Potent, selective inhibitors reveal that DAGLβ is a principal 2-AG biosynthetic enzyme and regulates inflammatory processes in mouse peritoneal macrophages. The endocannabinoid 2-arachidonoylglycerol (2-AG) is biosynthesized by diacylglycerol lipases DAGLα and DAGLβ. Chemical probes to perturb DAGLs are needed to characterize endocannabinoid function in biological processes. Here we report a series of 1,2,3-triazole urea inhibitors, along with paired negative-control and activity-based probes, for the functional analysis of DAGLβ in living systems. Optimized inhibitors showed high selectivity for DAGLβ over other serine hydrolases, including DAGLα (∼60-fold selectivity), and the limited off-targets, such as ABHD6, were also inhibited by the negative-control probe. Using these agents and Daglb−/− mice, we show that DAGLβ inactivation lowers 2-AG, as well as arachidonic acid and eicosanoids, in mouse peritoneal macrophages in a manner that is distinct and complementary to disruption of cytosolic phospholipase-A2. We observed a corresponding reduction in lipopolysaccharide-induced tumor necrosis factor-α release. These findings indicate that DAGLβ is a key metabolic hub within a lipid network that regulates proinflammatory responses in macrophages.

Significance: Many rare human genetic disorders are caused by mutations in genes that code for proteins of poorly characterized function. Determining the functions of these proteins is critical for understanding and devising potential treatments for human diseases. In this article, we discover using a combination of mouse genetic models, selective inhibitors, and lipid profiling that the DDHD2 enzyme, mutations of which cause a neurological disease termed complex hereditary spastic paraplegia (HSP), acts as a major brain triglyceride hydrolase. Mice lacking DDHD2 have elevated brain triglycerides and lipid droplet accumulation in neurons. We have thus discovered that the brain possesses a specialized pathway for triglyceride metabolism, disruption of which leads to biochemical and cellular changes that may contribute to complex HSP.