Potential conflict of interest: Nothing to report.
Hepatic ischemia-reperfusion (I/R) injury occurs in the settings of transplantation, trauma, and elective liver resection. Ischemic preconditioning has been used as a strategy to reduce inflammation and organ damage from I/R of the liver. However, the mechanisms involved in this process are poorly understood. We examined the role of the phosphatidylinositol 3 (PI3) kinase/Akt-signaling pathway during hepatic ischemic preconditioning (IPC). Prior to a prolonged warm ischemic insult, BALB/c mice were subjected to a 20-minute IPC period consisting of 10 minutes of ischemia and 10 minutes of reperfusion. Mice undergoing IPC demonstrated a significantly greater level and earlier activation of Akt in the liver compared with control animals. IPC also resulted in markedly less hepatocellular injury and improved survival compared with control animals. Akt activation associated with hepatic IPC suppressed the activity of several modulators of apoptosis, including Bad, glycogen synthase kinase β, and caspase-3. In addition, IPC also inhibited the activities of c-Jun N-terminal kinase and nuclear factor κB after I/R. Pretreatment of mice with PI3 kinase inhibitors completely abolished Akt phosphorylation and the protective effects seen with IPC. In conclusion, these results indicate that the PI3 kinase/Akt pathway plays an essential role in the protective effects of IPC in hepatic I/R injury. Modulation of this pathway may be a potential strategy in clinical settings of ischemic liver injury to decrease organ damage. Supplementary material for this article can be found on the HEPATOLOGY website (http://interscience.wiley.com/jpages/0270-9139/suppmat/index.html). (HEPATOLOGY 2006;44:573–580.)
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Ischemia and reperfusion (I/R) injury is a pathophysiological process whereby hypoxic organ damage is accentuated following return of blood flow and oxygen delivery to the compromised tissue. Transient episodes of hepatic ischemia occur during solid organ transplantation, trauma, hypovolemic shock, and elective liver resection, when inflow occlusion or total vascular exclusion is used to minimize blood loss. The pathophysiology of liver I/R injury includes both direct cellular damage as the result of the ischemic insult as well as delayed dysfunction and damage resulting from activation of inflammatory pathways. Activation of the inflammatory pathways involves Toll-like receptor 4 signaling and the nuclear factor high mobility group box 1.1–3 Histopathological changes include cellular swelling, vacuolization, endothelial cell disruption, neutrophil infiltration, and hepatocellular necrosis.4, 5
Early ischemic preconditioning (IPC) is a strategy used clinically to prevent I/R injury. It consists of a brief period of ischemia followed by a short interval of reperfusion before a more sustained ischemic insult. The preconditioning phenomenon is based on the principle that cells primed by various kinds of stress trigger defense mechanisms against subsequent lethal injury of the same or different type.5 IPC has been shown to protect the liver against I/R injury in experimental models6–9 as well as in patients undergoing hepatectomy.10 Tumor necrosis factor, adenosine, nitric oxide, heat shock proteins, insulin-like growth factor, and cell adhesion molecules have been implicated in mechanisms that limit I/R-induced injury following IPC.6, 7, 11, 12 However, the signaling pathway activated by IPC that prevent I/R-induced damage is incompletely characterized.
The phosphatidylinositol 3 (PI3) kinase/Akt pathway is an important survival pathway involved in protection against various stressors. Activation of this pathway promotes cell survival in part by its modulation of various downstream elements involved in apoptosis.13, 14 Akt phosphorylates Bad, blocking the proapoptotic action of this prodeath Bcl-family protein.15 Akt has also been found to phosphorylate and inactivate glycogen synthase kinase β (GSK-3β). GSK-3β is a protein involved in the regulation of cellular metabolism, and phosphorylation of GSK-3β by Akt has been found to suppress caspase-3 activity and block apoptosis.16, 17 Recent studies have shown that activation of the Akt pathway plays a protective role during I/R.18–20 However, the mechanism by which Akt activation results in protection from I/R is not clear. In the present study, we show that mice undergoing IPC were protected from hepatic I/R injury through reactive oxygen species (ROS)-mediated activation of Akt. This occurred in association with the inhibition of I/R-induced c-Jun N-terminal kinase (JNK), Bad, and GSK-3β phosphorylation.
