{alpha}-Lipoic acid preconditioning reduces ischemia-reperfusion injury of the rat liver via the PI3-kinase/Akt pathway

Christian Müller,1,* Friedrich Dünschede,2,* Elke Koch,1 Angelika M. Vollmar,1 and Alexandra K. Kiemer1

1Department of Pharmacy, Center of Drug Research, University of Munich, 81377 Munich; and 2Department of Surgery, University of Mainz, 55101 Mainz, Germany

Submitted 7 January 2003 ; accepted in final form 11 June 2003


    ABSTRACT
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 DISCLOSURES
 REFERENCES
 
In liver resection and transplantation ischemia-reperfusion injury (IRI) is one of the main causes of organ dys- or nonfunction. The aim of the present study was to determine whether {alpha}-lipoic acid (LA) is able to attenuate IRI. Rat livers were perfused with Krebs-Henseleit buffer with or without LA (±wortmannin), followed by ischemia (1 h, 37°C) and reperfusion (90 min). Efflux of lactate dehydrogenase (LDH) and purine nucleoside phosphorylase (PNP) and hepatic ATP content were determined enzymatically. Activation of NF-{kappa}B and activating protein 1 (AP-1) was examined by EMSA, and protein phosphorylation was examined by Western blot. Caspase-3-like activity served as an indicator for apoptotic processes. Animals treated intravenously with 500 µmol LA were subjected to 90 min of partial no-flow ischemia followed by reperfusion for up to 7 days. Preconditioning with LA significantly reduced LDH and PNP efflux during reperfusion in isolated perfused rat livers. ATP content was significantly increased in LA-treated livers. Postischemic activation of NF-{kappa}B and AP-1 was significantly reduced in LA-pretreated organs. Preconditioning with LA significantly enhanced Akt phosphorylation. It showed neither effect on endothelial nitric oxide synthase nor on Bad phosphorylation. Importantly, simultaneous administration of wortmannin, an inhibitor of the phosphatidylinositol (PI)3-kinase/Akt pathway, blocked the protective effect of LA on IRI, demonstrating a causal relationship between Akt activation and hepatoprotection by LA. Interestingly, despite activation of Akt, LA did not reduce postischemic apoptotic cell death. The efficacy of LA treatment in vivo was shown by reduced GST plasma levels and improved liver histology of animals pretreated with LA. This study shows for the first time that the PI3-kinase/Akt pathway plays a central protective role in IRI of the rat liver and that LA administration attenuates IRI via this pathway.

adenosine 5'-triphosphate; transcription factors; p38 mitogen-activated protein kinase; wortmannin; phosphatidylinositol 3-kinase


ISCHEMIA-REPERFUSION INJURY (IRI) is a major problem in liver resection, liver transplantation, and hemorrhagic shock. The pathomechanisms of IRI can be divided into incidents during ischemia and events occuring during reperfusion. Major pathophysiological features of ischemic liver cell injury comprise depletion of ATP, disturbance of natrium-calcium homeostasis, and activation of phospholipase A2 (3, 5, 21). Reperfusion of livers leads to an aggravation of ischemic liver cell damage: reactive oxygen species (ROS) derived from activated Kupffer cells or neutrophils and consequently activation of proinflammatory, redox-sensitive transcription factors, such as NF-{kappa}B and AP-1, may contribute to hepatic reperfusion injury (1, 12, 44). In the past few years especially, preconditioning interventions have been developed as protective strategies against hepatic IRI. Among them are hyperthermic (36), ischemic (30, 42), and pharmacological preconditioning (5, 20).

Our attention focused on the potential of preconditioning with {alpha}-lipoic acid (LA) on IRI of the rat liver. Besides its well-described antioxidant effects, LA exhibits distinct regulatory action on signal-transduction processes playing a central role in tissue damage and protection. In this context, the potential of LA to regulate stress-related signaling pathways, such as NF-{kappa}B, on the one hand (22, 31) and to activate cytoprotective protein kinases on the other hand has recently been reported (24, 41).

Naturally occuring LA is found as a prosthetic group in {alpha}-keto acid dehydrogenase complexes of mitochondria and therefore plays a fundamental role in metabolism (29). Furthermore, LA is established in the therapy of diabetic polyneuropathy (10) and has been described as a therapeutic agent in a number of conditions related to liver disease, including alcohol-induced damage, mushroom poisoning, metal intoxification, CCl4 poisoning, and hyperdynamic circulation in biliary cirrhosis (6, 7, 26).

