We investigated whether two kinases critical for survival during periods of energy deficiency in anoxia-intolerant mammalian species, AMP-activated kinase (AMPK), and protein kinase B (AKT), are equally important for hypoxic/anoxic survival in the extremely anoxia-tolerant crucian carp (Carassius carassius). We report that phosphorylation of AMPK and AKT in heart and brain showed small changes after 10 days of severe hypoxia (0.3 mg O2/l at 9°C). In contrast, anoxia exposure (0.01 mg O2/l at 8°C) substantially increased AMPK phosphorylation but decreased AKT phosphorylation in carp heart and brain, indicating activation of AMPK and deactivation of AKT. In agreement, blocking the activity of AMPK in anoxic fish in vivo with 20 mg/kg Compound C resulted in an elevated metabolic rate (as indicated by increased ethanol production) and tended to reduce energy charge. This is the first in vivo experiment with Compound C in a nonmammalian vertebrate, and it appears that AMPK plays a role in mediating anoxic metabolic depression in crucian carp. Real-time RT-PCR analysis of the investigated AMPK subunit revealed that the most likely composition of subunits in the carp heart is α2, β1B, γ2a, whereas a more even expression of subunits was found in the brain. In the heart, expression of the regulatory γ2-subunit increased in the heart during anoxia. In the brain, expression of the α1-, α2-, and γ1-subunits decreased with anoxia exposure, but expression of the γ2-subunit remained constant. Combined, our findings suggest that AMPK and AKT may play important, but opposing roles for hypoxic/anoxic survival in the anoxia-tolerant crucian carp.
anoxia tolerance in vertebrates is limited to a few species, the most extreme being the crucian carp (Carassius carassius) and the freshwater turtles (genera Trachemys, Chrysemys, and Chelydra). These ectothermic animals can survive for months without oxygen at temperatures near 0°C (43, 50). A key to survival in prolonged anoxia is the successful matching of ATP demand to the limited ATP production from anaerobic glycolysis. The anoxia-tolerant species appear to have evolved different strategies to meet this challenge (32). The turtle is strongly metabolically depressed during anoxia, with a drastically suppressed rate of ATP-turnover. Anoxic Carassius also decreases metabolic rate, but to a more moderate extent (62). The anoxic carp also upregulates glycolysis to match ATP supply and demand (33), but has evolved a unique strategy of producing ethanol as the major end product of anaerobic glycolysis (56). Ethanol is released to the surrounding water to avoid severe acidosis (42), and liver glycogen content is likely the limiting factor for anoxic survival (41). Thus, accurate control over energy status is essential for surviving prolonged anoxia.
In anoxia-intolerant mammalian species, AMP-activated kinase (AMPK), has been ascribed a key role in maintaining cellular and systemic energy balance (16, 49, 68). AMPK is activated by AMP and inactivated by ATP (i.e., the ATP-to-AMP ratio), acting as an energy sensor (16). In general, AMPK turns on ATP-producing pathways and decreases ATP-consuming pathways (7), may increase glycolysis (35), increases glucose uptake by the myocardium (52), and slows down energy consuming pathways like protein translation (24).
AMPK is a heterotrimeric protein formed by α-, β-, and γ-subunits where all subunits are required for a functional kinase. In mammals, the catalytic α-subunit has two isoforms (α1 and α2). Both isoforms have a conserved Ser/Thr kinase-domain in the activation loop, which contains a conserved phosphorylation residue at Thr172 essential for controlling AMPK activity (19). Furthermore, the COOH-terminal region of the α-subunit is required for making a complex with the β- and γ-subunits (10). The β-subunit, which serves to connect the α- and the γ-subunits (16) and contains a glycogen binding domain (23, 46), also has two isoforms in mammals (β1 and β2). Interestingly, a glycogen debranching enzyme is associated with the β-subunit and can regulate AMPK activity in mouse skeletal muscle (53). The γ-subunit has three isoforms in mammals (γ1, γ2, and γ3). It contains the AMP/ATP binding domain (16) and has been suggested to contain an internal autoinhibitory sequence (16), where AMP-binding inhibits the autodephosphorylation of AMPK (55). In humans, mutations in the γ2-gene (known as the PRKAG2 gene) are associated with hypertrophic cardiomyopathy and cardiac glycogen overload (1, 15).
Another signaling pathway that has been linked to mammalian cell survival during energy deficiency is the phosphatidylinositol 3-kinases (PI3-Ks) protein kinase B (AKT) pathway (34, 45). AKT is highly conserved in evolution (13) and is important during ischemia-reperfusion injury in both brain and heart (38). A strong protective stimuli against ischemia-reperfusion injury is ischemic preconditioning (39), and blocking AKT abrogates ischemic preconditioning-induced protection (17). AKT is believed to be activated by reactive oxygen species through a PKG-dependent mechanism acting on the mitochondria and the mitochondrial permeability transition pore (11). Furthermore, AKT reduces apoptosis (6) and promotes cell growth through activation of the mammalian activator of rapamycin (mTOR) (34). Interestingly, the mTOR complex is inactivated by AMPK (67), suggesting opposing roles for AKT and AMPK in cell proliferation. Indeed, AKT has been indicated to lower AMPK activity in the mouse heart by inhibiting phosphorylation of the α-subunit (30). Still, activation of both AKT and AMPK are important for maintaining glucose metabolism during oxidative stress in mouse cardiomyocytes (22). As glycolysis is the only substrate for ATP during anoxia, glucose metabolism is essential for anoxic survival in the crucian carp.
