br Materials and methods br Results
Materials and methods
Discussion Until very recently, the anti-atherogenic activity of niacin was attributed almost exclusively to its impact on HDL and other plasma lipids, perhaps mediated via its action on GPR109A expressed in adipose or other tissues. However, recent clinical studies of synthetic GPR109A agonists have failed to demonstrate clinically meaningful changes in lipid profiles. Two recent discoveries have also introduced significant doubts about this premise. First, it was demonstrated that the lipid-modifying properties of niacin are not mediated through GPR109A, but rather through another, unknown mechanism . Second, niacin possesses a distinct immune cell mediated anti-atherogenic activity, which is dependent on GPR109A and independent of HDL and other plasma lipid modulation . To date, no direct mechanistic link between increased HDL levels and reductions of atherosclerotic disease has been established. Furthermore, failure to demonstrate tangible clinical benefits of HDL elevation in recent clinical trials of CETP inhibitors torcetrapib and dalcetrapib raises serious questions as to whether such a mechanistic link really exists, or whether low HDL simply serves as a biomarker for higher cardiovascular risk with no causal relationship . Thus, the relative contributions of both HDL elevation and direct macrophage (and potentially other immune cell) mediated anti-atherogenic properties in the clinical effectiveness of niacin as an anti-atherogenic agent are currently uncertain. A direct effect of niacin on the atherosclerotic plaque through modulation of resident immune calcifediol may potentially be a major component in niacin's mechanism of action in improving cardiovascular outcomes. A more complete understanding of the molecular mechanisms of niacin's action on immune cells thus becomes important. The current study is directed to investigation of distinct GPR109A-mediated signaling pathways in macrophages, as compared to those in adipocytes and Langerhans cells, which may mediate niacin's biological effects. The results support a mechanistic hypothesis for niacin and GPR109A-mediated modulation of reverse cholesterol transport in macrophages involving two synergistic signaling pathways; the release of prostanoids (mediated by calcium, ERK1/2 signaling, and perhaps other signaling pathways) that can act in an autocrine/paracrine manner to stimulate Gs-coupled prostanoid receptors and the potentiation of cAMP accumulation through an unorthodox Gi–βγ protein mediated mechanism. Multiple signaling pathways are activated by niacin in adipocytes, Langerhans cells and macrophages. Several distinct lines of evidence suggest that these signaling pathways are GPR109A dependent. First, they are activated by niacin, but not its isomer isoniacin, which has very low affinity for GPR109A. Second, activation of these pathways is blocked by pertussis toxin, indicating that they are mediated through a Gi protein coupled GPCR. Lastly, they are activated not only by niacin, but also by other structurally distinct GPR109A agonists with a rank order of potencies matching those measured in recombinant GPR109A assays. In all three cell types, GPR109A signaling involves similarities as well as striking differences. In both Langerhans cells and macrophages, niacin activates signaling through calcium and ERK1/2, both of which are implicated in release of prostanoids such as PGD2 and PGE2. Prostanoids released from Langerhans cells and macrophages are critical intermediates in the biological processes mediated by GPR109A in these cells, i.e., flushing and regulation of reverse cholesterol transport, respectively. In adipocytes, niacin does not activate the calcium signaling pathway. The most striking GPR109A signaling difference observed in adipocytes versus macrophages was observed in the modulation of intracellular cAMP. In adipocytes, niacin activation of GPR109A activates Gαi which mediates inhibition of adenylyl cyclase in a classical manner to reduce intracellular cAMP levels. This leads to inhibition of hormone sensitive lipase and inhibition of lipolysis, resulting in reductions in free fatty acid production. In macrophages GPR109A does not exert its activity through Gαi, but instead through the Gβγ subunits of the heterotrimeric Gi protein complex. As a result, niacin activation of GPR109A does not lead to inhibition of cAMP, but instead yields further amplification of cAMP accumulation when additional Gs stimuli are present. Moreover, under certain conditions, niacin administration alone can increase intracellular cAMP levels in macrophages as a result of the synergistic action of GPR109A-medtiated prostanoid release and activation of the adenylyl cyclase interacting Gβγ subunit. Thus the increases in levels of cAMP in these cells were eliminated by blocking prostanoid release (COX inhibition) and by Gi inactivation by PTX treatment. Interestingly, a small increase in cAMP levels in a monocytic cell line in response to niacin has been observed previously , . Though neither the mechanism nor GPR109A dependence of the change in cAMP levels was investigated, it likely occurred through mechanisms similar to those described in macrophages here.