Acetaminophen (APAP) is the most common cause of acute liver failure in the United States, accounting for 46% of all cases. APAP hepatotoxicity involves the active participation of signal transduction pathways that activate c-jun-N-terminal kinase (JNK). Inhibition of JNK prevents APAP-induced liver injury even in the presence of extensive glutathione (GSH) depletion and covalent binding. We have proposed a two-hit hypothesis to mitochondria as the central mechanism mediating APAP-induced liver injury. APAP is metabolized to NAPQI by CYP2e1, which depletes GSH, leading to covalent binding in cytoplasm and mitochondria (first hit). Mitochondrial GSH depletion and covalent binding increase the generation of mitochondrial (reactive oxygen species [ROS]) that activate JNK, through upstream MAP kinase pathways. Activated JNK translocates to mitochondria binding to Sab (second hit), an outer membrane protein, which is phosphorylated by JNK and is required for toxicity. JNK binding to Sab on mitochondria leads to further enhancement of ROS generation by a mechanism that is not yet understood; the enhanced ROS is important in sustaining JNK activation and inducing the mitochondrial permeability transition (MPT) to mediate hepatocyte necrosis. JNK signaling is essential for APAP-induced programmed necrosis, and other signaling proteins such as GSK-3β, MLK-3, and ASK-1 that mediate APAP-induced liver injury acting upstream to modulate JNK signaling.[4, 6-8]
Aside from MAPK, other signaling pathways may be activated by ROS. Previously, we have shown that hydrogen peroxide-induced necrosis of primary mouse hepatocytes is modulated by activation of protein kinase C (PKC) and subsequent inactivation of AMP-activated kinase (AMPK). PKC inhibitors significantly protect against H2O2-induced hepatocyte necrosis through activation of an AMPK kinase survival pathway. There are at least 11 known isoforms of PKC, which are divided into three major classes: the classical group (α, βI, βII, and γ), which can be activated by diacylglycerol (DAG), calcium, or phorbol esters; the novel group (δ, ε, η, and θ), which can be activated by DAG but is insensitive to calcium; and the atypical group (ζ and λ/ι), which are insensitive to calcium, DAG, and phorbol esters. Distribution of PKC isoforms in different organs and tissues is variable.[11, 12] Five isoforms (α, βII, δ, ε, ζ) have been shown to be present in the liver. Whether PKC activation or inhibition protects or promotes cell death may depend on the model of injury, cell type, and which isoforms are involved.[14, 15] PKC also plays a role in energy homeostasis, insulin signaling, and glucose metabolism. Inhibition of atypical PKC has been shown to cause activation of AMP-activated kinase (AMPK), a key regulator of energy homeostasis in cultured endothelial cells.
AMPK is another serine-threonine kinase with a heterotrimeric complex consisting of catalytic α subunit and two regulatory subunits (β and γ) and serves as an important energy sensor in cells responding to the AMP:ATP ratio.[17, 18] Phosphorylation at the Thr 172 site in the α subunit is essential for AMPK activation. AMPK activation promotes ATP production by switching off anabolic processes and turning on catabolic pathways. AMPK not only regulates energy homeostasis but also has cytoprotective effects in hepatocytes by inhibition of apoptosis, regulation of mitochondrial biogenesis, protection against mitochondrial injury, and activation of autophagy.[19-25]
AMPK activates autophagy through inhibition of mammalian target of rapamycin complex 1 (mTORC1). It has also recently been shown that APAP treatment inhibits mTORC1 and leads to activation of autophagy. Induction of autophagy is presumed to protect against APAP hepatotoxicity by removal of injured mitochondria. Autophagy is regulated by the autophagy-related proteins (Atg), which form protein complexes during assembly, docking, and degradation of the autophagosome. Recently, it has been shown that knockout of Atg7, a ubiquitin E1-like enzyme required for autophagosome formation, in mice increased susceptibility to APAP-induced liver injury.
The roles of PKC and AMPK in APAP hepatotoxicity have not been previously explored. In the present study, we explore how broad-spectrum PKC inhibitors and silencing of PKC-α modulate AMPK, the master energy regulator in hepatocytes, and JNK signaling to mediate APAP-induced liver injury.