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ATP-consuming processes in hepatocytes of river lamprey Lampetra fluviatilis on the course of prespawning starvation
Comparative Biochemistry and Physiology, Part A 201 (2016) 95–100
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ATP-consuming processes in hepatocytes of river lamprey Lampetra fluviatilis on the course of prespawning starvation Natalia I. Agalakova ⁎, Irina V. Brailovskaya, Svetlana A. Konovalova, Sergei M. Korotkov, Elena A. Lavrova, Anatolii A. Nikiforov Sechenov Institute of Evolutionary Physiology and Biochemistry, Russian Academy of Sciences, 44 Thorez av., Sankt-Petersburg 194223, Russia
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Article history: Received 23 November 2015 Received in revised form 5 July 2016 Accepted 5 July 2016 Available online 9 July 2016 Keywords: Lamprey Hepatocytes Prespawning starvation Metabolic depression Mitochondrial membrane potential Protein synthesis Na+-K+-pump Aminotransferases
a b s t r a c t The work was performed to establish which of the major ATP-consuming processes is the most important for surviving of hepatocytes of female lampreys on the course of prespawning starvation. The requirements of protein synthesis and Na+-K+-ATPase for ATP in the cells were monitored by the changes in mitochondrial membrane potential (MMP) in the presence of corresponding inhibitors from the peak of metabolic depression (January– February) to the time of recovery from it (March–April) and spawning (May). Integrity of lamprey liver cells was estimated by catalytic activities of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in blood plasma. In January–February, the share of ATP necessary for protein synthesis was 20–22%, whereas before spawning it decreased to 8–11%. Functioning of Na+-K+-pump required 22% of cellular ATP at the peak of metabolic depression, but 38% and 62% of ATP in March–April and May, respectively. Progression of prespawning period was accompanied by 3.75- and 1.6-fold rise of ALT and AST activities in blood plasma, respectively, whereas de Ritis coefficient decreased from 2.51 ± 0.34 to 0.81 ± 0.08, what indicates severe damage of hepatocyte membranes. Thus, the adaptive strategy of lamprey hepatocytes to develop metabolic depression under conditions of energy limitation is the selective production of proteins necessary for spawning, most probably vitellogenins. As spawning approaches, the maintenance of transmembrane ion gradients, membrane potential and cell volume to prevent premature cell death becomes the priority cell function. © 2016 Elsevier Inc. All rights reserved.
1. Introduction Many vertebrate species of different phylogenetic levels, which living cycle includes seasonal oxygen deprivation, hypothermia, hibernation, aestivation, anhydrobiosis, etc., are able to survive the natural phenomenon of reversible metabolic depression (Boutilier, 2001; Hochachka and Lutz, 2001; Storey and Storey, 2004). Although the defense strategies are divergent and broad array of adaptations were evolved by different species, a key mechanism of survival is an ability to fundamentally suppress ATP utilization and O2 demand to such hypometabolic state which balances the reduced capacity of cells to produce ATP. The intensity of ATP-demanding processes such as biosynthesis, protein turnover, membrane ion transport and secretion are reduced or almost suspended, leading to overall reduction in ATP consumption. Among the species able to reorganize their cellular metabolic rate, lamprey Lampetra fluviatilis is a unique example. Anadromous migration of these monocyclic animals is accompanied by prolonged
⁎ Corresponding author. E-mail address: [email protected] (N.I. Agalakova).
http://dx.doi.org/10.1016/j.cbpa.2016.07.002 1095-6433/© 2016 Elsevier Inc. All rights reserved.