Few studies have investigated AMPK and AKT in nonmammalian vertebrates and little is known of their physiological roles. AKT is known to be activated after retinal injury in goldfish (29) and is phosphorylated in the frog (Rana perezi) liver during hypoxia (3). Interestingly, it was recently shown that the total level of AKT was increased in anoxic turtle (Trachemys scripta) brain, and the phosphorylation was higher and lower compared with normoxic control at 1 and 4 h of anoxia, respectively (37). AMPK has been shown to be phosphorylated in the liver of hypoxic frogs (Rana perezi), but not in the liver or skeletal muscle of anoxic wood frogs (Rana sylvatica) (51). As AMPK appears to be a universal energy sensor, balancing ATP production with demand, and AKT seems to play a vital role in cell protection and repair, we hypothesized that these kinases may be important for anoxic survival and subsequent reoxygenation in crucian carp. We, therefore, compared levels of total and phosphorylated AMPK and AKT in hypoxic and anoxic crucian carp heart and brain, probably the most anoxia-sensitive organs in mammals. Recently, we reported that the crucian carp heart pumps normally after 5 days of anoxia (59), and due to the importance of AMPK and AKT after mammalian myocardial ischemia, we wanted to disclose their activation in the carp. Since the regulation of AMPK could be at the transcriptional level or with change in subunit composition, we also examined the expression levels of the multiple AMPK subunits in carp heart and brain following anoxia exposure and reoxygenation using real-time RT-PCR. Moreover, we assessed the physiological actions of AMPK in vivo by blocking AMPK-activity in normoxic and anoxic carp with the reversible pharmacological antagonist Compound C. Whole animal ethanol production (a measure of anoxic metabolic rate), as well as brain [ATP], [ADP], [AMP], and energy charge were compared. Combined, our findings suggest that AMPK and AKT may play important but opposing roles for hypoxia/anoxia survival in the anoxia-tolerant crucian carp.
MATERIAL AND METHODS
Crucian carp were caught in Tjernsrud Pond, Oslo Community, Norway, in June. They were kept in 750-liter tanks continuously supplied with dechlorinated, aerated Oslo tap water. Temperature varied seasonally (ranging from 9 to 6°C between individual series, but constant during each series), but the photoperiod was held constant at 12:12-h light-dark. Fish were fed commercial carp food on a daily basis. All experiments were approved by the Norwegian Animal Health Authority.
Hypoxia/Anoxia Exposures for Western Blot Analysis and Real-Time RT-PCR
Tissues utilized for Western blot analysis and real-time RT-PCR were obtained from carp that had been randomly selected to be exposed in the dark to different oxygen regimens (see below) in a flow-through (0.5–1.0 ml/s), circular, 25-liter polyvinyl chloride tank sealed with an airtight lid. Fish were allowed at least 18 h to acclimate to the tank prior to hypoxia/anoxia exposure. The desired oxygen concentration was obtained by bubbling the incoming water with air or nitrogen in a 1,500-mm Plexiglas column. Oxygen concentrations and temperatures were continuously monitored with an oxygen electrode. After exposure, fish were killed by stunning with a sharp blow to the head, the spinal cord and dorsal aorta were severed, and tissues were sampled. Three exposure series were conducted.
Series 1: 10 days of hypoxia (0.3 mg O2/l) at 9 ± 1°C.
Carp weighing 33 ± 12 g (mean ± SD) were randomized into the following three exposure groups: 1) normoxic controls (N10; n = 7), 2) 10 days of hypoxia (H10; n = 6), and 3) 10 days of hypoxia followed by 10 days of reoxygenation (R10; n = 6).
Brain and heart were excised within 30 and 60 s, respectively, snap frozen in liquid nitrogen, and stored at −80°C. Only Western blot analysis was performed with these tissues.
Series 2: 7 days of anoxia (0.01 mg O2/l) at 8 ± 1°C.
Crucian carp weighing 40 ± 15 g (mean ± SD) were randomized into the following four exposure groups: 1) normoxic controls (N7; n = 13), 2) 1 day of anoxia (A1; n = 13), 3) 7 days of anoxia (A7; n = 13), and 4) 7 days of anoxia followed by 7 days of reoxygenation (R7; n = 12).
Heart, brain, and liver were sampled and stored at −80°C. Hearts and brains from six or seven fish were randomly selected for either Western blot analysis or real-time RT-PCR. Due to technical difficulties, the final inclusion of the brain for Western blot analysis and PCR, respectively, was N7 (n = 7, n = 5), A1 (n = 6, n = 5), A7 (n = 7, n = 6), and R7 (n = 5, n = 4).
