Indirect AMP-Activated Protein Kinase Activators Prevent Incision-Induced Hyperalgesia and Block Hyperalgesic Priming, Whereas Positive Allosteric Modulators Block Only Priming in Mice

AMP-activated protein kinase (AMPK) is a multifunctional kinase that negatively regulates the mechanistic target of rapamycin (mTOR) and mitogen-activated protein kinase (MAPK) signaling, two signaling pathways linked to pain promotion after injury, such as surgical incision. AMPK can be activated directly using positive allosteric modulators, as well as indirectly through the upregulation of upstream kinases, such as liver kinase B1 (LKB1), which is a mechanism of action of metformin. Metformin's antihyperalgesic effects occur only in male mice, raising questions about how metformin regulates pain sensitivity. We used metformin and other structurally distinct AMPK activators narciclasine (NCLS), ZLN-024, and MK8722, to treat incision-induced mechanical hypersensitivity and hyperalgesic priming in male and female mice. Metformin was the only AMPK activator to have sex-specific effects. We also found that indirect AMPK activators metformin and NCLS were able to reduce mechanical hypersensitivity and block hyperalgesic priming, whereas direct AMPK activators ZLN-024 and MK8722 only blocked priming. Direct and indirect AMPK activators stimulated AMPK in dorsal root ganglion (DRG) neuron cultures to a similar degree; however, incision decreased phosphorylated AMPK (p-AMPK) in DRG. Because AMPK phosphorylation is required for kinase activity, we interpret our findings as evidence that indirect AMPK activators are more effective for treating pain hypersensitivity after incision because they can drive increased p-AMPK through upstream kinases like LKB1. These findings have important implications for the development of AMPK-targeting therapeutics for pain treatment. SIGNIFICANCE STATEMENT: Nonopioid treatments for postsurgical pain are needed. Our work focused on whether direct or indirect AMP-activated protein kinase (AMPK) activators would show greater efficacy for inhibiting incisional pain, and we also tested for potential sex differences. We conclude that indirect AMPK activators are likely to be more effective as potential therapeutics for postsurgical pain because they inhibit acute pain caused by incision and prevent the long-term neuronal plasticity that is involved in persistent postsurgical pain. Our work points to the natural product narciclasine, an indirect AMPK activator, as an excellent starting point for development of therapeutics.