(7–8 months) synchonia, genetically programmed and hormonally regulated behavior phenomenon of natural starvation (Larsen, 1980). The digestive system is atrophied, and the vitality of lampreys during all phases of prespawning period is maintained due to mobilization of glycogen, lipids and proteins mainly from muscle tissue (Emelyanova et al., 2004; Emel'yanova et al., 2007). However, the supply of hepatocytes with substrates is limited, because blood enters the liver through portal vein which undergoes degenerative changes. Hepatocyte metabolism is exclusively aerobic and based on oxidation of fatty acids, but the lipid drops accumulated in hepatocytes are not used until spring (Savina and Gamper, 1998; Gamper and Savina, 2000; Emel'yanova et al., 2007). As a result, to the middle of winter the hepatocytes develop deep metabolic depression, with endogenous respiration rate, oxidative phosphorylation and ATP content exhibiting considerable decline in comparison to values observed at the beginning of spawning migration (Savina and Gamper, 1998; Gamper and Savina, 2000; Gamper et al., 2001). When the spawning approaches, hepatocyte metabolism is activated again, the rates of endogenous respiration and ATP synthesis are restored and almost reach autumn values. In spite of metabolic depression, liver cells of female lampreys synthesize vitellogenins, precursors of the yolk proteins, which titer in the blood was confirmed to be constant throughout the spawning period (Mewes et al., 2002).
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Therefore, the strategy of lamprey hepatocytes to develop metabolic depression is not associated with unfavorable environmental factors (hypoxia or hypothermia), but serves to preserve energy resources for spawning. The goal of present work was to reveal the cellular processes being the most important for surviving of lamprey hepatocyte until spawning under conditions of energy limitation induced by prespawning starvation. For this purpose, we made an attempt to evaluate the sensitivity of major ATP-consuming processes to seasonal decrease in ATP production. In the cells of mammals and some other studied vertebrate species, major ATP-demanding processes under standard metabolic rate are protein synthesis (consuming 24–30% of ATP coupled to oxygen consumption) and activity of Na+-K+-ATPase (19–28%). The remaining is spent by Ca2+-ATPase activity, gluconeogenesis, protein degradation, ureagenesis, mitochondrial proton leak, mRNA synthesis, etc. (Buttgereit and Brand, 1995; Hochachka et al., 1996; Rolfe and Brown, 1997; Wieser and Krumschnabel, 2001). Biosynthesis of proteins and activity of sodium pump are also most sensitive to energy deprivation. Taking this into account, we monitored the requirement of protein synthesis and Na+-K+-pump in lamprey hepatocytes for ATP on the course of prespawning starvation, from the peak of metabolic depression (January–February) to the time of recovery from it (March–April) and spawning (May). We also evaluated the possible gluconeogenic capacity of liver cells at different energy states. Next, we measured the catalytic activities of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in lamprey blood plasma to monitor the integrity of liver cells at different seasons of prespawning period. The share of ATP necessary for the maintenance of examined cellular processes were evaluated by the rate of their inhibition measured as the changes in potential on inner mitochondrial membrane - MMP (Δψm). MMP is a key parameter controlling mitochondrial respiration and ATP synthesis, exerting a significant effect on the maximal ATPgenerating capacity. MMP accounts for the majority of the protonmotive driving force, which is in turn the central intermediate of aerobic energy production. Resting MMP reflects the balance between energy supply and ATP consumption. The study was based on two assumptions. First, acute inhibition of individual ATP-consuming processes causes a consequential drop in ATP demand and triggers a proportional drop in MMP due to decrease in the rate of electron transport by respiratory chain after administration of inhibitor. Second, an inhibition of one ATP-demanding process does not alter the rates of other processes. Since alterations of MMP values in lamprey hepatocytes on the course of prespawning starvation completely follows the dynamics of ATP production (Zubatkina et al., 2011; Savina et al., 2011), each of these parameters can be reliable indicator of cell energy state and enables studying the relationships between energy metabolism and activities of cellular processes requiring ATP. 2. Material and methods 2.1. Animals Lampreys (Lamperta fluviatilis L.) were caught in the Neva river in November–December after the beginning of their prespawning migration and maintained until May in the tanks with dechlorinated and permanently aerated water at 2–6 °C. The experiments were performed on females. 2.2. Solutions and chemicals The standard medium used for isolation and incubation of hepatocytes contained (mM): 137 NaCl, 5 KCl, 0.8 MgSO4, 0.44 KH2PO4, 0.33 Na2HPO4, 5 NaHCO3, 10 HEPES, 5 glucose, pH 7.6. All buffer salts, cycloheximide, emetine hydrochloride, sodium phenylpyruvate, alpha-cyano-4-hydroxycinnamate, ouabain, collagenase (type VIII), HEPES, FCCP (carbonyl cyanide-4-(trifluoromethoxy)
phenylhydrazone), DMSO (dimethyl sulfoxide) were purchased from Sigma (USA). FCCP was dissolved in ethanol, emetine and sodium phenylpyruvate - in distilled water. Tetramethyl rhodamine methyl ester (TMRM) was from Fluka. Stock solution of TMRM was prepared on DMSO, aliquoted and stored at −20 °C in the dark.