Series 3: 7 or 21 days of anoxia (0.01 mg O2/l) at 6 ± 1°C.
Crucian carp weighing 10.7 ± 4.0 g, six individuals in each group (mean ± SD). Four exposure groups: 1) 7 days normoxic controls, 2) 7 days of anoxia, 3) 21 days normoxic control, 4) 21 days of anoxia.
Heart and brain were sampled, but, due to small fish size, only brain proteins were extracted.
In Vivo Compound C Experiment
Compound C (20 mg/ml) was dissolved in 100 mM HCl adjusted to pH 5.5 with NaOH. Thirty-two crucian carp weighing 44 ± 9 g (mean ± SD) were randomized into the following four experimental groups: 1) normoxia sham injected (100 mM HCl adjusted to pH 5.5 with NaOH); 2) normoxia Compound C injected (20 mg/kg), 3) 24 h anoxia sham injected, 4) 24 h anoxia Compound C injected.
Fish were weighed and placed in a polyvinyl chloride aquarium (450 mm × 230 mm × 120 mm divided into 110 mm × 110 mm × 120 mm cells to house individual fish). Each cell could be separately bubbled with either air or N2 to achieve the required normoxic or anoxic conditions. Fish were then allowed a 24-h acclimation period under normoxic conditions before receiving either sham or Compound C injections (∼200 μl ip). One hour following injection, fish of the anoxia exposure treatment groups were made anoxic for 24 h by bubbling their cells with N2 (0 oxygen within 1 h). The oxygen concentration was monitored continuously and did not exceed 0.01 mg O2/1 during the experiment. After 22 h of anoxia, air and N2 bubbling was stopped, and at 24 h, 10-ml water samples were acquired from each cell for the subsequent measurement of water ethanol concentration. Also at 24 h, fish were euthanized as described above, and the brains quickly removed, immediately frozen in liquid nitrogen, and stored at −80°C for later quantification of adenylates with HPLC.
Western Blot Analysis
Frozen heart and brain samples were transferred to an ice-cold lysis buffer containing 210 mM sucrose, 40 mM NaCl, 30 mM HEPES, 5 mM EDTA, 100 μM sodium orthovanadate, 1% Tween-20. In addition, one tablet complete EDTA-free protease inhibitor (Roche) and 250 μl phosphatase inhibitor cocktail 1 were added to 50 ml of extraction buffer (20 mg tissue/ml extraction buffer). The tissue was homogenized using a Polytron PT 1200 homogenizer. Lysates were subsequently centrifuged at 12,000 g and 4°C for 10 min to remove insoluble material. Next, 1% SDS was added to the supernatant, and the samples were vortexed for 15 min at room temperature. The samples were then frozen in liquid nitrogen and stored for later analysis. Protein content was determined by Micro BCA protein assay kit (Pierce, Rockford, IL). Protein (20 μg/lane) was subjected to 10% SDS-PAGE, and the fractionated products were electrophoretically transferred onto a hybond-P membrane (Amersham Biosciences Europe, Freiburg, Germany). The membrane was incubated for 1 h with 5% skimmed milk in Tris-buffered saline (20 mM Trizma-base and 140 mM NaCl) containing 0.1% Tween 20 (TBST) to block nonspecific reactions. The membrane was then incubated over night with primary antibodies both phosphospecific [1:1,000 for AMPK (Thr172) and AKT (Ser473)] and total protein of AMPK (1:1,000) and AKT (1:2,000; Cell Signaling Technology). After being washed with TBST, the membrane was incubated for 1 h with secondary antibody (1:2,500, goat anti-rabbit; Southern Biotech) conjugated to horseradish peroxidase. After being washed, the immunoreactions were visualized by chemiluminescence (ECL+; Amersham Biosciences Europe), and pictures were taken with ImageReader LAS-1,000 (Fujifilm Europe). The densitometry of each band was investigated using ImageQuant (Amersham Biosciences Europe). The membranes were stained with Commasie blue (Bio-Rad Laboratories) and scanned (CanonScan Lide 35), and equal loading was investigated using ImageQuant on the scanned membranes. Membranes with uneven blotting were removed from the analysis.
