full agonists induce Erk1/2 and Ca^2 + signaling, release of prostanoids, upregulation of cholesterol transporters ABCA1 and ABCG1 and stimulation of reverse cholesterol transport in GPR109A dependent manner. A mechanism is presented in which signals from the autocrine action of released prostanoids and G_i protein mediated cAMP augmentation are integrated leading to modulation of reverse cholesterol transport regulatory components. These studies provide key insights into mechanisms by which GPR109A may influence cholesterol efflux in macrophages; a process that may be at least partially responsible for niacin's anti-atherosclerotic activity. MK-0354 does not induce niacin-like GPR109A signaling in macrophages, suggesting that biased agonists devoid of the flushing side-effect may also lack properties required for macrophage-mediated anti-atherosclerotic effects.     

3-Nitro-4-amino benzoic acids and 6-amino nicotinic acids are highly selective agonists of GPR109b

A series of 3-nitro-4-substituted-aminobenzoic acids were prepared and found to act as potent and highly selective agonists of the orphan human GPCR GPR109b, a low affinity receptor for niacin. No activity was observed at the closely homologous high affinity niacin receptor, GPR109a. A second series, comprising 6-amino-substituted nicotinic acids was, also prepared and several analogues showed comparable activity to the nitroaryl series.