2.3. Preparation of hepatocytes Hepatocytes were isolated by a modified collagenase method (Mommsen et al., 1994). The liver was perfused for 2–3 min in situ with the standard medium through the ventral hepatic vein, then carefully minced with a razor blade and incubated for 1 h at room temperature in the same medium supplemented with 0.1% collagenase type VIII. After incubation, the digested tissue was passed through nylon mesh and the cells were washed three times by centrifugation in the standard medium supplemented with 1.5 mM CaCl2 and 0.5% bovine serum albumin (BSA). The final cell pellet was suspended in the same medium at concentration of 4–6 millions of cells per ml and stored on ice. Cell viability of hepatocytes, evaluated by Trypan blue, was 90–95%.
2.4. Determination of mitochondrial membrane potential Mitochondrial membrane potential was determined by flow cytometry using fluorescent dye tetramethyl rhodamine methyl ester (TMRM). TMRM is a lipophilic cationic compound accumulating in the mitochondrial membranes space in inverse proportion to mitochondrial membrane potential according to the Nernst equation. The hepatocytes were incubated for 30 min in the standard medium with tested inhibitors at room temperature and then loaded for 30 min with TMRM (500 nM) in the dark. After loading the cells were washed twice by centrifugation for 3 min at 1000g with fresh medium. Because TMRM is rapidly lost from the cells in the absence of external probe, the hepatocytes were placed in incubation medium containing 50 nM TMRM to allow continuous reequilibration of fluorophore across the plasma membrane and immediately subjected for flow cytometry. An analysis of orange TMRM fluorescence was performed on EPICS-XL (Beckman Coulter, USA) at wavelength of 575 ± 15 nm. The intensity of TMRM fluorescence was set on logarithmic scale. The data were first plotted using SSC (side scatter) vs FSC (forward scatter) to reveal cell size and complexity, and the gate was applied to isolate the population of cells with normal volume and remove the debris along the axes. Then the data were re-plotted using FSC and TMRM and the gate was used to remove dying cells with low TMRM fluorescence. Finally, the data were collected and gated so that only hepatocytes with high TMRM fluorescence were used for analysis. 30,000 events were collected for each sample. Mean fluorescence intensity in arbitrary units obtained for control samples (without inhibitors) in each experiment were taken as 100%. The fluorescence of samples in the presence of inhibitors was expressed as the percentage of control. According to the Nernst equation, intracellular distribution of cationic mitochondrial probe (TMRM) reflects the differences in the transmembrane potential across both the plasma membrane and inner mitochondrial membrane, i.e., besides mitochondria, TMRM is accumulated in the cytoplasm as well. The specificity of TMRM fluorescence for MMP was assessed using protonophore FCCP. FCCP completely depolarized the mitochondria, thus causing a collapse in MMP and, respectively, loss of TMRM signal. After determination of TMRM fluorescence, 40 μM FCCP was added to each sample, and the fluorescence was determined again in 10 min. TMRM fluorescence in the presence of FCCP (negative control) was quite similar in all samples and did not exceed 14–18 arbitrary units, whereas without FCCP it was 200– 300 units. For analysis, TMRM signal in the presence of FCCP was subtracted from the total TMRM signal in the sample.