Due to size constraints on the gels, only 14 individuals can be analyzed at the same time. The first Western blot analysis was done on brains from the N7 and A7 exposure (series 2) groups. Therefore, proteins from the brains in the N7 and A7 groups and the A1 and R7 groups were extracted at different times. Although all extracted protein was remeasured after the second extraction, it is possible that differences may exist between these extractions, which may explain the 50% reduction in total AMPK in the A1 and R7 brain protein groups. The monoclonal antibody recognizes both α1- and α2-subunits, and the gene expression data of these subunits show a similar reduction for the A1 group as the protein data. We have no good explanation of why the total AMPK protein data for the A7 group is higher than the A1 group. It may be that expression changes after 1 day in anoxia, resulting in new proteins. Although at a reduced state, the anoxic crucian carp has de novo synthesis of protein (57). Nonetheless, we have measured the protein amount in our samples several times and obtained the same protein concentration. In addition, total AKT (see Fig. 2D) and total ERK1/2 (data not shown) do not differ in amount of total protein between the N7-A7 and A1-R7 groups, arguing that the amount is similar. As the total level of AMPK does not change after 7 days in series 1 and after 7 and 21 days of anoxia in the series 2, we suggest that the amount of AMPK is unaltered in sustained anoxia and that AMPK activity is regulated through phosphorylation. To avoid such a confounding factor in our analysis, changes in brain AMPK and AKT are expressed as ratios between phosphorylated and total protein kinase (see Fig. 2, C and D). When three groups are compared (hypoxia groups), we express all changes as ratios to avoid loading errors (see Statistics). In the heart, the α2-subunit is dominant, and both the gene expression data and the protein data support an unaltered amount of AMPK. Only phosphorylated protein is reported to reduce the amount of included blots.
Cloning of AMPK Genes
Gene-specific primers for partial cloning of the AMPK subunits α1, α2, β1a, β1b, γ1, γ2a, and γ2b are listed in Table 1, and were designed from zebrafish (Danio rerio) sequences using Primer3 (http://frodo.wi.mit.edu/primer3/input.htm). Zebrafish sequences were retrieved using the NCBI Homepage (www.ncbi.nlm.nih.gov/) or the Ensembl Genome Browser (www.ensembl.org/index.html). Sequence alignments were performed using GeneDoc (version 22.214.171.124, www.psc.edu/biomed/genedoc/) and ClustalX (version 1.81, ftp://ftp-igbmc.u-strasbg.fr/pub/ClustalX/).
Total RNA was extracted from untreated crucian carp brain tissue using Trizol reagent (Invitrogen, Carlsbad, CA), whereupon the quality and quantity was assessed using a 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA) and an ND-1000 UV-Vis Spectrophotometer (NanoDrop Technologies, Rockland, DE).
For EST cloning, 1 μg of total RNA was DNase I-treated (Sigma-Aldrich, St. Louis, MO) and reverse transcribed using oligo(dT)18 and Superscript III in reaction volumes of 20 μl (Invitrogen). PCR was performed on 1/25 dilutions of the resulting cDNA using Platinum Taq (Invitrogen). The following PCR program was used 1) 94°C for 10 min, 2) 94°C for 30 s, 3) 50°C for 1 min, 4) 72°C for 1 min, 5) repeat steps 2–4 44x, 6) 72°C for 10 min, and 7) hold at 4°C. The resulting PCR products were cloned using pGEM-T Easy Vector System I (Promega, Madison, WI) and CaCl2-competent cells (TOP10 F′; Invitrogen). Positive clones were checked for inserts of correct size using PCR and were sequenced using T7 primers (ABI-lab, University of Oslo, Oslo, Norway). All procedures were carried out according to each manufacturer's protocol.
RNA Extraction and cDNA Synthesis for Real-Time RT-PCR
Total RNA for real-time RT-PCR experiments was extracted from 42 mg brain tissue and 5 mg heart tissue using 15 μl Trizol per milligram. The extractions were performed in accordance with the protocol previously outlined (12), adding 20 pg external RNA control gene (MW2060) per milligram tissue. The quality and quantity of the total RNA was assessed as described for the cloning.
One microgram total RNA was DNase I-treated and reverse-transcribed using oligo(dT)18 and Superscript III, as previously described. For each fish, duplicate cDNA syntheses were performed. Negative RT controls were performed as random checks on ∼10% of the samples. cDNA reactions were diluted to 1:25 using autoclaved MilliQ water. All procedures were carried out according to each manufacturer's protocol.
Real-time RT-PCR was performed on a Lightcycler 2.0 (Roche Diagnostics, Basel, Switzerland). Calculations of normalized levels of gene expression were performed using Eq. 1 (1) where Tar is target gene, Con is control gene (MW2060), E is priming efficiency, and Cp is crossing point. MW2060 was used as an RNA control gene in all real-time RT-PCR data sets. E values were calculated for each real-time RT-PCR reaction using LinRegPCR software (48), but in the final calculations, average priming efficiencies (Emean) were used, calculated separately for each primer pair and based on all real-time RT-PCR reactions. Cp values were obtained for each reaction using the Lightcycler 2.0 software and were defined as the second derivative maximum. Real-time RT-PCR primers were designed using Primer3 and are presented in Table 1.