In the present study, we examined whether preconditioning with LA has protective potential in hepatic IRI. In this context, our special interest focused on the characterization of mechanisms in LA-mediated hepatoprotection. Thereby, we identified, for the first time, that the phosphatidylinositol 3-kinase (PI3-K)/Akt pathway plays a central protective role in IRI of the liver.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 DISCLOSURES
 REFERENCES
 
Materials. Racemic LA (1,2-dithiolane-3-pentanoic acid, thioctic acid) was a gift from ASTA Medica (Frankfurt am Main, Germany). All other materials were purchased from either Sigma (Deisenhofen, Germany) or Merck (Darmstadt, Germany).

Animals. Sprague-Dawley rats, weighing 220–280 g, were purchased from Charles River (Sulzfeld, Germany), BrownNorway rats (175–200 g body wt) were from Harlan (Paderborn, Germany). The animals had free access to chow [Harlan Teklad Global Diet or standard diet (Altromin 1314; Altromin, Lage, Germany)] and water up to the time of experiments.

The research involving animals adhered to the Guiding Principles for the Care and Use of Animals. All animals received humane care according to the criteria outlined in the Guide for the Care and Use of Laboratory Animals prepared by the National Academy of Sciences and published by the National Institutes of Health (NIH publication 86-23, revised 1985), and the study was registered with the local animal welfare committee.

Liver perfusion. Isolated rat liver perfusions were performed with male Sprague-Dawley rats as described by Bilzer et al. (5) including 30 min of perfusion, a 60-min ischemic period (37°C), and reperfusion for up to 90 min in a nonrecirculating system. LA (10 or 50 µM) was applied in a typical preconditioning protocol by being infused 20 min before ischemia. At the indicated times, i.e., before ischemia, at the end of ischemia, and after 45 and 90 min of reperfusion, livers were snap-frozen and stored at –80°C until further analysis (Fig. 1). Perfusate probes were collected for immediate determination of lactate dehydrogenase (LDH) activity. In an additional experiment, livers were perfused in the presence of the PI3-K inhibitor wortmannin (WM; 100 nM, Alexis Biochemicals, Grünberg, Germany) and LA (50 µM) for 20 min before ischemia.



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Fig. 1. Experimental protocol. Livers were perfused for 30 min (Pre-I) in the absence or presence of 50 µM {alpha}-lipoic acid (LA), which was added 20 min before ischemia. After 60 min of ischemia (WI; 37°C), livers were reperfused (R) for 45 or 90 min. In additional experiments, livers were perfused for 30 min in the presence of 100 nM wortmannin (WM) and 50 µM LA, which were given 20 min before ischemia. After WI (37°C), livers were reperfused for 90 min. Arrows indicate time points when liver samples were taken.

 

Determination of LDH, purine nucleoside phosphorylase, {alpha}-GST, and ATP. Sinusoidal efflux rates of LDH and purine nucleoside phosphorylase (PNP) were measured as indicators of liver cell damage (4, 13). The activity of LDH and PNP in perfusates, {alpha}-GST in serum, and hepatic ATP content in acidic liver homogenates [ratio of perchloric acid 6% (wt/vol) to tissue weight: 4:1] were analyzed according to standard methods (2).