All animal experiments were conducted in accordance with local institutional guidelines for the care and use of laboratory animals. Anesthetized male BALB/c mice (25-30 g) were subjected to no-flow ischemia of 70% of the liver with a small vascular clip; the remaining 30% of the liver was removed just before reperfusion was reinstituted. The mice were divided into the following experimental groups as shown in Supplementary Fig. 1: (a) an ischemic I/R group (control) in which animals were subjected to ischemia for 90 minutes without IPC; (b) an IPC + I/R group in which animals were subjected to 90 minutes of ischemia after IPC that consisted of 10 minutes of ischemia followed by 10 minutes of reperfusion; (c) an IPC+I/R+wortmannin group in which wortmannin (50 nmol/kg) was injected intravenously after 90 minutes of ischemia that was preceded by IPC consisting of 10 minutes of ischemia and 10 minutes of reperfusion; (d) an IPC+I/R+LY294002 group in which LY294002 (10 μmol/kg) was injected intravenously after 90 minutes of ischemia that was preceded by IPC consisting of 10 minutes of ischemia and 10 minutes of reperfusion; (e) an I/R+wortmannin group in which wortmannin (50 nmol/kg) was injected intravenously after 90 minutes of ischemia; (f) an I/R+LY294002 group in which LY294002 (10 μmol/kg) was injected intravenously after 90 minutes of ischemia. To examine the mechanism of Akt activation, we injected 0.2 mg/kg of ZM241385 (adenosine A2 recepter blocker) or 2,000 U/kg of superoxide dismutase (SOD) intravenously 5 minutes before IPC (n = 4-6). Liver tissues were harvested at different time points during ischemia and after reperfusion. Blood samples were harvested at 6 hours after reperfusion for the assessment of serum alanine aminotransferase (ALT) levels as an indicator of hepatocellular injury. The survival rates after 60 (n = 6), 90 (n = 17), and 120 (n = 6) minutes of ischemia were measured on day 7.
SDS-PAGE and Western Blotting.
Liver samples were homogenized and centrifuged as previously described.21 Protein lysates were loaded (25 μg) and fractionated using 12% polyacrylamide gel containing SDS. Specific bands were then visualized via Western blotting using antibodies recognizing phosphorylated-Akt (Ser473), total Akt, phosphorylated-Bad (Ser112), total Bad, phosphorylated GSK-3β, total GSK-3β, cleaved caspase-3, phosphorylated JNK, and β-actin. The intensity of Akt phosphorylation was quantified using NIH image software (Version 1.62, National Institutes of Health, Bethesda, MD) and presented as a relative value to the mean value before ischemia.
Liver specimens were fixed in 10% buffered formalin solution, embedded in paraffin, sectioned at 5-μm thickness, and stained with hematoxylin-eosin. Histological changes were scored in a blind fashion from 0 to 3 based on the degree of cytoplasmic vacuolization, sinusoidal congestion, sinusoidal derangement, and necrosis of parenchymal cells using modified Suzuki classification as described by Takeda et al.22 For immunohistochemical analysis of the liver, sections were deparaffinized and dehydrated in graded alcohol solutions. Phospho-specific (Ser473) Akt antibody staining of sections was performed according to the manufacturer's instructions.
Terminal dUTP Nick-End Labeling.
Sections of formalin-fixed, paraffin-embedded liver tissue were deparaffinized in xylene and dehydrated through a graded series of alcohols ending with 15 minutes of incubation in phosphate-buffered saline. Terminal dUTP nick end labeling (TUNEL) assay was performed using a commercial kit (Boehringer Mannheim Co., Indianapolis, IN) according to the manufacturer's recommended protocol. TUNEL-positive hepatocytes were then counted blindly and expressed as a percentage of the total hepatocytes, excluding the surrounding massive necrosis lesion.
Electrophorectic Mobility Shift Assays.