All real-time RT-PCR reactions were performed with reaction volumes of 10 μl, using Lightcycler Faststart DNA MasterPLUS SYBR Green I (Roche Diagnostics, Basel, Switzerland) and Lightcycler Capillaries (Roche Diagnostics). Five microliters of 1:25-diluted cDNA were used as the template (prepared as described for the cloning), and the following real-time RT-PCR program was used 1) 94°C for 10 min, 2) 94°C for 10 s, 3) 60°C for 12 s, 4) 72°C for 8 s, 5) repeat steps 2–4 39×. Two real-time RT-PCR reactions were performed for each gene for each cDNA synthesis. Since two cDNA syntheses were performed for each total RNA sample, a total of four real-time RT-PCR reactions were performed on each gene for each sample of total RNA. The mean value of all four reactions was used in the gene expression analyses. All real-time RT-PCR reactions were performed using primer concentrations of 100 nM and all primer pairs were found to amplify the desired cDNA species. The latter was verified by melting curve analyses (Lightcycler 2.0 software), gel electrophoresis, and cloning/sequencing. Primer efficiencies and average Cp values for the different primer pairs are summarized in Table 1. It should be noted that three primer pairs were tested for each gene to find primers that worked well with the outlined real-time RT-PCR protocol (primer concentration of 100 nM and annealing temperature of 60°C). The primer pairs that displayed distinct melting curves and the highest Cp values were chosen. This was done as an alternative to optimization of primer concentrations/annealing temperatures. All procedures were carried out according to the manufacturer's protocol.
Frozen heart and brain tissue were weighed (30 ± 8 mg and 132 ± 19 mg, respectively) and homogenized in 2.5 ml 6% perchloric acid. After centrifugation at 15,000 g for 10 min, 1 ml of the supernatant was neutralized with 0.75 ml of 0.85 M K2CO3 and centrifuged again as described above. Contents of ATP, ADP, and AMP were analyzed as described by Van der Boon et al. (63). The HPLC apparatus consisted of a reversed-phase column (4 mm × 120 mm, Nucleosil 120, C18 3 μm; Machery-Nagel, Düren, Germany), a SpectroMonitor 4100 detector, and a ConstaMetric III pump (both from LDC Analytical, Riviera Beach, FL). The mobile phase buffer contained 215 mM NaH2PO4, 2.5 mM tetrabutylammonium bromide (pH 6.25), and 1.5% acetonitrile. The energy charge was calculated as ([ATP] + 0.5 [ADP]) / ([ATP] + [ADP] + [AMP]) (26).
Water ethanol was measured enzymatically using alcohol dehydrogenase (ADH) (29). Briefly, 200 μl water samples were added to a 1.5 ml cuvette containing 1 ml distilled water, 90 units of ADH, and 300 μl of reaction mixture (1 M Tris buffer with 0.3 mM EDTA and 6.25 mM NAD+; pH 9.5). The absorbance was measured at 340 nm before and after the reaction had stopped. [Ethanol] was calculated from the amount of NADH produced using a millimolar extinction coefficient of 6.22. Ethanol excretion is presented as micromole ethanol per gram of fish.
Drugs and Chemicals
Unless otherwise stated, chemicals were purchased from Sigma.
Western Blot Analysis
Four group comparison (anoxic heart).
Each gel was loaded with three individuals from each group and normalized to the mean of the control group (N7). The change in intensity for each sample was calculated for each sample in percent compared with the mean of the control group. The setup and calculations were performed twice giving a total of six biological replicates per group. Due to differences in average intensity between blots, it is not possible to calculate ratios between total protein and phosphospecific densitometry (see below). Hence only phosphorylated protein kinases were analyzed when four groups were loaded on each gel. However, total protein kinase blots were performed with the same premade sample buffer against both AKT and AMPK to ensure even protein concentration and to check whether total kinase levels changed with exposure. Statistics were performed using a Kruskal-Wallis test with a Dunn posttest (all columns vs. control, N7).
Two-group comparison (all groups in hypoxia and anoxic brain).
Each gel was loaded with six or seven hearts from each group and this was done twice for each comparison to allow immunoblotting against both total and phosphospecific antibodies. The sample buffer was prepared for both gels to avoid errors during sample preparation. The arbitrary unit from the densitometry was then calculated as a ratio between the phosphorylated and nonphosphorylated form of each protein kinase for each sample. This ratio was normalized by averaging the control group and set to zero. Changes in each heart are expressed as change compared with the mean from the control group (N7). Statistical differences between the three groups were calculated using a Kruskal-Wallis test with a Dunn posttest.
The expression of the different subunits was normalized to the control group (N7) and set to zero. Statistical analysis with Kruskal-Wallis and a Dunn posttest (all columns vs. control, N7).
Phosphorylated Adenylates and Ethanol Production
To investigate differences between individual groups for the phosphorylated adenylates a one-way ANOVA with a Dunn posttest was performed (reported in Fig. 6). To reveal the effect of Compound C in anoxia, a two-way ANOVA with a Bonferroni posttest was performed (see results). A similar approach was used for evaluation of ethanol production between groups.
AMPK phosphorylation in heart was not significantly affected by 10 days of hypoxia (0.3 mg O2/1), although it tended to increase (series 1, Fig. 1A). AKT phosphorylation was not affected by hypoxia exposure (Fig. 1B). However, after 10 days of reoxygenation, AMPK phosphorylation in heart was significantly reduced compared with the hypoxic level, and AKT phosphorylation was increased compared with both the hypoxic and control level by 154 ± 3% (Fig. 1, A and B). There were no changes in total protein kinase for AMPK or AKT among exposure groups (Fig. 1, A and B).
