Elevated cAMP Protects against Diclofenac-Induced Toxicity in Primary Rat Hepatocytes: A Protective Effect Mediated by the Exchange Protein Directly Activated by cAMP/cAMP-Regulated Guanine Nucleotide Exchange Factors

Abstract

Background: Chronic consumption of the nonsteroidal anti-inflammatory drug diclofenac may induce drug-induced liver injury (DILI). The mechanism of diclofenac-induced liver injury is partially elucidated and involves mitochondrial damage. Elevated cAMP protects hepatocytes against bile acid-induced injury. However, it is unknown whether cAMP protects against DILI and, if so, which downstream targets of cAMP are implicated in the protective mechanism including the classical protein kinase A (PKA) pathway or alternative pathways like the exchange protein directly activated by cAMP (EPAC). Aim: Investigate whether cAMP and/or its downstream targets protect against diclofenac-induced injury in hepatocytes. Methods: Rat hepatocytes were exposed to 400 µmol/L diclofenac. Apoptosis and necrosis were measured by caspase-3 activity assay and Sytox green staining respectively. Mitochondrial membrane potential (MMP) was measured by JC-10 staining. mRNA and protein expression were assessed by qPCR and Western blot, respectively. The cAMPelevating agent forskolin, the pan-phosphodiesterase inhibitor IBMX and EPAC inhibitors CE3F4 and ESI-O5 were used to assess the role of cAMP and its effectors, PKA or EPAC. Results: Diclofenac exposure induced apoptotic cell death and loss of MMP in hepatocytes. Both forskolin and IBMX prevented diclofenac-induced apoptosis. EPAC inhibition, but not PKA inhibition abolished the protective effect of forskolin and IBMX. Forskolin and IBMX preserved the MMP while both EPAC inhibitors diminished this effect. Both EPAC1 and EPAC2 were expressed in hepatocytes and localized in mitochondria. Conclusion: cAMP elevation protects hepatocytes against diclofenac-induced cell death, a process primarily involving EPACs. The cAMP/EPAC pathway may be a novel target for treatment of DILI.

Metformin protects against diclofenac-induced toxicity in primary rat hepatocytes by preserving mitochondrial integrity via a pathway involving EPAC

“…This study shows that metformin prevents DF hepatotoxicity and that this protection is related to cAMP-EPAC-2 signaling. This statement is supported by previous research [3]. Metformin has been demonstrated to decrease cAMP generation, a process linked to AMPK and EPAC1/2 signaling [19, 36,40,45].…”

“…This model has previously been described and induces predominantly apoptotic cell death in primary rat hepatocytes. The optimal concentration for the experiments was defined previously [3,16,48]. Apoptotic cell death determined by caspase-3 activity assay peaked at 400 µmol/L and 12 h exposure.…”

“…It was previously shown that both EPACs are expressed in rat hepatocyte mitochondria [3] and since MnSOD is exclusively located in the mitochondria and an early elevation of cAMP levels correlated with mitochondrial protection [1,31], the role of metformin in preservation of MnSOD expression in an EPAC-dependent manner was investigated. Hepatocytes were either incubated with diclofenac, with or without ESI-05 or CE3FA4, or in the presence or absence of metformin.…”

“…Primary rat hepatocyte mitochondria express both EPAC1 and EPAC2 [3]. It was demonstrated in a previous study that elevating cAMP levels prevented DF-induced toxicity in hepatocytes, and the protective effect was mediated by EPACs partly by preservation of mitochondrial integrity [3].…”

“…Via its effectors EPAC and PKA, cAMP is implicated in various processes, including but not limited to inhibition of apoptosis [3,23], increased cell proliferation in response to partial hepatectomy [53] and protection against several noxious stresses in the liver, e.g., bile acids [6,12,17,27].…”