Nuclear factor κB (NF-κB) DNA binding activity was measured via electrophoretic mobility shift assay using nuclear extracts from liver tissues as previously described.1
Statistical analysis was performed by analysis of variance and all of the data were expressed as the mean ± SEM. Differences between two dependent groups were evaluated with an unpaired Student t test. Comparisons among multiple groups were performed with one-way ANOVA followed by Bonferroni post hoc tests. Animal survival was compared using the Fisher exact test. A P value of less than .05 was considered statistically significant.
Protective Effects of IPC Are Blocked by PI3 Kinase Inhibitors.
The protective effects of IPC were evaluated in a mouse model of hepatic I/R. ALT levels were significantly lower 6 hours after reperfusion in animals subjected to IPC (Fig. 1). Administration of selective PI3-kinase inhibitors, wortmannin, and LY294002 at the time of reperfusion prevented the protective effect of the IPC in animals subjected to I/R. Administration of wortmannin or LY294002 to the animals subjected to I/R (no IPC) did not significantly alter the ALT levels compared with I/R alone.
Akt Phosphorylation After Reperfusion Induced by IPC.
We examined changes in Akt phosphorylation in the liver following I/R with and without IPC. As shown in Fig. 2A, strong Akt phosphorylation was detected 1 hour after reperfusion in the livers subjected to IPC but not the I/R alone livers. The phosphorylation caused by IPC was completely abolished by the treatment with wortmannin. These results were confirmed by immunohistochemical staining with Akt phosphospecific antibody. As shown in Fig. 2B-1, preischemic cells did not stain positive for phosphoylated Akt antibody. However, in the IPC+I/R group, significant phosphorylation was seen in the intermediate and central zones of the hepatic parenchyma (Fig. 2B-2). This phosphorylation was diminished by the addition of wortmannin.
Time Course of Akt Phosphorylation During Ischemia and After Reperfusion.
We examined changes in Akt phosphorylation during ischemia and after reperfusion in mouse livers. As shown in Supplementary Fig. 2A, Akt was phosphorylated between 2 and 3 hours after reperfusion in animals subjected to I/R without IPC. However, animals undergoing IPC displayed Akt phosphorylation much earlier, starting at 15 minutes after reperfusion. Phosphorylation of Akt caused by IPC was completely abolished by the addition of wortmannin. The level of total Akt protein expression was not significantly changed after reperfusion (Supplementary Fig. 2).
Survival After Reperfusion.
To determine the significance of the PI3 kinase/Akt pathway in the protection seen with IPC, we examined the effects of IPC and its inhibition with wortmanin in mice undergoing ischemia for 60, 90, and 120 minutes (Fig. 3). There was no difference in the groups of mice undergoing 60 minutes of ischemia (all animals survived). After 90 minutes of ischemia, IPC improved the survival from 29% (5/17) to 76% (13/17). Treatment with wortmannin abolished the protective effects of IPC. In animals subjected to 120 minutes of liver ischemia, IPC was not effective in improving survival. These results suggest that Akt activation contributes to survival after 90 minutes of ischemia, because pretreatment with wortmannin is able to abolish the protective effects of IPC.
The histological appearance of the liver was examined by hematoxylin-eosin staining 4 hours after reperfusion (Fig. 4). In the I/R without IPC groups, significant levels of cytoplasmic vacuolization, sinusoidal congestion, and massive necrosis of parenchymal cells were seen (Fig. 4A-1). However, in the group that received IPC, the parenchymal appearance was near normal (Fig. 4A-2). In the evaluation of the histological features of I/R injury, the IPC+I/R group had significantly lower scores of cytoplasmic vacuolization and massive necrosis compared with the I/R group (Fig. 4B). Wortmannin prevented the protective effect of IPC with increased cytoplasmic vacuolization and hepatocellular necrosis.
Because activation of the PI3-kinase/Akt pathway can promote cell survival in part by modulating various downstream elements involved in apoptosis, we performed TUNEL staining to detect DNA degradation after I/R. Several TUNEL-positive cells were seen 4 hours after hepatic I/R (Fig. 5A -1). The majority of these cells were associated with the necrotic lesions seen on hematoxylin-eosin staining. The number of TUNEL-positive cells was reduced by IPC. Wortmannin reversed the effects of IPC, returning the level of TUNEL-positive cells to that seen in the I/R control group. Because both apoptotic and necrotic cells are detected via TUNEL assay,23, 24 we also examined the number of TUNEL-positive cells in nonnecrotic areas of the liver. The number of positive cells in the IPC+I/R group was lower than that in the I/R control group (Fig. 5B). Wortmannin administration in the IPC+I/R group returned the number of positive cells to the level seen in the I/R group.
