Dihydropyrazine activates the CAMKK2-AMPK-ULK1 signaling pathway in human hepatoma HepG2 cells

Dihydropyrazines (DHPs) are glycation intermediates produced by nonenzymatic glycation reactions in vivo and in foods. We previously reported that 3-hydro-2,2,5,6-tetramethylpyrazine (DHP-3) activates endoplasmic reticulum (ER) stress and inhibits autophagy, although its effect on autophagy initiation remained unclear. In this study, we examined the effect of DHP-3 on the autophagy initiation pathway in HepG2 cells. DHP-3 exposure resulted in activation of UNC-51-like kinase 1 (ULK1), the catalytic subunit of the ULK complex essential for autophagy initiation. Notably, phosphorylation of AMP-activated protein kinase (AMPK), an upstream activator of ULK1, was enhanced without a corresponding increase in the ADP/ATP ratio. Among the upstream kinases regulating AMPK, phosphorylation of calcium/calmodulin-dependent protein kinase kinase 2 (CAMKK2) was increased, whereas that of liver kinase B1 (LKB1) remained unchanged. These findings suggest that DHP-3 activates the CAMKK2-AMPK-ULK1 signaling pathway, thereby inducing autophagy initiation.