Proteolytic Cleavage of AMPKα and Intracellular MMP9 Expression Are Both Required for TLR4-Mediated mTORC1 Activation and HIF-1α Expression in Leukocytes

LPS-induced TLR4 activation alters cellular bioenergetics and triggers proteolytic cleavage of AMPK alpha and HIF-1 alpha expression in leukocytes. In human leukocytes, and more specifically neutrophils, AMPK alpha cleavage yields 55- and 35-kDa protein fragments. In this study, we address the mechanism by which AMPK alpha is cleaved and its relevance to human health. Our data indicate that AMPK alpha cleavage is linked to MMP9 expression and that both are required for mammalian target of rapamycin complex-1 and S6K1 activation and HIF-1 alpha expression in LPS-stimulated human and mice leukocytes. Three key observations support this conclusion. First, no changes in AMPK alpha and TLR4 signaling intermediates (mammalian target of rapamycin complex-1/S6 kinase 1/HIF-1 alpha) were detected in LPS-stimulated MMP9-deficient mice leukocytes. Second, rMMP9 cleaved human AMPK alpha ex vivo, producing degradation products similar in size to those detected following LPS stimulation. Third, MMP9 inhibitors prevented AMPK alpha degradation and HIF-1 alpha expression in LPS-activated human leukocytes, whereas AMPK activators blocked MMP9 and HIF-1 alpha expression. Significantly, AMPKa degradation, MMP9, and TLR4 signaling intermediates were all detected in leukocytes from patients with type 2 diabetes mellitus and patients following cardiopulmonary bypass surgery. Plasma from these two patient cohorts induced AMPK alpha cleavage and TLR4 signaling intermediates in healthy donor leukocytes and either a TLR4 inhibitor or polymyxin prevented these outcomes. Detection of AMPK alpha degradation, MMP9 expression, and TLR4 signaling intermediates described in this study in leukocytes, the most readily available human cells for clinical investigation, may provide a powerful tool for further exploring the role of TLR4 signaling in human diseases and lead to identification of new, context-specific therapeutic modalities for precision medicine.