NLRP3 inflammasome was recently shown to play a key role in Western diet-induced systemic inflammation and long-lasting trained immunity in myeloid cells. Saturated fatty acids (SFAs) are sterile triggers able to induce the assembly of the NLRP3 inflammasome in macrophages, leading to IL-1 secretion while unsaturated ones (UFAs) prevent SFAsmediated NLRP3 activation. Unlike previous studies, we do not see any ROS or ER stress involvement in SFAs-mediated NLRP3 activation. Rather we show that SFAs need to enter the cells and to be activated into acyl-CoA to lead to NLRP3 activation in human macrophages. However, their -oxidation is dispensable. Instead, they are channeled towards phospholipids but redirected towards lipid droplets containing triacylglycerol in the presence of UFAs. Lipidomic analyses and Laurdan fluorescence experiments demonstrate that SFAs induce a dramatic saturation of phosphatidylcholine correlated with a loss of membrane fluidity, both events inhibited by UFAs. This SFA-induced membrane remodeling promotes a disruption of the plasma membrane Na, K-ATPase, instigating a K + efflux essential and sufficient for NLRP3 activation. This work opens novel therapeutic avenues to interfere with Western diet-associated diseases such as those targeting some enzymes of the glycerolipid pathway.

eEF2K [eEF2 (eukaryotic elongation factor 2) kinase] phosphorylates and inactivates the translation elongation factor eEF2. eEF2K is not a member of the main eukaryotic protein kinase superfamily, but instead belongs to a small group of so-called α-kinases. The activity of eEF2K is normally dependent upon Ca2+ and calmodulin. eEF2K has previously been shown to undergo autophosphorylation, the stoichiometry of which suggested the existence of multiple sites. In the present study we have identified several autophosphorylation sites, including Thr348, Thr353, Ser366 and Ser445, all of which are highly conserved among vertebrate eEF2Ks. We also identified a number of other sites, including Ser78, a known site of phosphorylation, and others, some of which are less well conserved. None of the sites lies in the catalytic domain, but three affect eEF2K activity. Mutation of Ser78, Thr348 and Ser366 to a non-phosphorylatable alanine residue decreased eEF2K activity. Phosphorylation of Thr348 was detected by immunoblotting after transfecting wild-type eEF2K into HEK (human embryonic kidney)-293 cells, but not after transfection with a kinase-inactive construct, confirming that this is indeed a site of autophosphorylation. Thr348 appears to be constitutively autophosphorylated in vitro. Interestingly, other recent data suggest that the corresponding residue in other α-kinases is also autophosphorylated and contributes to the activation of these enzymes [Crawley, Gharaei, Ye, Yang, Raveh, London, Schueler-Furman, Jia and Cote (2011) J. Biol. Chem. 286, 2607–2616]. Ser366 phosphorylation was also detected in intact cells, but was still observed in the kinase-inactive construct, demonstrating that this site is phosphorylated not only autocatalytically but also in trans by other kinases.

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We previously investigated whether inhibition of AMP-metabolizing enzymes could enhance AMP-activated protein kinase (AMPK) activation in skeletal muscle for the treatment of type 2 diabetes. Soluble 5′-nucleotidase II (NT5C2) hydrolyzes IMP and its inhibition could potentially lead to a rise in AMP to activate AMPK. In the present study, we investigated effects of NT5C2 deletion in mice fed a normal-chow diet (NCD) or a high-fat diet (HFD). On a NCD, NT5C2 deletion did not result in any striking metabolic phenotype. On a HFD however, NT5C2 knockout (NT5C2 −/−) mice displayed reduced body/fat weight gain, improved glucose tolerance, reduced plasma insulin, triglyceride and uric acid levels compared with wild-type (WT) mice. There was a tendency towards smaller and fewer adipocytes in epididymal fat from NT5C2 −/− mice compared to WT mice, consistent with a reduction in triglyceride content. Differences in fat mass under HFD could not be explained by changes in mRNA expression profiles of epididymal fat from WT versus NT5C2 −/− mice. However, rates of lipolysis tended to increase in epididymal fat pads from NT5C2 −/− versus WT mice, which might explain reduced fat mass. In incubated skeletal muscles, insulin-stimulated glucose uptake and associated signalling were enhanced in NT5C2 −/− versus WT mice on HFD, which might contribute towards improved glycemic control. In summary, NT5C2 deletion in mice protects against HFD-induced weight gain, adiposity, insulin resistance and associated hyperglycemia.