Preparation of nuclear extracts and EMSA. Preparation of nuclear extracts and DNA binding activity of NF-{kappa}B and AP-1 was performed by EMSA as described previously (23). Briefly, tissue samples were homogenized in 3 ml of ice-cold hypotonic buffer A [in mM: 10 HEPES (pH 7.9), 10 KCl, 0.1 EDTA, 0.1 EGTA, 1 DTT, and 0.5 PMSF] with a Dounce homogenizer. The homogenate was transferred to a polypropylene centrifuge tube and, after a 10-min incubation on ice, centrifuged at 1,000 g for 10 min at 4°C. The cell pellet was suspended in 1.4 ml of ice-cold buffer A, and 90 µl of a 10% solution of Nonidet P-40 were added followed by 10 s of vigorous vortexing. The suspension was incubated on ice for 10 min and then centrifuged at 12,000 g for 30 s at 4°C. The supernatant was removed, and the nuclear pellet was extracted with 200 µl of hypertonic buffer B [20 mM HEPES (pH 7.9), 0.4 M NaCl, 1 mM EDTA, 1 mM EGTA, 1 mM DTT, and 1 mM PMSF] by shaking at 4°C for 30 min. The extract was centrifuged at 12,000 g for 10 min at 4°C, and the supernatant was frozen at –70°C. The protein concentration was determined by the method of Lowry using BSA as a standard. For EMSA, two double-stranded oligonucleotide probes containing a consensus binding sequence for either NF-{kappa}B (5'-AGT TGA GGG GAC TTT CCC AGG C-3') or AP-1 (5'-CGC TTG ATG AGT CAG CCG GAA-3') (Promega, Heidelberg, Germany) were 5'-end-labeled with [{gamma}32P]ATP (3,000 Ci/mmol, Amersham, Braunschweig, Germany) using T4 polynucleotide kinase (Promega). Ten micrograms of nuclear protein were incubated in a 15-µl reaction volume containing 10 mM Tris·HCl (pH 7.5), 5 x 104 counts/min radiolabeled oligonucleotide probe, 2 µg poly(dIdC), 4% glycerol, 1 mM MgCl2, 0.5 mM EDTA, 50 mM NaCl, and 0.5 mM DTT for 20 min at room temperature. Nucleoprotein-oligonucleotide complexes were resolved by electrophoresis in a 4.5% nondenaturing polyacrylamide gel in 0.25 x Tris-borate-EDTA at 100 V. The gel was autoradiographed with an intensifying screen at –80°C overnight. Specificity of the DNA-protein complex was confirmed by competition with a 100-fold excess of unlabeled NF-{kappa}B and AP-1 sequences, respectively. Signal detection and quantification was performed by phosphorimaging (Cyclone Storage Phosphor Screen; Canberra-Packard, Dreieich, Germany).

Phospho-p38 MAPK, -Akt, -endothelial nitric oxide synthase, and -Bad Western blots. Deep-frozen livers were homogenized in ice-cold lysis buffer (137 mM NaCl, 20 mM Tris, 2 mM EDTA, 10% glycerol, 2 mM Na-pyrophosphate, 1% Triton X-100, 20 mM Na-{beta}-glycerophosphate, 10 mM NaF, 2 mM Na-orthovanadate, 1 mM phenylmethylsulfonyl fluoride) supplemented with Complete (Roche Diagnostics, Mannheim, Germany) and were centrifuged, and protein concentrations were determined by the Lowry assay. Western blots were performed using 100–200 µg of protein and anti-phospho-Akt, -phospho-p38 MAPK, -phospho-Bad, and -phospho-endothelial nitric oxide synthase (eNOS) polyclonal rabbit antibodies (Cell Signaling, New England Biolaboratories, Frankfurt am Main, Germany) followed by incubation in horseradish peroxidase-conjugated anti-rabbit IgG antibody (Dianova, Hamburg, Germany). Finally, the immunoreactive bands were visualized by Renaissance chemiluminescence reagent plus (New England Nuclear, Köln, Germany). Detection and quantification were performed with a Kodak image station (New England Nuclear). Reprobing of the blots was carried out with antibodies against the unphosphorylated forms of p38 MAPK (Calbiochem, Bad Soden, Germany), Bad, and Akt (Cell Signaling, New England Biolaboratories) to exclude loading differences.

Caspase-3-like activity. Caspase-3-like activity was determined according to Thornberry (37) with modifications as reported by Hentze et al. (17). Generation of free fluorescent 7-amino-4-trifluoromethylcoumarin (Sigma) was measured kinetically after incubation at 37°C (Fluostar; BMG, Offenburg, Germany). Protein concentrations of the corresponding samples were quantified with the Pierce assay (Pierce, Rockford, IL). Control experiments confirmed that the activity was linear with time and with protein concentration. LA in final concentrations up to 50 µM was demonstrated not to interfere with the assay (data not shown).

Analysis of LA. Contents of LA in liver and perfusate were determined by HPLC (data not shown). An influence of LA on the applied assays could be excluded due to control experiments, in which LA in maximal final concentrations did not interfere with the performed reactions (data not shown).

In vivo model. Male Brown-Norway rats were subjected to 90 min of partial no-flow ischemia followed by 1 h, 4 h, or 7 days reperfusion. The group of the controls was sham operated. The animals in the treatment group received a bolus dose of 500 µmol LA (1 ml) intravenously into the inferior vena cava 15 min before ischemia. The control group received 1 ml of NaCl.

