Acetaminophen (APAP) overdose is the leading cause of acute liver failure in developed countries. APAP is metabolized in the liver by cytochromes P450 (CYPs) to N-acetyl-p-quinone imine (NAPQI), which depletes glutathione (GSH) and covalently modifies intracellular proteins. NAPQI-induced GSH depletion and protein binding in the mitochondria of hepatocytes induces oxidative stress and the generation of reactive oxygen species (ROS, i.e., superoxide, hydrogen peroxide, and peroxynitrite).[1, 2] Mitochondrial ROS activates mitogen-activated protein kinases (MAPK), which in turn activate JNK and cause its translocation to the mitochondria.[3] Dr. Kaplowitz's group4 has shown previously that translocated JNK binds to Sab, a scaffold protein on the outer mitochondrial membrane of mitochondria, and this binding leads to sustained increase in mitochondrial ROS generation. Sustained mitochondrial oxidative stress and peroxynitrite formation result in formation of the mitochondrial permeability transition (MPT) pore and the consequent release of intermembrane proteins, including cytochrome c, endonuclease G, and apoptosis-inducing factor (AIF). Together, these events contribute to mitochondrial DNA damage, nuclear DNA fragmentation, plummeting of adenosine triphosphate (ATP) production, and collapse of membrane potential, events which culminate in oncotic necrosis of hepatocytes.[5] Factors expressed and/or released by dying hepatocytes can activate nonparenchymal cells to produce circulating factors that contribute both to the progression of hepatocellular necrosis and to its resolution.[6-10] Hepatocellular protective processes are also activated during the ensuing injury, among which is autophagy, which can promote cell survival.[11]

Some of the MAPK signaling elements that mediate JNK activation during APAP pathogenesis are known, but less is understood about other pathways that might contribute to hepatocellular necrosis. The study by Saberi et al. in this issue[12] aimed to identify JNK upstream regulators, which could serve as potential therapeutic targets for APAP overdose. Much of the study focused on AMP-activated kinase (AMPK) and on protein kinase C (PKC). Eleven isoforms of PKC exist; these have been classified as “classical,” “novel,” or “atypical” depending on their response to various activators.[13] Using mouse hepatocytes, the authors found that APAP treatment decreased the activation (phosphorylation) of AMPK and that broad-spectrum PKC inhibitors increased AMPK activation and reduced cytotoxicity without affecting APAP-induced JNK activation. A more selective inhibitor of classical PKCs also afforded protection but did reduce JNK activation. This suggested that atypical PKCs contribute to cytotoxicity through AMPK inhibition and independently of JNK. Also, markers of autophagy were enhanced by broad-spectrum PKC inhibitors, suggesting a role for AMPK activation in mediating the hepatoprotective effect of the PKC inhibitors.

APAP treatment caused translocation of PKCα, a classical PKC, to mitochondria and phosphorylation of mitochondrial proteins that are PKCα substrates. In contrast to the results with broad-spectrum PKC inhibition, selective inhibition or silencing of PKCα failed to affect AMPK but decreased JNK activation and its mitochondrial translocation, ameliorated mitochondrial dysfunction, and reduced cytotoxicity in hepatocytes, suggesting a JNK-dependent role in APAP cytotoxicity for this particular classical PKC isoform. Conversely, silencing of JNK reduced PKCα translocation and activity, suggesting a dysregulated amplification cycle involving these two kinases. The interpretation of these results was that a hepatotoxic dose of APAP leads to the activation of novel or atypical PKCs, and this inhibits AMPK activation, thereby reducing the cell survival influence of autophagy. These events occur independently of JNK activation. In contrast, activation of a classical PKC (i.e., PKCα) enhances JNK activation, which reciprocally further activates PKCα, thereby contributing to the initiation of mitochondrial cell death pathways.

A potential issue with interpretation involves the large concentration of APAP (25 mM) used for most of the in vitro studies. This concentration is larger than plasma concentrations that would occur in most studies of APAP toxicity in mice, in which 300-400 mg/kg are typically used, and are above plasma concentrations attained in clinical cases of human APAP overdose. The concern is that pathways to cell death from APAP could be different at small and large drug concentrations. If so, this could influence appropriate interpretation of the results and their relevance to human poisonings. The same question pertains to the large doses (up to 800 mg/kg) used in some of the mouse studies. Despite this potential shortcoming, the results of this study from Dr. Kaplowitz's laboratory provide new insight into pathways to hepatocellular injury from APAP overdose.