IPC Modulates Downstream Effecters of Akt.
There are multiple targets of the PI3-kinase/Akt pathway that may mediate the ability of this cascade to promote survival during I/R injury. Bad and GSK-3β are 2 proteins involved in cell death that have been shown to be regulated by Akt activity. Upon activation of Akt, Bad and GSK-3β are phosporylated, resulting in their inactivation and the promotion of cell survival. As shown in Fig. 6, phosphorylated levels of Bad and GSK-3β protein levels are only slightly increased after I/R compared with sham animals. However, IPC results in higher phosphorylation levels of both proteins. In addition, IPC prevents the cleavage of caspase-3, an effector of apoptosis. These effects of IPC were all inhibited by Wortmannin treatment.
JNK and NF-κB Is Suppressed by Ischemic Preconditioning.
To further elucidate the mechanism of IPC protection, we examined the activity of JNK and NF-κB via Western blotting and electrophoretic mobility shift assay, respectively. Sustained JNK activation has been shown to lead to hepatocyte necrosis.25 Although NF-κB activation can inhibit apoptosis, its upregulation can also lead to the production of inflammatory mediators that contribute to cellular toxicity.26 As shown in Fig. 7A, IPC resulted in decreased JNK phosphorylation 1 hour after reperfusion compared with control animals undergoing I/R without IPC. However, mice treated with Wortmannin prior to IPC demonstrated levels of JNK phosphorylation similar to control I/R animals. Similar findings were seen with NF-κB (Fig. 7B).
Protective Effects of IPC Through the Akt Pathway Are Attenuated by ROS Scavenger But Not by Adenosine A2A Receptor Blocker In Vivo.
To examine the mechanism of Akt activation, mice were given either SOD or ZM241385 (selective adenosine A2A receptor antagonist) before IPC. As shown in Fig. 8, SOD pretreatment diminished the protective effects of IPC and resulted in the inhibition of Akt phosphorylation after reperfusion caused by IPC. However, ZM241385 pretreatment resulted in a significant increase in hepatic injury after I/R. Treatment of mice undergoing sham procedure with ZM241385 did not cause liver damage. Interestingly, IPC with ZM241385 was associated with increased Akt phosphorylation and protection from further hepatic injury.
Ischemic preconditioning has been shown to protect against ischemic stress in a variety of tissues, including skeletal muscle, spinal cord, retina, intestine, brain, heart, and liver.5 Previous studies have shown the PI3-kinase/Akt pathway to be involved in the ischemic tolerance seen in heart and in neurons.27–30 In addition, recent studies have also demonstrated a protective role of Akt activation against ischemic injury in the liver.18, 19 However, the mechanism of the protection conferred by IPC remains unclear. Therefore, the aim of our study was to determine if the PI3-kinase/Akt pathway mediates the protective effects of IPC in the liver. We found that (1) mice undergoing a short period of ischemia prior to a prolonged ischemic insult were protected from hepatic injury; (2) IPC resulted in increased Akt phosphorylation, which was associated with (3) subsequent deactivation of various proapoptotic factors such as Bad and GSK-3β and downregulation of JNK and NF-κB activities after I/R.; (4) the protective effects of ischemic preconditioning were abolished with PI3 kinase inhibition; and (5) ROS contribute to the mechanism of Akt activation by IPC.
The PI3 kinase/Akt pathway is involved in transmitting signals by growth factors for the promotion of cell growth and survival. Carini et al.19 reported that Akt is activated by IPC during ischemia and results in increased hepatocyte viability during hypoxia in vitro. However, our results in vivo show that Akt is not phosphorylated during ischemia. Moreover, we found that only I/R leads to an activation of Akt starting between 60 and 120 minutes after reperfusion. The major effect of IPC was an almost immediate increase in Akt phosphorylation following reperfusion. Thus, the timing of the activation of Akt pathway appears to be critical to provide protection. This finding also suggests that the events injurious to the liver in I/R commence within the first hour upon reperfusion. The timing of Akt activation in I/R after IPC suggests the liver is primed for Akt activation upon reperfusion. How this priming effect occurs is unclear.