Under ether anesthesia, laparotomy was performed and the blood supply to the left lateral and the left part of median lobe of the liver was occluded with an atraumatic vascular clamp for 90 min. The body temperature was monitored and maintained at 37 ± 0.3°C by a heating lamp. Reperfusion was initiated by removal of the clamp; the wound was closed in layers, and the animal was allowed to recover.

Groups of animals were killed under anesthesia at the end of different times during reperfusion (1 h, 4 h, or 7 days). After the animal was anesthetized, a blood sample (2–3 ml) was collected from the intrathoracal inferior vena cava with a heparinized syringe. The animal was then killed by exsanguination, and liver samples were collected. Samples were fixed in glutaraldehyde (1 mM) for histological analysis and stained with methylene blue according to standard protocols.

Statistical analysis. All data are expressed as means ± SE (n = no. of organs). Unless stated otherwise, all experiments were performed with n = 5 organs per treatment group. Statistical significance between groups was determined with one sample or Student's t-test. A P value <0.05 was considered statistically significant. Statistical analyses were performed with GraphPad Prism (Version 3.02, GraphPad Software, San Diego, CA).


    RESULTS
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 DISCLOSURES
 REFERENCES
 
LA attenuates postischemic sinusoidal efflux of LDH. To investigate an effect of LA preconditioning on IRI, LA was administered 20 min before the ischemic period as shown in Fig. 1. Liver cell damage was determined by measuring release of the cytosolic enzymes LDH and PNP into perfusate. LDH and PNP efflux (Fig. 2) were significantly reduced in livers treated with 50 µM LA compared with untreated controls, whereas preconditioning with 10 µM LA only slightly but not significantly decreased enzyme release during reperfusion (data not shown). Interestingly, LA even decreased early enzyme washout in the first minutes of reperfusion.



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Fig. 2. Influence of LA preconditioning on LDH and PNP efflux. Livers were perfused for 30 min in the absence (Co) or presence of 50 µM LA, which was added 20 min before ischemia. After ischemia (60 min, 37°C), livers were reperfused for up to 90 min. Analysis of lactate dehydrogenase (LDH; A) and purine nucleoside phosphorylase (PNP; B) activities in perfusate was carried out at different reperfusion time points as described in MATERIALS AND METHODS. Data are expressed as enzyme activity in mU·min1·g liver, and they show means ± SE of n = 5 experiments in each group. #P < 0.05 (10 µM), ***P < 0.001, **P < 0.01, and *P < 0.05 (50 µM) represent significant differences in the values among untreated and LA-treated livers.

 

LA preconditioning reduces NF-{kappa}B and activating protein 1 activation. It is known that the redox-sensitive transcription factors NF-{kappa}B and activating protein 1 (AP-1) are activated in IRI with the consequence of an increased expression of proinflammatory mediators (12, 18). Therefore, the effect of LA pretreatment on NF-{kappa}B and AP-1 activity was investigated. After 90 min of reperfusion, NF-{kappa}B activation was significantly decreased in LA-preconditioned livers (Fig. 3A). AP-1 DNA binding activity was significantly reduced after 60 min of ischemia (WI) and 90 min of reperfusion (Fig. 3B).



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Fig. 3. Effect of LA preconditioning on NF-{kappa}B and activating protein 1 (AP-1) activation in livers after WI and reperfusion (R). Livers were untreated (Co) or preconditioned with 50 µM LA 20 min before ischemia. After WI, livers were reperfused for up to 90 min. Binding activity of nuclear protein to the radiolabeled consensus binding sequences of NF-{kappa}B(A) and AP-1 (B) was assessed by EMSA (see MATERIALS AND METHODS) after WI and 45 and 90 min of R. Data show 1 representative gel shift experiment for each time point on the left side. Bars on the right side show densitometric analysis of the shifts, whereby NF-{kappa}B/AP-1 activation of LA-preconditioned livers is expressed as x-fold of the values in the correlating untreated group. Data are shown as means ± SE of n = 3 livers in each group, analyzed in 2 independent shifts. ***P < 0.001, **P < 0.01, and *P < 0.05 represent significant differences in the values between untreated and LA-pretreated livers.

 

Elevated ATP content in LA-pretreated livers. ATP content of livers is an indicator of their energetic status. As known from the literature (3, 21), ATP concentrations decreased during ischemia and reperfusion (Fig. 4). Importantly, preconditioning with LA significantly increased ATP contents after WI and at the end of the reperfusion period compared with control livers.



