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 Gßγ 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 Gi 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.

Differential phosphorylation of perilipin 1A at the initiation of lipolysis revealed by novel monoclonal antibodies and high content analysis.

Lipolysis in adipocytes is regulated by phosphorylation of lipid droplet-associated proteins, including perilipin 1A and hormone-sensitive lipase (HSL). Perilipin 1A is potentially phosphorylated by cAMP(adenosine 3',5'-cyclic monophosphate)-dependent protein kinase (PKA) on several sites, including conserved C-terminal residues, serine 497 (PKA-site 5) and serine 522 (PKA-site 6). To characterize perilipin 1A phosphorylation, novel monoclonal antibodies were developed, which selectively recognize perilipin 1A phosphorylation at PKA-site 5 and PKA-site 6. Utilizing these novel antibodies, as well as antibodies selectively recognizing HSL phosphorylation at serine 563 or serine 660, we used high content analysis to examine the phosphorylation of perilipin 1A and HSL in adipocytes exposed to lipolytic agents. We found that perilipin PKA-site 5 and HSL-serine 660 were phosphorylated to a similar extent in response to forskolin (FSK) and L-γ-melanocyte stimulating hormone (L-γ-MSH). In contrast, perilipin PKA-site 6 and HSL-serine 563 were phosphorylated more slowly and L-γ-MSH was a stronger agonist for these sites compared to FSK. When a panel of lipolytic agents was tested, including multiple concentrations of isoproterenol, FSK, and L-γ-MSH, the pattern of results was virtually identical for perilipin PKA-site 5 and HSL-serine 660, whereas a distinct pattern was observed for perilipin PKA-site 6 and HSL-serine 563. Notably, perilipin PKA-site 5 and HSL-serine 660 feature two arginine residues upstream from the phospho-acceptor site, which confers high affinity for PKA, whereas perilipin PKA-site 6 and HSL-serine 563 feature only a single arginine. Thus, we suggest perilipin 1A and HSL are differentially phosphorylated in a similar manner at the initiation of lipolysis and arginine residues near the target serines may influence this process.

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.

Langerhans cells release prostaglandin D2 in response to nicotinic acid.

Nicotinic acid, used for atherosclerosis treatment, has an adverse effect of skin flushing. The flushing mechanism, thought to be caused by the release of prostaglandin D(2) (PGD(2)), is not well understood. We aimed to identify which cells mediate the flushing effect. Nicotinic acid receptor (GPR109A) gene expression was assessed in various tissues and cell lines. Cells expressing GPR109A mRNA were further assayed for PGD(2) release in response to nicotinic acid. Of all samples, only skin was able to release PGD(2) upon stimulation with nicotinic acid. The responsive cells were localized to the epidermis, and immunocytochemical studies revealed the presence of GPR109A on epidermal Langerhans cells. CD34+ cells isolated from human blood and differentiated into Langerhans cells (hLC-L) also showed GPR109A expression. IFNgamma treatment increased both mRNA and plasma membrane expression of GPR109A. IFNgamma-stimulated hLC-Ls released PGD(2) in response to nicotinic acid in a dose-dependant manner (effector concentration for half-maximum response=1.2 mM+/-0.7). Acifran, a structurally distinct GPR109A ligand, also increased PGD(2) release, whereas isonicotinic acid, a nicotinic acid analog with low affinity for GPR109A, had no effect. These results suggest that nicotinic acid mediates its flushing side effect by interacting with GPR109A on skin Langerhans cells, resulting in release of PGD(2).