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2.5. Determination of ALT and AST activity The activities of ALT and AST in lamprey blood plasma were determined by enzymatic reactions using commercial colorimetric assay kits (Sigma). 2.6. Statistics The obtained results were processed by SigmaPlot software package version 11.0 (Jandel Scientific). The statistical differences were assessed using one-way ANOVA for many groups followed by Tukey's test. The level of significance was established at p ≤ 0.05. The data are presented as arithmetic means ± SE. 3. Results 3.1. Effects of protein synthesis inhibitors on MMP in lamprey hepatocytes on the course of prespawning starvation The metabolic cost of protein synthesis in lamprey hepatocytes on the course of prespawning starvation was evaluated using two distinct inhibitors of protein synthesis, cycloheximide (CHX) and emetine (EME), at the doses of 10-fold concentration range to reveal the minimal saturating concentration. Treatment of the cells with 1 μM CHX at all three seasons examined caused similar ~7% decrease in the intensity of TMRM fluorescence (Fig. 1). However, inhibitory potential of higher CHX concentrations depended on the time of prespawning starvation. During January– February the effect of CHX was clearly concentration-dependent, with 10 and 100 μM showing ~18 and 20% decrease, respectively, being statistically significant in comparison to 1 μM. At other time points, March– April and May, the decline in MMP induced by 10 and 100 μM was not statistically different from that caused by 1 μM, overall not exceeding 8%. Different inhibition profile of MMP was observed in lamprey hepatocytes exposed to the same range of EME concentrations (Fig. 2). In January–February, EME produced the similar dose-dependent decline in MMP as CHX, with 100 μM causing ~22% suppression. However, no effect on MMP was observed for all three EME doses in March–April. In
Fig. 1. Effect of protein synthesis inhibitor cycloheximide (CHX) on mitochondrial membrane potential (MMP) in lamprey hepatocytes on the course of prespawning starvation. Isolated lamprey hepatocytes were incubated for 30 min with 1, 10 and 100 μM of cycloheximide (CHX) at room temperature, then loaded for 30 min with 500 nM of tetramethyl rhodamine methyl ester (TMRM) in the dark, and flow cytometry analysis of orange TMRM fluorescence was performed in FL2. The data are calculated as percentages to corresponding controls taken as 100%. Presented are arithmetic means with standard errors. Number of animals: January–February – 12, March–April – 10, May – 7. *p b 0.001, #p b 0.05 in comparison to corresponding control.
Fig. 2. Seasonal dynamics of MMP suppression by protein synthesis blocker emetine (EME). Lamprey hepatocytes were incubated for 30 min with 1, 10 and 100 μM of emetine (EME) at room temperature, loaded for 30 min with 500 nM of TMRM and subjected for flow cytometry analysis. The data are calculated as percentages to corresponding controls taken as 100%. Shown values are arithmetic means ± SE (number of animals is indicated on Fig. 1). *p b 0.001, #p b 0.05 in comparison to corresponding control.
May, 1 and 10 μM EME did not affect MMP, whereas 100 μM induced ~11% decrease. 3.2. Seasonal dynamics of MMP inhibition associated with Na+-K+-ATPase activity The portion of ATP production necessary for the maintenance of Na+-K+-ATPase activity was examined using different concentrations of specific sodium pump inhibitor, ouabain. Previous experiments have shown that 2 mM ouabain inhibited 96% of Na+-K+-pump activity, measured as Rb+ influx to the cells in the medium where K+ was replaced by Rb+ (Gamper et al., 2001). A clear seasonal dynamics was revealed for Na+-K+-pump-dependent MMP changes (Fig. 3). During January–February, incubation of hepatocytes with 2 mM ouabain decreased the intensity of TMRM fluorescence by 22% in average. In March–April, MMP sensitive to highest of applied ouabain dose
Fig. 3. Effect of Na+-K+-pump inhibitor ouabain on MMP in lamprey hepatocytes at different seasons of prespawning period. Experimental procedure and number of animals as on Fig. 1. Presented values are arithmetic means ± SE. *p b 0.001 in comparison to corresponding control.