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Fig. 4. Effect of LA-preconditioning ATP content in liver. Livers were perfused in the absence (Co) or presence of 50 µM LA, which was added 20 min before ischemia (WI, 60 min, 37°C), followed by R for up to 90 min. ATP content in liver was analyzed after 30 min of perfusion (Pre-I), after 60 min of warm ischemia (WI), and after 45 and 90 min of R as described in MATERIALS AND METHODS. Data are expressed as µmol/g liver wt and show means ± SE of n = 5 experiments. **P < 0.01 represents significant differences of the values between untreated and LA-pretreated livers.

 

LA preconditioning does not affect activation of p38 MAPK. There is evidence that ischemic and hypoxic preconditioning are mediated via the activation of p38 MAPK (8, 32, 35). To investigate p38 MAPK as a potential mediator of LA preconditioning, we determined the activation of this kinase. Pretreatment of livers with LA, however, exerted no significant effect on phosphorylation of p38 MAPK (Fig. 5). Thus p38 MAPK activation does not seem to be involved in LA preconditioning.



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Fig. 5. Influence of LA preconditioning on p38 MAPK phosphorylation. Livers were perfused for 30 min in the absence (Co) or presence of 50 µM LA, which was added 20 min before 60-min ischemia (WI). Western blot analysis with antibodies against phosphorylated (phospho-p38) and total p38 MAPK (tot-p38; see MATERIALS AND METHODS) was performed after 30 min of perfusion (Pre-I), after 60 min of WI, and after 45 or 90 min of R. One representative Western blot is shown for each time point.

 

LA preconditioning increases phosphorylation of Akt (protein kinase B). Activation of another kinase, Akt (protein kinase B), has been described as possessing cytoprotective potential and being involved in ischemic preconditioning of the heart (25, 28, 38). Figure 6 shows that pretreatment of livers with LA significantly increased activation of Akt at the end of ischemia and at 90 min of reperfusion.



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Fig. 6. LA preconditioning effect on Akt (protein kinase B) activation. Livers were perfused for 30 min in the absence (Co) or presence of 50 µM LA, which was added 20 min before 60 min ischemia (WI). Western blot analysis (see MATERIALS AND METHODS) was performed detecting phosphorylated (phospho-Akt) and total Akt (tot-Akt) after 30 min of perfusion (Pre-I), after 60 min of ischemia (WI), and after 45 or 90 min of R. One representative Western blot is shown for each time point.

 

Inhibiting the PI3-K/Akt kinase pathway abrogates the protective effect of LA preconditioning. To determine a causal relationship between Akt activation and hepatoprotection by LA preconditioning, we cotreated livers with LA and the inhibitor of the upstream PI3-K WM (100 nM) (9, 14, 38). Efficacy of WM treatment in inhibiting LA-induced Akt activation was confirmed by the use of phospho-Akt Western blots (Fig. 7B). LDH and PNP release were again taken as an indicator of cell damage. Application of WM alone for 20 min before ischemia did not affect postischemic cell damage (data not shown). Figure 7A shows that simultaneous treatment with WM blocked the protection of LA preconditioning. Therefore, these data provide evidence that LA preconditioning reduces IRI via the PI3-K/Akt kinase pathway.



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Fig. 7. LDH and PNP efflux after pretreatment with LA and WM. Livers were perfused for 30 min in the absence (Co) or presence of 50 µM LA and 100 nM WM, which were added simultaneously 20 min before ischemia. After 60 min of WI, livers were reperfused for 90 min. Analysis of LDH (A) and PNP activity (B) was carried out immediately after collecting perfusate as described in MATERIALS AND METHODS. Data are expressed as enzyme activity in mU·min1·g liver wt and show means ± SE of n = 5 livers in each group. ***P < 0.001, **P < 0.01, and *P < 0.05 represent significant differences between LA-treated and untreated livers. The efficiency of WM treatment was proven by phospho-Akt Western blots (see MATERIALS AND METHODS) of simultaneously pretreated livers with WM and LA compared with livers preconditioned with LA alone (C).