No change in AMPK phosphorylation occurred in the brain after 10 days of hypoxia, but there was a dephosphorylation after 10 days of reoxygenation compared with both normoxic and hypoxic fish (series 1, Fig. 1C). There was no significant change in phosphorylation of brain AKT with hypoxia exposure or reoxygenation.
Anoxia induced a large increase in phosphorylation of AMPK (by 202 ± 54% at 1 day of anoxia and by 177 ± 32% at 7 days of anoxia, series 2, Fig. 2A). After reoxygenation for 7 days, AMPK phosphorylation had returned to preanoxic levels (Fig. 2A). AKT phosphorylation was not affected by 1 day of anoxia, but by 7 days of anoxia, it was significantly dephosphorylated (−20 ± 11%). AKT remained dephosphorylated after 7 days of reoxygenation (−30 ± 11%) (Fig. 2B). There were no significant changes in total kinase in the heart.
As exemplified with the blot picture in Fig. 2C we found a reduction of total AMPK levels after 1 day of anoxia (−49 ± 4%, P < 0.01, data not shown) and reoxygenation (−65 ± 5%, P < 0.01, data not shown) compared with normoxic controls. Still, the pattern of AMPK phosphorylation in anoxia (expressed as ratio of phosphorylated to total protein kinase) was similar to the heart. Thus, AMPK phosphorylation increased by 419 ± 104% after 1 day of anoxia and by 204 ± 57% after 7 days of anoxia (series 2, Fig. 2C). However, unlike the heart, where AMPK phosphorylation returned to control normoxic levels after 7 days of reoxygenation, the degree of AMPK phosphorylation remained elevated in reoxygenated brains (Fig. 2C). In the long-term anoxia exposure series (series 3) no changes in total brain AMPK protein were observed after 7 or 21 days of anoxia. Yet, increased phosphorylation of brain AMPK was still observed. Specifically, brain AMPK phosphorylation was increased by 67 ± 9% (P < 0.001) after 7 days of anoxia and increased further to 100 ± 28% (P < 0.001) after 21 days of anoxia (data not shown).
No differences in total brain AKT were observed among any of the experimental groups, but we found that AKT phosphorylation decreased by −43 ± 28% after 7 days of anoxia and by −24 ± 6% with reoxygenation (series 2, Fig. 2D). In series 3, the decrease in AKT phosphorylation was similar (−30 ± 10% and −32 ± 18% after 7 and 21 days of anoxia days, respectively, P < 0.01; data not shown).
A pilot analysis was done on proteins extracted from liver samples from the same fish in series 1. Only phosphorylated proteins were investigated, and they revealed similar increases in AMPK phosphorylation after 1 and 7 days of anoxia, respectively (by 239 ± 157% and 219 ± 83%, P < 0.05, data not shown). Mirroring this increase was a dephosphorylation of AKT (−53 ± 47% and −74 ± 5% in the same groups, P < 0.05).
Normoxic and Anoxic Gene Expression of AMPK Subunits
When compared with mammalian sequences, crucian carp AMPK subunits showed high degrees of conservation. For example, the catalytic α1- and α2-subunits showed 100% amino acid conservation with mammalian species in the activation loops, indicating preserved functions. For β1- and γ2-subunits, two paralogous variants were found. These paralogs showed 91% and 95% similarity, and were treated as separate genes during real-time RT-PCR analyses.
The mRNA levels suggested differences in the subunit's composition between heart and brain tissue. In heart, α2 dominated over α1 by 10:1, whereas in brain, α1 and α2 showed similar levels of expression, (series 2, Fig. 3). With anoxia, only the α1-subunit showed lowered expression in the heart (α1; −52 ± 14%, Fig. 4A), whereas the two α-subunits displayed similar lowering of expression in brain (α1; −54 ± 23% and −52 ± 10% after 1 and 7 days of anoxia, respectively, Fig. 5, A and B). In reoxygenated hearts, the α-subunit expression returned to preanoxic levels, whereas in reoxygenated brains, it remained reduced (−55 ± 25% and −33 ± 37% for the α1-and the α2-isoform, respectively, Fig. 5A and B).
The β1b-paralog dominated over the β1a-paralog in heart tissue (by 25:1), whereas both β-subunits showed similar expression levels in brain (series 2, Fig. 3). In heart, the expression of β1a and β1b were lowered by −54.5 ± 8.2% and 49.2 ± 7.0% after 7 days of anoxia, respectively (Fig. 4, C and D), and the expression of β1b tended to increase in the reoxygenation group (Fig. 4D). In brain, the expression of the two β1-paralogs did not change significantly in response to anoxia, although they tended to be reduced (Fig. 5, C and D).