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Fig. 4. Seasonal dynamics of MMP suppression in the presence of sodium phenylpyruvate (SPP). Experimental procedure and number of animals as on Fig. 1. Average values ± SE are shown. *p b 0.001, #p b 0.05 in comparison to corresponding control.
accounted for 38% in average, while in May it increased to 62% of the total MMP value.
3.3. Effect of sodium phenylpyruvate and alpha-cyano-4-hydroxycinnamate on MMP in lamprey hepatocytes at different seasons of prespawning starvation To ascertain a possible share of gluconeogenesis in ATP turnover in lamprey hepatocytes, we used two chemicals known to impair glucose synthesis - sodium phenylpyruvate (SPP), an inhibitor of pyruvate carboxylase (Meijer and Williamson, 1974), and alpha-cyano-4hydroxycinnamate (CHC), blocker of the mitochondrial pyruvate transporter (Halestrap and Denton, 1975). Although 1 and 10 mM SPP decreased the intensity of TMRM fluorescence in comparison to control, no evident differences were revealed between the values of SPPsensitive MMP at three stages of prespawning starvation (Fig. 4). Overall, the effect of 10 mM SPP did not exceed 10%. An addition of increasing CHC concentrations diminished MMP in a dose-dependent manner
Fig. 6. Catalytic activity of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in lamprey blood plasma at different seasons of prespawning period. A – seasonal dynamics of ALT, B – seasonal dynamics of AST, C - de Ritis coefficient.
(~15% by highest concentration) as well, however, the inhibitory effect of CHC on MMP was equal at all three stages examined (Fig. 5). 3.4. ALT and AST activities in lamprey blood at different seasons of prespawning period The integrity of lamprey liver cells at the different seasons of prespawning period was monitored by estimating the catalytic activity of alanine aminotransferase (ALT, EC 2.6.1.2) and aspartate aminotransferase (AST, EC 2.6.1.1) in blood plasma. The prolonged starvation of animals was accompanied by progressive rise of both ALT and AST activities. Fig. 6 and Table 1 demonstrate that catalytic activity of ALT exhibited 3.75-fold increase from November to May, whereas that of AST showed only 1.6-fold rise. Accordingly, de Ritis coefficient diminishes in N 3 times. Table 1 Averaged catalytic activity of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in lamprey blood plasma at the different seasons of prespawning period. Enzyme activity is expressed as units/l (mean ± SEM).
Fig. 5. Effect of alpha-cyano-4-hydroxycinnamate (CHC) on MMP in lamprey hepatocytes on the course of prespawning starvation. Experimental procedure and number of animals as on Fig. 1. Presented values are arithmetic means ± SE. *p b 0.001, #p b 0.05 in comparison to corresponding control.
Month
ALT
AST
de Ritis coefficient (AST/ALT)
November–December (n = 8) February–March (n = 7) May (n = 5)
16 ± 2 34 ± 5⁎ 60 ± 4⁎
37 ± 3 45 ± 3⁎ 48 ± 3⁎
2.51 ± 0.34 1.43 ± 0.19 0.81 ± 0.08⁎
n – number of experiments. ⁎ The differences are statistically significant (p b 0.05) in comparison to October values.