Akt is known to be activated in a number of cell types by cellular stresses, such as UVB radiation, anticancer drug administration, heat shock, and oxidative insults.27, 31–34 Recent studies have shown that adenosine or ROS may mediate the protective effects against liver I/R injury through Akt activation.18, 19 Our study suggests that ROS may play a role in priming the liver for Akt activation during IPC. Animals pretreated with SOD displayed inhibition of Akt activation and abrogation of the protective effects of IPC. However, adenosine does not appear to mediate the protective effects of hepatic IPC, because use of an adenosine A2A–specific antagonist did not demonstrate a similar effect. The mechanisms of how ROS may mediate Akt activation remains to be elucidated. ROS have been shown to induce Akt activation through PI3K membrane recruitment to its substrate site, thereby enabling PI3K to maximize its catalytic efficiency.35 Increased PI3K activity results in the production of phosphatidylinositol 3,4,5-triphosphate, which in turn binds to Akt, thereby causing Akt to translocate to the plasma membrane, where it is phosphorylated by PI-(3,4,5)P3-dependent kinase 1 and 2.36, 37 IPC may also activate Akt by inhibiting phosphatase and TENs in homologue on chromosome 10 (PTEN), a negative regulator of Akt. Studies have shown that ROS play an important role by oxidizing and inactivating PTEN protein, thus allowing for Akt phosphorylation.38, 39 In addition to ROS, changes in intracellular calcium levels and glucose and/or amino acid deprivation associated with IPC have been suggested to activate Akt.21, 40 Thus, there are multiple possible mechanisms by which the liver can be primed for Akt activation upon reperfusion after IPC.
Activation of the PI3-kinase/Akt pathway promotes cell survival in part by its modulation of various downstream elements involved in apoptosis.13, 14 We show that Bad and GSK-3β, 2 proteins involved in cell death, are inhibited by Akt activity as a result of IPC. Indeed, IPC-treated animals have decreased numbers of cells undergoing apoptosis in the liver after I/R compared with control I/R animals. The role of apoptosis as a mechanism of cell death after warm I/R of the liver remains controversial, because both necrosis and apoptosis can result in TUNEL-positive cells.23, 24, 41 Malhi et al.41 reported that apoptosis and necrosis were frequently the consequence of the same initiating factors and signaling pathways and that both death modes frequently coexist in liver pathology. Therefore, apoptotic and necrotic cell death are not necessarily distinct and independent events and the pathways leading to necrosis and apoptosis can be shared, a phenomenon described as necrapoptosis or aponecrosis.5, 22, 41 In our study, we have also found that JNK and NF-κB activation after ischemia is inhibited by IPC. Both molecules have been shown to mediate the production of inflammatory cytokines and tissue necrosis. Thus, Akt-mediated downregulation of JNK and NF-κB may contribute to the decreased necrotic cell death seen after IPC.
Although IPC-induced Akt activation may promote antiapoptotic events, another mechanism of protection may be its regulation of cellular metabolism after reperfusion. Akt has been shown to regulate several processes involved in metabolism. These include effects on the activity of glycogen synthase16 and the induction of GLUT1 synthesis.42 Some studies have shown GLUT1 expression to be rapidly and markedly upregulated in response to hypoxia.43, 44 Indeed, increased glucose uptake and glycolysis during ischemia and early reperfusion are associated with improved postischemic recovery of the heart.45 Therefore, another possibility for Akt-mediated protection is the upregulation of glucose transport to maintain the cellular energy status, thereby providing protection against energy depletion after ischemia.
In conclusion, our study demonstrates that the PI3 kinase/Akt pathway plays an essential role in the protective effects of IPC in hepatic I/R injury. The Akt pathway may be an important target for the development of novel therapeutic strategies for protection against ischemic insults.