The γ2a-subunit was more expressed than the γ2b-subunit in heart tissue (by 5:1), whereas they showed similar levels of expression in brain tissue (series 2, Fig. 3). In the anoxic heart, there was an increase in both γ2a and γ2b after 7-days anoxia (by 58 ± 54% and 194 ± 76%, respectively, Fig. 4, F and G). The γ1-subunit tended to be reduced after 1-day anoxia in brain (−37 ± 21%) and was significantly reduced after 7-days anoxia (−59 ± 9%) (Fig. 5E).
Given the present data, the most likely subunit composition in the crucian carp heart is α2, β1B, and γ2a. No obvious subunit composition can be given for the brain (Fig. 5).
Effect of Compound C on Brain Phosphorylated Adenylates
A two-way ANOVA analysis of the data (with anoxia and Compound C as the independent variables) showed that Compound C had a suppressing effect on brain [ATP] and energy charge (P < 0.05) and anoxia increased [ADP] and [AMP] (series 4, two-way ANOVA, P < 0.05). The statistical results of a one-way ANOVA analysis are shown in Fig. 6. This analysis revealed that anoxia reduced energy charge in both sham- and Compound C-treated groups compared with normoxic control (P < 0.01 and P < 0.001, respectively). Moreover, AMP was higher in the brain of anoxic Compound C-treated fish (0.22 ± 0.10 μmol/g) compared with normoxic control (0.13 ± 0.06 μmol/g; P < 0.05).
Effect of Compound C on Ethanol Production
Compound C significantly increased the ethanol production of anoxic carp (series 4). After 24 h of anoxia, sham-injected carp had excreted 7.1 ± 1.2 μmol ethanol/g, whereas Compound C-injected fish excreted 9.4 ± 0.9 μmol ethanol/g (P < 0.05; two-way ANOVA, Bonferroni posttest). Normoxic sham- and Compound C-injected fish excreted < 0.04 μmol ethanol/g.
We found a large increase in phosphorylation of the energy-sensitive AMPK in crucian carp heart and brain during anoxia. This was evident already after 1 day of anoxia and the phosphorylation level was equally high after 21 days of anoxia in the brain. Unlike anoxia, prolonged severe hypoxia did not significantly affect the phosphorylation status of AMPK. Moreover, we have identified and partially cloned different subunits of AMPK, including two α-subunits (α1 and α2), two paralogs of the β1-subunit (β1a and β1b), and three γ-subunits (γ1, γ2a, and γ2b). The expression of these subunits varied between heart and brain, but also between different anoxic regimens. Blocking AMPK with Compound C increased ethanol production in anoxia, indicating a role of AMPK in suppressing metabolic rate. In contrast to the strong phosphorylation of AMPK, AKT was dephosphorylated during anoxia in the brain and tended to be reduced in the anoxic heart.
Severe hypoxia did not significantly change phosphorylation of either AMPK or AKT. In nature, hypoxia occurs prior to anoxia, and we could, therefore, expect molecular changes in kinase phosphorylation in hypoxia, especially for an energy sensor like AMPK. However, as the crucian carp has evolved to deal with anoxia, these mechanisms might not be activated before oxygen is totally absent. Indeed it has been shown that the large glycogen stores in the liver are not mobilized by starvation but by anoxia (41). It is, therefore, interesting to note that the AMPK phosphorylation is high already after 1 day of anoxia but shows no significant increase at hypoxic conditions that would kill other fish species.
AMP has been shown to allosterically change AMPK, decreasing the autodephosphorylation (55). We observed a small increase in [AMP] in crucian carp brain during anoxia, which is in agreement with previous findings (26). The reported [AMP], in which 50% of the AMPK is activated in rat hearts, is between 2 and 30 μM (14). It is, therefore, likely that the observed increase in [AMP] may activate AMPK in the crucian carp. Although deprived of oxygen, the crucian carp maintains energy balance in brain (26, 64) and heart (64) and can keep a normoxic cardiac output during at least 5 days of anoxia (59). Our results indicate a role for AMPK in maintaining energy balance in anoxic crucian carp. In isolated rat myocytes, AMPK increased glycolysis by activating 6-phosphofructo-2-kinase (35) and decreased glycogen synthesis in skeletal muscle (27). During anoxia, the crucian carp is solely dependent on glycolysis for ATP production, fueled by glycogen breakdown in the liver (41, 43). Interestingly the β-subunit of AMPK is associated with a glycogen debranching enzyme in rats (53). As AMPK phosphorylation showed a twofold increase in the liver of anoxic crucian carp, it is tempting to speculate that this kinase is important in glycogen debranching also in the fish.
Blocking AMPK with the reversible antagonist Compound C increased ethanol production, the end product of anaerobic metabolism in the crucian carp (40). This indicates that AMPK suppressed energy consumption during anoxia. Both anoxia and Compound C decreased brain energy charge, and the lowest level was seen in anoxic fish treated with Compound C, supporting a role for AMPK in maintaining energy charge. To our knowledge, this is the first use of Compound C in a nonmammalian in vivo system. Our dose of Compound C (20 mg/kg) injected intraperitoneally reduces AMPK phosphorylation in rat brains after middle cerebral artery occlusion (36), and 12 μM blocks AMPK in isolated perfused mouse hearts (25). AMPK subunits have been conserved during the course of evolution (16), a notion that is supported by the partial clones obtained in this study. They typically showed > 90% amino acid conservation compared with mammals, and it is, therefore, likely that the observed effect of Compound C in the crucian carp is caused by AMPK inhibition. However, as with other pharmacological interventions, we cannot rule out any unspecific effects of Compound C in the crucian carp.