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4. Discussion 4.1. Protein synthesis Protein synthesis was established to be the ATP consumer most sensitive to energy deprivation. Thus, the protein synthesis is suppressed under anoxia in hepatocytes of western painted turtle Chrysemys picta bellii (Land et al., 1993; Land and Hochachka, 1994), goldfish Carassius auratus and trout Oncorhynchus mykiss (Krumschnabel et al., 2000), under hypoxia in the liver of goldfish Carassius auratus (Jibb and Richards, 2008) and under hypoxia/hypercapnia in hepatopancreas of Pacific whiteleg shrimp Litopenaeus vannamei (Hardy et al., 2013). Aestivation reduces the rate of protein synthesis in liver of Australian desert frog Neobatrachus centralis (Fuery et al., 1998) and hepatopancreas of snail Helix apersa (Pakay et al., 2002), daily torpor is accompanied by protein synthesis suppression in the liver of Djungarian hamster (Phodopus sungorus) (Berriel Diaz et al., 2004). The situation in lamprey hepatocytes is different. Thus, in January– February, in spite of deep metabolic depression, protein synthesis remains one of the prominent ATP-utilizing processes accounting for consumption of 20–22% total energy produced in the cells (Figs. 1–2). This proportion is comparable to that reported for vertebrate cells under standard metabolic rate (Buttgereit and Brand, 1995; Hochachka et al., 1996; Rolfe and Brown, 1997). An ability of female lamprey hepatocytes to maintain the energy-costly process of protein synthesis is most probably associated with the production of vitellogenins (VTGs), hepatic phospho-glyco-lipoprotein precursors of the yolk proteins secreted to bloodstream and transported to developing oocytes. VTGs are the only proteins crucially required to be synthesized at this period of lamprey life. Recently, Mewes et al. (2002) have shown that VTG titer in the blood of female lampreys Lampetra fluviatilis is high throughout an entire prespawning period, which confirms that the deposition of VTG into maturing oocytes occurs directly before spawning. In contrast, in March–April and May, when energy metabolism is markedly activated, but the maturation of oocytes is terminated, the portion of ATP necessary for protein synthesis is reduced to 7–8% (Figs. 1–2). Thus, the adaptive strategy of lamprey hepatocytes to develop metabolic depression is not only to survive until spawning but also allow the selective production of proteins necessary for oocyte maturation. 4.2. Sodium pump Suppression of the Na+-K+-ATPase activity is another adaptive mechanism aimed to survive the periods of energy limitation by diminishing ATP demands. Active ion pumping is reduced in response to oxygen deficit in the brain cells of goldfish (Carassius auratus) (Wilkie et al., 2008) and freshwater turtles (Trachemys scripta) (Hylland et al., 1997), in hepatocytes of trout Oncorhynchus mykiss (Krumschnabel et al., 2000). Hibernation was shown to be the cause of Na+ pump suppression in the red blood cells of black bear (Chauhan et al., 2002) and muscles of frogs Rana temporaria (Boutilier, 2001). In contrast, Na+-K+-ATPase activity was stable in anoxic brain of crucian carp Carassius carassius (Hylland et al., 1997) and goldfish hepatocytes (Krumschnabel et al., 2000). Another example of surviving is Na+-K+-ATPase in hepatocytes of western painted turtle Chrysemys picta bellii, which in response to anoxic incubation reduced ATP demand by 75%, but in percentage terms this suppression is less than for overall ATP turnover, as a result, Na+ pump becomes the dominant energy sink of the cell consuming 75% of cellular ATP (Buck and Hochachka, 1993). In lamprey hepatocytes even at the depth of metabolic depression (January–February) the functioning of Na+-K+-pump requires 22% of total cellular ATP (Fig. 3), being within the range described for the standard metabolic rate. This indicates that Na-K-pump energy supply during metabolic depression is kept at appropriate level. The stability of active cation transport was confirmed earlier by work of Gamper et al. (2001), reporting that Na+-K+-pump in lamprey hepatocytes is capable of