The findings point to some interesting foci for future investigation. For example, the results suggest that p-AMPK up-regulation protects against APAP-induced liver injury by inducing autophagy. AMPK is a metabolic master switch, which upon activation inhibits anabolic, energy-consuming pathways and stimulates energy-producing, catabolic pathways.[14, 15] Thus, the main function of AMPK is to regulate cellular metabolic pathways, such as glucose uptake, fatty acid beta-oxidation, and mitochondrial biogenesis. Other functions of AMPK activation have been reported, such as activating autophagy through inhibiting mammalian target of rapamycin complex 1 (mTORC1), a protein complex that functions as an energy and redox sensor and controller of protein synthesis.[16-18] The current study demonstrated that enhanced AMPK by broad-spectrum PKC inhibitors was associated with an increase of autophagy markers. Identifying a link between AMPK and autophagy in APAP-induced hepatocellular injury is a novel finding. It will be of interest to substantiate this link by using AMPK activators, such as AICA-ribonucleotide (AICAR) and metformin, and to examine whether autophagy can be enhanced further.

Another implication of the study is that inhibiting a novel or atypical PKC results in up-regulation of p-AMPK during APAP pathogenesis. However, the particular isoform of PKC remains to be identified, as the study used broad-spectrum PKC inhibitors. It has been established that AMPK activation is regulated by phosphatase 2Cα and two upstream kinases, i.e., LKB1 and calmodulin-dependent protein kinase kinase-beta (CaMKKβ), which directly phosphorylate AMPK.[19] Accordingly, it is likely that PKC is involved indirectly in AMPK phosphorylation. The only studies linking PKC and AMPK reported that atypical PKCzeta up-regulates p-AMPK through a specific phosphorylation of LKB1, thereby promoting its nucleocytoplasmic translocation.[20, 21] To substantiate the conclusion that inhibiting atypical PKC leads to up-regulation of p-AMPK, additional studies to investigate the effects of PKC inhibition on LKB1, CaMKKβ, and phosphatase 2Cα are warranted. The energy-sensing capability of AMPK derives from its ability to detect and react to the changes in AMP/ATP ratio. Modulation of the AMP/ATP ratio by PKC inhibitors is another avenue to be investigated.

In summary, Kaplowitz and colleagues provide evidence that PKC family members contribute to hepatocellular injury from APAP and that particular PKC isoforms likely have different roles (Fig. 1). Classical PKCs (e.g., PKCα) likely promote injury by acting through JNK, whereas novel and/or atypical PKCs act independently of JNK to inhibit an AMPK pathway that bolsters cell survival. These findings add to the list of signaling kinases that play various roles in the pathogenesis of APAP-induced liver injury.

  • Cynthia Ju, Ph.D.1 and Robert A. Roth, Ph.D.2

  • 1Skaggs School of Pharmacy and Pharmaceutical Sciences University of Colorado Aurora, CO 2Department of Pharmacology and Toxicology Center for Integrative Toxicology Michigan State University East Lansing, MI


Figure 1. Working hypothesis based on the results of the study. APAP is bioactivated by CYPs to NAPQI (top left), which causes mitochondrial oxidative stress. This activates an MAPK pathway, culminating in JNK phosphorylation and translocation to the mitochondrial membrane, where it associates with Sab and prompts MPT pore formation and the release from the mitochondria of factors that cause hepatocellular necrosis. Activation of (unidentified) novel and/or atypical PKCs inhibits AMPK activation, which reduces autophagy, perhaps through a mechanism involving mTORC1 inhibition. Inasmuch as autophagy promotes cell survival, this enhances necrosis. Classical PKCs (e.g., PKCα) are also activated, at least in part due to JNK activation. Reciprocal activation of classical PKCs and JNK promote sustained activation of these two kinases (dotted blue lines). Pathways demonstrated in the studies by Saberi et al. are highlighted in green.

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