 

Phosphorylation of Akt downstream targets. To determine which targets are phosphorylated by LA-induced Akt activation, we focussed on two prime targets: eNOS and Bad. eNOS has previously been shown to play a protective role in hepatic IRI (19), and the phosphorylation of Bad represents a typical antiapoptotic cell survival event (11). However, LA-treated livers showed neither increased phosphorylation of eNOS nor of Bad (Fig. 8). This might point to a novel target being responsible for LA-mediated hepatoprotection.



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Fig. 8. Phosphorylation of Akt downstream targets endothelial nitric oxide synthase (eNOS) and Bad. Livers were perfused for 30 min in the absence (Co) or presence of 50 µM LA, which was added 20 min before 60 min ischemia (WI). Western blot analysis (see MATERIALS AND METHODS) was performed detecting phosphorylated eNOS (A), phosphorylated Bad (phospho-Bad), or total Bad (tot-Bad) after 60 min of WI and after 45 or 90 min of R. Because phosphorylated eNOS is located in the membrane, whereas unphosphorylated eNOS (tot-eNOS) resides in the cytoplasm, normalization on tot-eNOS was omitted. One representative Western blot is shown for each time point.

 

LA preconditioning does not alter caspase-3-like activity. Due to the fact that Akt is typically described as an antiapoptotic kinase (9), we determined the impact of LA pretreatment on apoptotic processes. Occurrence of postischemic apoptotic cell death as a marker for the outcome of livers stressed by ischemia and reperfusion is controversially discussed (16, 27, 33). We assessed caspase-3-like activity as an indicator of apoptotic cell death. In fact, we observed elevated caspase-3-like activity after 45 and 90 min of reperfusion, showing the occurrence of apoptosis in our model of IRI. However, LA preconditioning did not significantly affect caspase-3-like activity during ischemia and reperfusion (Fig. 9).



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Fig. 9. Caspase-3-like activities in LA-preconditioned livers. Livers were untreated (Co) or preconditioned with 50 µM LA. Sixty minutes of WI were followed by R for up to 90 min. Caspase-3-like activity was determined as described in MATERIALS AND METHODS. Data are expressed as x-fold caspase-3-like activity of untreated livers after 60 min of WI and show means ± SE of n = 5 experiments. n.s., No statistical significance between untreated and LA-pretreated livers.

 

LA protects against hepatic IRI in vivo. To investigate the in vivo relevance of our observation, we pretreated rats before ischemia with 500 µmol LA. Interestingly, livers of LA-pretreated animals showed significantly reduced serum {alpha}-GST levels at 4 h of reperfusion compared with untreated controls (Fig. 10A). Additionally, LA-preconditioned organs showed a markedly improved liver histology (Fig. 10B). This experiment therefore represents a proof of principle that LA pretreatment represents a promising novel approach in reducing ischemia-reperfusion injury.



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Fig. 10. LA protects against ischemia-reperfusion injury (IRI) in vivo. Livers were untreated (Co) or preconditioned with 500 µmol LA. Ninety minutes of ischemia were followed by R for the indicated times. A: data show serum {alpha}-GST levels at 1 and 4 h of R. The dashed line shows {alpha}-GST levels of sham-treated animals. **P < 0.01 represents significant differences between LA-treated and untreated livers at 4 h of R. B: data show typical histology of sham-treated animals (a) or animals undergoing ischemia and R. Livers after 1 h of R are shown in b (Co) and c (LA). Sections of organs after 7 days of R are shown in d, e (Co), and f (LA).

 


    DISCUSSION
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 DISCLOSURES
 REFERENCES
 
LA is widely used in the therapy of diabetic polyneuropathy. In this context, the substance is documented in numerous clinical trials to be well tolerated (10). Concerning diabetic polyneuropathy, the beneficial effects of LA seem to be partially derived from its anti-oxidative properties. More recently, the findings of Konrad et al. (24) suggested that LA increases glucose uptake by specifically activating molecular signaling cascades of the cell.

This work shows for the first time that preischemic treatment with this well-established antidiabetic drug attenuates hepatic IRI. We also describe here for the first time that the survival kinase Akt is causally involved in protection from IRI. This assumption is based on two findings. First, LA increased phosphorylation of Akt, and second, the PI3-K inhibitor WM abrogated the protective effect of LA preconditioning by preventing activation of Akt. In addition, we observed effects conferred by LA, which should also be discussed in terms of its beneficial role in attenuating IRI.

Preconditioning with LA led to an increased ATP content in liver tissue during ischemia. The potency of LA to increase ATP levels has been reported before in rat heart mitochondria (43). Because ATP depletion occurring during ischemia (15, 21) leads to liver cell damage, the increased energy pool by LA might contribute to the protective effect of LA preconditioning.