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successful functioning within the wide range of cellular ATP (2.5– 1 nmol per 10−6 cells) and is suppressed only when ATP content drops below 0.8 nmol per 10−6 cells. However, the recovery of lamprey hepatocytes from metabolic depression (March–April) is accompanied by dramatic increase in ATP portion necessary for activity of Na+-K+-ATPase (38%) (Fig. 3). Before spawning (May), the greatest part of ATP produced in lamprey hepatocytes (62%) is spent for transport of Na+ and K+ across plasma membrane, most probably associated with the necessity to maintain ion balance of the cells. Earlier, metabolic depression of lamprey hepatocytes has been shown to be accompanied by a moderate increase in cytosolic Ca2+ content (Konovalova et al., 2011a) and decrease in intracellular pH (Konovalova et al., 2011b), which are considered to give these cells an opportunity of surviving in low-energy state for a few months. However, such ion changes, when reach the threshold concentrations, inevitably lead to disturbances in cellular ion and water balance and induce cell death (Shirokova, 2007). Indeed, the number of lamprey hepatocytes undergoing both apoptosis and necrosis increases following progression of prespawning starvation (Konovalova et al., 2012). Thus, enhanced activity of Na+-K+-ATPase can indicate a compensatory response to reestablish transmembrane ion gradients, membrane potential and cell volume, being the priority cell function associated with the necessity to prevent their premature death. Indirect evidence supporting degenerative damage of lamprey hepatocyte membranes following progression of prespawning starvation is the alterations in ALT and AST activities in blood plasma (Fig. 6, Table 1). These enzymes are released from the cells at a constant rate, and their levels in the blood reflect the equilibrium between the normal level of hepatocytes programmed death (apoptosis) and the clearance of enzymes from plasma. In mammals, altered activity of these two aminotransferases is known to be a sensitive marker of liver inflammation or disease, whereas de Ritis ratio (de Ritis coefficient AST/ALT) gives a possibility to estimate the character and degree of hepatocellular injury (Botros and Sikaris, 2013). ALT resides predominantly in the liver, with negligible quantities found in other organs, thus being a more specific indicator of hepatocellular damage. ALT is a cytoplasmic enzyme, and an increased ALT activity is observed not only upon damage of hepatocytes membrane but also due to increase in its permeability. AST presents in both hepatocyte cytoplasm and mitochondria, therefore, its increase always indicates severe cell damage. In our study, elevation of ALT and AST catalytic activities alongside with progressive decrease in de Ritis coefficient confirm the damage of hepatocytes associated with development of apoptosis and necrosis as the spawning approaches. 4.3. Influence of gluconeogenesis inhibitors Gluconeogenesis, the metabolic pathway generating glucose from non-carbohydrate carbon substrates such as pyruvate, lactate, glycerol, and glucogenic amino acids, is a major mechanisms to keep normal blood glucose levels during periods of fasting or starvation. In this study, sodium phenylpyruvate (SPP) and alpha-cyano-4hydroxycinnamate (CHC), the inhibitors of gluconeogenic pathway from pyruvate, diminished MMP by 10–15% during the period of energy limitation (Figs. 4–5). Unexpectedly, after the recovery of hepatocytes from metabolic depression, when intracellular ATP content rises to the levels able to support gluconeogenesis, the degree of MMP inhibition by SPP and CHC remains the same. From one hand, it would indicate that the rate of gluconeogenesis in lamprey hepatocytes is stable during an entire period of natural starvation, with the share of ATP supply close to that revealed in vertebrate cells under standard metabolic rate. On the other hand, previous studies established that lamprey liver is not a “glucostat”. Thus, gluconeogenesis in lamprey hepatocytes during winter months of prespawning starvation was shown to be depressed, since at existing ATP concentrations the carboxylation of pyruvate is not occurred, hence, the synthesis of glucose from amino acids is not possible (Savina and Derkachev, 1983). Moreover, liver metabolism is
