Regulation of H₂O₂-induced necrosis by PKC and AMP-activated kinase signaling in primary cultured hepatocytes

Recent studies have suggested that, in certain cases, necrosis, like apoptosis, may be programmed, involving the activation and inhibition of many signaling pathways. In this study, we examined whether necrosis induced by H₂O₂ is regulated by signaling pathways in primary hepatocytes. A detailed time course revealed that H₂O₂ treated to hepatocytes is consumed within minutes, but hepatocytes undergo necrosis several hours later. Thus, H₂O₂ treatment induces a "lag phase" where signaling changes occur, including PKC activation, Akt (PKB) downregulation, activation of JNK, and downregulation of AMP-activated kinase (AMPK). Investigation of various inhibitors demonstrated that PKC inhibitors were effective in reducing necrosis caused by H ₂O₂ (∼80%). PKC inhibitor treatment decreased PKC activity but, surprisingly, also upregulated Akt and AMPK, suggesting that various PKC isoforms negatively regulate Akt and AMPK. Akt did not appear to play a significant role in H₂O₂-induced necrosis, since PKC inhibitor treatment protected hepatocytes from H₂O₂ even when Akt was inhibited. On the other hand, compound C, a selective AMPK inhibitor, abrogated the protective effect of PKC inhibitors against necrosis induced by H₂O₂. Furthermore, AMPK activators protected against H₂O₂-induced necrosis, suggesting that much of the protective effect of PKC inhibition was mediated through the upregulation of AMPK. Work with PKC inhibitors suggested that atypical PKC downregulates AMPK in response to H₂O₂. Knockdown of PKC-α using antisense oligonucleotides also slightly protected (∼22%) against H ₂O₂. Taken together, our data demonstrate that the modulation of signaling pathways involving PKC and AMPK can alter H ₂O₂-induced necrosis, suggesting that a signaling "program" is important in mediating H₂O₂-induced necrosis in primary hepatocytes.