Based on the gene expression profiles obtained in this study, the most likely subunit composition in the crucian carp heart is α2, β1B, and γ2a. The α2-subunit dominates in the rat heart, whereas the γ2 is slightly more expressed than the γ1 (31). The same study found that ischemia phosphorylates both isoforms of these subunits in rat heart, although the α2 more than the α1. It has also been shown that exercise activates the α2 more than α1 in rat skeletal muscle (9), possibly due to greater sensitivity to [AMP] (54). Compared with the subunit expression observed in the crucian carp heart, the brain showed a more even expression of the different subunits. In the mouse brain, the α1 and γ1 dominate (61), whereas in the chicken brain the distribution of subunits is similar to that of crucian carp (47).
Expression of the γ2-subunit, which holds the AMP/ATP binding site and acts as a regulator of the kinase (8), increased in the anoxic heart. A mutation in human gene PRKAG2, encoding the γ2-subunit results in massive glycogen building and cardiac hypertrophy (1, 15). Interestingly, overexpressing the PRKAG2 gene in mice protects against ischemia reperfusion damage, probably as an effect of increased consumption of glycogen and increased glycolysis during ischemia (44). The crucian carp has seasonal variation in the glycogen content in both heart and brain (65, 66), and it is likely that these stores contribute to anoxic survival in addition to the large liver glycogen store (41). To access the circulating glucose, AMPK may increase uptake by translocation of glucose transporters (2, 60). Moreover, activation of AMPK may be a common mechanism for insulin-independent glucose transport in skeletal muscle during metabolic stress (20). The ability of insulin to induce redistribution of GLUT4 is abolished by wortmannin, an inhibitor of AKT (21). AMPK has also been shown to increase myocardial glucose uptake independent of the insulin-AKT pathway (52).
One important function of AMPK is to slow down energy-consuming pathways (7, 16) and it also inhibits protein translation (67). If AMPK acts as a brake, the survival kinase AKT acts as an accelerator for cell repair, growth, and proliferation and as a regulator of metabolism in mammals (34, 38). AKT is also activated after ischemia-reperfusion injury in the heart (18) and brain (38), and blocking AKT abolishes the protective effect of ischemic preconditioning in the heart (17). Moreover, AKT seems to be transiently phosphorylated in the initial phase of anoxia in warm-acclimated turtles (37). The observed reduction in AKT phosphorylation during long-term anoxia in the crucian carp may seem counterintuitive given its role as an important survival kinase in ischemic mammalian systems. However, stimulation of cell growth and proliferation is energy consuming and is probably not beneficial for an animal adapted for long-term survival in anoxia. Another point is that one of the triggers of AKT phosphorylation is believed to be reactive oxygen species (11), and they might not be available during anoxia. One of the targets of AKT in mammals is mTOR via inhibition of the tuberous sclerosis complex (TSC1)/TSC2 complex (34, 38), a complex that AMPK activates (24). TSC2 plays an essential role in controlling cell size in response to energy limitation and to protect cells from glucose deprivation-induced apoptosis (24). Moreover, AMPK-dependent phosphorylation of TSC2 is important for TSC2 function in cell size regulation and cell survival under conditions of energy starvation (24). There is also emerging evidence for a direct crosstalk between AKT and AMPK. In myeloma cells (4) and immortalized hippocampal neurons (28), AMPK can inhibit AKT. Moreover, there is evidence that AKT negatively regulates phosphorylation of AMPK (30). A possible mechanism is that AKT directly phosphorylates AMPK α1/α2 at Ser485-491 and that this can prevent activation at Thr172 (58). In contrast, it has been shown that AMPK can positively regulate AKT and stimulate glucose uptake in isolated rat cardiomyocytes (5). More studies are clearly needed to disclose the interactions between AMPK and AKT in brain and heart physiology.
The anoxic crucian carp heart and brain are good models for studying adaptive mechanisms, allowing survival without oxygen and counteracting subsequent reoxygenation injury. In this study, we found that the metabolic sensor AMPK was strongly phosphorylated in anoxic crucian carp heart and brain, whereas there was a dephosphorylation of AKT. Blocking AMPK in anoxic crucian carp increased ethanol production, indicating increased metabolic rate. AMPK and AKT may have opposing effects during anoxia, since AMPK mainly work to restore energy balance, whereas AKT has its central role in cell growth and repair, which may have to be suppressed to save energy during long-term anoxia. The increased ethanol production and reduced energy charge during AMPK blockade and the strong sustained phosphorylation of AMPK both in short- and long-term anoxia indicate a role for this kinase in the anoxic survival strategy of crucian carp.
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