Abstract
LPS-induced TLR4 activation alters cellular bioenergetics and triggers proteolytic cleavage of AMPKα and HIF-1α expression in leukocytes. In human leukocytes, and more specifically neutrophils, AMPKα cleavage yields 55- and 35-kD protein fragments. Here we address the mechanism by which AMPKα is cleaved, and it’s relevance to human health. Our data indicate that AMPKα cleavage is linked to matrix metalloproteinase (MMP9) expression and that both are required for mTORC1 and S6K1 activation, and HIF-1α expression, in LPS stimulated human and mice leukocytes. Three key observations support this conclusion. First, no changes in AMPKα and TLR4 signaling intermediates (mTORC1/S6K1/ HIF-1α) were detected in LPS-stimulated MMP9-deficient mice leukocytes. Second, recombinant MMP9 cleaved human AMPKα
ex vivo
, producing degradation products similar in size to those detected following LPS-stimulation. Third, MMP9 inhibitors prevented AMPKα degradation and HIF-1α expression in LPS-activated human leukocytes, while AMPK activators blocked MMP9 and HIF-1α expression. Significantly, AMPKα degradation, MMP9, and TLR4 signaling intermediates were all detected in leukocytes from Type 2 diabetes mellitus (T2DM) patients and patients following cardiopulmonary bypass (CPB) surgery. Plasma from these two patient cohorts induced AMPKα cleavage and TLR4 signaling intermediates in healthy donor leukocytes and either a TLR4 inhibitor or polymyxin prevented these outcomes. Detection of AMPKα degradation, MMP9 expression, and TLR4 signaling intermediates described herein 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.