This study was designed to test the hypothesis that ω-3 PUFAs antagonize macrophage inflammation through activation of AMPK/SIRT1 pathways. The plausibility of this hypothesis was driven by several prior findings on ω-3 PUFAs' anti-inflammatory effects and AMPK and SIRT1 as novel cellular mediators linking nutrient metabolism and inflammation. First, we and others have previously shown that ω-3 PUFAs antagonize macrophage inflammation 
. However, the cellular mechanisms underlying the anti-inflammatory effects of ω-3 PUFAs are not completely understood. Second, we recently demonstrated that two nutrient sensors AMPK and SIRT1 negatively regulate macrophage inflammation 
. It would be interesting to know whether these two nutrient sensors also respond to ω-3 PUFAs, a group of beneficial nutrients commonly seen in supplementation (fish oil) and dietary sources (e.g. fish), and mediate their anti-inflammatory effects. Indeed, we found that ω-3 PUFAs activate AMPK and SIRT1 pathways that in turn deacetylate the NF-κB subunit p65 and down-regulate its signaling, leading to suppression of pro-inflammatory gene expression.
The most significant finding in our study is that the anti-inflammatory effect of ω-3 PUFAs may be mediated through activation of AMPK/SIRT1 pathways, which results in down-regulation of NF-κB signaling by deacetylating its subunit p65. We previously demonstrated that AMPK and SIRT1 can detect excess nutrients in diet-induced obesity and serve as negative regulators of nutrient stress-induced inflammation 
. We found that AMPK signaling in adipose tissue and macrophages are substantially down-regulated by inflammatory stimuli LPS and in diet-induced obesity 
. To test whether the down-regulation of AMPK signaling might be physiologically significant and contributes to obesity-induced inflammation, we explored the role of AMPK in regulation of macrophage inflammation in both gain- and loss- of function studies. We showed that AMPK activates SIRT1 to suppress macrophage inflammation 
. The underlying mechanism includes the ability of AMPK and SIRT to deacetylate NF-κB, whose acetylation status affect NF-κB activity and signaling 
. Based on these observations, we determined: 1) whether AMPK/SIRT1 not only detects excess unhealthy nutrients (e.g. saturated lipids) associated with obesity, but also responds to healthy nutrients (e.g. ω-3 PUFAs) beneficial for prevention and treatment of obesity-associated metabolic disorders; and 2) whether activation of AMPK/SIRT1 in response to ω-3 PUFAs antagonizes macrophage inflammation via antagonism of NF-κB signaling by deacetylating p65. We found that ω-3 PUFAs increase expression, phosphorylation and activity of the major isoform α1AMPK in macrophages, which further leads to SIRT1 over-expression. Our data suggest that AMPK indeed responds to ω-3 PUFAs. It is noteworthy that other anti-inflammatory/anti-oxidants such as polyphenols can also activate AMPK 
. It appears that both inflammatory and anti-inflammatory signals converge on AMPK that in turn exerts its actions to regulate inflammation. To study the consequence of AMPK/SIRT1 activation by ω-3 PUFAs, we examined the NF-κB acetylation and signaling. We found that the ω-3 PUFA DHA mimics the effect of SIRT1 to deacetylate NF-κB, and SIRT1 mediates the effect of DHA in deacetylation of NF-κB and inhibition of its signaling. ω-3 PUFAs' anti-inflammatory functions have been extensively investigated and a number of potential mechanisms have been proposed. For instance, ω-3 PUFAs can competitively inhibit the conversion of arachidonate to pro-inflammatory lipid intermediates 
. ω-3 PUFAs have also been shown to serve as endogenous ligands for PPARγ 
, a known signal that has anti-inflammatory function. Serhan's and his colleagues have also identified the anti-inflammatory lipid mediators such as resolvins and protectins that mediate ω-3 PUFAs' effects 
. More recently, Olefsky's group has reported a novel G-protein coupled receptor GPR120 that mediates the potent anti-inflammatory actions and insulin sensitizing effects of ω-3 PUFA. It is not clear how ω-3 PUFA regulation of AMPK/SIRT1 would fit and interact with the other pathways to regulate inflammation. It is possible that these pathways may be intertwined. For example, as a ligand, ω-3 PUFAs can activate PPARγ that has been shown to activate AMPK 
. It is also noteworthy that although we demonstrate that anti-inflammatory effect of ω-3 PUFAs is mediated through antagonism of NF-κB signaling, we do not exclude the possibility that ω-3 PUFAs may also act on other inflammatory pathways such as JNK and iKK, which appears to be down-regulated by ω-3 PUFAs in previous reports 
. It is conceivable that all these pathways may not be mutually exclusive, and probably have crosstalk.
In summary, we first demonstrate that ω-3 PUFAs suppress LPS-induced cytokine expression in macrophages. The anti-inflammatory effect of ω-3 PUFAs is likely mediated through antagonism of NF-κB signaling. We then demonstrate that AMPK/SIRT1 pathways are downstream signals that mediate ω-3 PUFAs' anti-inflammatory effects. ω-3 PUFAs activates AMPK signaling by increasing its protein levels, which further leads to increased SIRT1 protein expression. More importantly, DHA mimics the effect of SIRT1 on deacetylation of NF-κB, and the full capacity of DHA to deacetylate NF-κB and inhibit its signaling and downstream cytokine expression requires SIRT1. We conclude that ω-3 PUFAs negatively regulate macrophage inflammation by deacetylating NF-κB, which acts through activation of AMPK/SIRT1 pathway. AMPK and SIRT1, two classic energy sensors that play key roles in regulating energy metabolism, may serve as novel cellular mediators for the anti-inflammatory effects of ω-3 PUFAs.