Furthermore, LA preconditioning markedly reduced the activation of two pivotal redox-sensitive transcription factors, namely NF-{kappa}B and AP-1. LA has been described before to possess the ability to inhibit NF-{kappa}B binding activity in various cell culture models, supporting our data (22, 34). Activation of transcription factors, such as NF-{kappa}B and AP-1, has been linked to the pathophysiology of IRI by activating inflammatory cascades leading to organ damage (1, 12, 40, 44). Thus inhibition of these proinflammatory mediators might contribute to a protective effect. However, in our study, LA attenuates these transcription factors mainly at late reperfusion time points. Therefore, these mechanisms are most likely not essential for the early protective effect of LA, which is seen already in the first minutes of the reperfusion period. This assumption is confirmed by the fact that WM abrogated LA-mediated tissue protection, whereas it did not abrogate the inhibitory action of LA on NF-{kappa}B and AP-1 (data not shown).

Focusing on early events responsible for the protective effect of LA preconditioning, we investigated the activation of p38 MAPK. This protein kinase is known to be activated within minutes by phosphorylation and has been described as mediating protection in ischemic preconditioning of hearts (39) and livers (32, 35). However, LA did not activate p38 MAPK in our model of IRI. Thus we suggest that LA does not confer its protection via this kinase, although LA has previously been shown to activate p38 MAPK in L6 GLUT4myc myotubes (24). These different observations might represent a cell type-dependent phenomenon.

The most meaningful effect of LA observed in this study is activation of Akt. To our knowledge, no information is available on a potential cytoprotective action of Akt in hepatic IRI. A few reports describe a role for Akt in ischemic preconditioning of hearts (25, 28, 38), and LA has been reported to activate Akt, an effect that was connected to its antihyperglycemic activity (24, 41). We report here for the first time that activation of this survival kinase, as induced by LA pretreatment, is causally involved in attenuating hepatic IRI. Inhibition of PI3-K by WM abrogated the protective effect of LA, indicating that LA-induced activation of Akt is pivotal for LA-mediated hepatoprotection.

Among potential cytoprotective downstream targets of Akt, phosphorylation of Bad (11) or eNOS (19) might play an important role, and the cytoprotective effect of Akt has been mainly attributed to its antiapoptotic potential (9). Interestingly, however, hepatoprotection by LA does not seem to be involved in antiapoptotic processes of our model of IRI, and neither Bad nor eNOS phosphorylation was increased in LA-treated organs. This observation suggests a completely new mechanism by which LA exerts antinecrotic but not antiapoptotic action during hepatic ischemia and reperfusion.

This study shows for the first time that LA has the potential to reduce IRI of the rat liver. Most importantly, the potential clinical relevance of our findings is supported by the fact that LA preconditioning also protects against IRI in vivo. Thus LA preconditioning might represent a new pharmacological approach in attenuating hepatic IRI. Even more importantly, we show that preconditioning with LA is mediated via the PI3-K/Akt kinase pathway. Therefore, our data reveal activation of Akt as a novel target mediating protection from hepatic IRI.


    DISCLOSURES
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 DISCLOSURES
 REFERENCES
 
This work was supported by the Deutsche Forschungsgemeinschaft (FOR 440/1–2: KI 702/2). A. K. Kiemer is a recipient of the "Bayerischer Habilitationsförderpreis."


    ACKNOWLEDGMENTS
 
We thank Dr. C. Wicke, ASTA Medica, Frankfurt am Main, Germany for providing LA and its analysis. The excellent technical assistance of C. Niemann, W. Rödl, R. Socher, B. Weiss, and A. Kraft is gratefully acknowledged. Thanks to Dr. A. Baron for support in liver perfusions, to K. Ladetzki-Baehs for support in Western blots, and Dr. M. Bilzer for helpful discussions.


    FOOTNOTES
 

Address for reprint requests and other correspondence: A. K. Kiemer, Dept. of Pharmacy, Center of Drug Research, Univ. of Munich, Butenandtstr. 5-13, 81377 Munich, Germany (E-mail: Alexandra.Kiemer{at}cup.uni-muenchen.de).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

* C. Müller and F. Dünschede contributed equally to this work. Back


    REFERENCES
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 DISCLOSURES
 REFERENCES
 

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