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suppressed to such negligible level that hepatectomized animals do not differ from the sham operated ones for several weeks, but the level of glucose in blood are maintained within normal range for prolonged time (Larsen, 1978). All this indicate that the effects of SPP and CHC cannot be linked with gluconeogenesis. Most probably, this portion of cellular ATP is consumed for the maintenance of redox-potential of the cytoplasm, which occurs due to functioning of “basal” or “futile” processes implicated in the transfer of reducing equivalents across mitochondrial membrane. The existence of three such cycles, associated with recirculation of malate and pyruvate across the mitochondrial membrane, was described in the rat hepatocytes (Meijer and Williamson, 1974) and rat kidney proximal tubules (Rognstad and Katz, 1972; Watford et al., 1980). All these cycles include pyruvate carboxylase and pyruvate transporter, so they can be suppressed in the presence of SPP and CHC. The physiological role of such cycles is not known, apparently they may have a role in metabolic regulation or may be used to maintain thermal homeostasis. In conclusion, the results of present work clearly show that the pattern of ATP utilization by the major cellular processes in hepatocytes of lampreys Lampetra fluviatilis on the course of prespawning starvation differs from that described for the cells of other vertebrates under hypoxia or anoxia (Hochachka et al., 1996; Rolfe and Brown, 1997). First, although the total absolute rates of ATP-demanding processes in lamprey hepatocytes might be quantitatively decreased at the peak of metabolic depression, their percentage proportions are unaltered and comparable to that revealed in vertebrate cells under standard metabolic rate, reflecting an ability of these cells to suppress ATP-consuming processes in coordination with reduction of total ATP production. Second, the development of metabolic depression is the adaptive strategy of lamprey hepatocytes in order not only to survive until spawning, but provide the selective production of proteins necessary for oocyte maturation. Acknowledgements The work was financially supported by Russian Fund for Basic Research (project 13-04-00011). References Berriel Diaz, M., Lange, M., Heldmaier, G., Klingenspor, M., 2004. Depression of transcription and translation during daily torpor in the Djungarian hamster (Phodopus sungorus). J. Comp. Physiol. B. 174, 495–502. Botros, M., Sikaris, K.A., 2013. The de ritis ratio: the test of time. Clin. Biochem. Rev. 34, 117–130. Boutilier, R.G., 2001. Mechanisms of cell survival in hypoxia and hypothermia. J. Exp. Biol. 204, 3171–3181. Buck, L.T., Hochachka, P.W., 1993. Anoxic suppression of Na+-K+-ATPase and constant membrane potential in hepatocytes: support for channel arrest. Am. J. Phys. 265, R1020–R1025. Buttgereit, F., Brand, M.D., 1995. A hierarchy of ATP-consuming processes in mammalian cells. Biochem. J. 312, 163–167. Chauhan, V.P., Tsiouris, J.A., Chauhan, A., Sheikh, A.M., Brown, W.T., Vaughan, M., 2002. Increased oxidative stress and decreased activities of Ca2+/Mg2+-ATPase and Na+/K+ATPase in the red blood cells of the hibernating black bear. Life Sci. 71, 153–161. Emelyanova, L.V., Koroleva, E.M., Savina, M.V., 2004. Glucose and free amino acids in the blood of lampreys (Lampetra fluviatilis L.) and frogs (Rana temporaria L.) under prolonged starvation. Comp. Biochem. Physiol. A 138, 527–532. Emel'yanova, L.V., Savina, M.V., Belyaeva, E.A., Brailovskaya, I.V., 2007. Peculiarities of functioning of liver mitochondria of the river lamprey Lampetra fluviatilis and the common frog Rana temporaria at periods of suppression and activation of energy metabolism. J. Evol. Biochem. Physiol. 43, 564–572. Fuery, C.J., Withers, P.C., Hobbs, A.A., Guppy, M., 1998. The role of protein synthesis during metabolic depression in the Australian desert frog Neobatrachus centralis. Comp. Biochem. Physiol. A Mol. Integr. Physiol. 119, 469–476. Gamper, N.L., Savina, M.V., 2000. Reversible metabolic depression in hepatocytes of lamprey (Lampetra fluviatilis) during pre-spawning: regulation by substrate availability. Comp. Biochem. Physiol. B 127, 147–154.
Gamper, N.L., Savina, M.V., Brailovskaya, I.V., Vereninov, A.A., 2001. Respiration rates, ATP content and ionic regulation in hepatocytes of starving lamprey during the prespawning period of their life cycle. J. Fish Biol. 58, 230–239. Halestrap, A.P., Denton, R.M., 1975. The specificity and metabolic implications of the inhibition of pyruvate transport in isolated mitochondria and intact tissue preparations by alpha-cyano-4-hydroxycinnamate and related compounds. Biochem. J. 148, 97–106. Hardy, K.M., Burnett, K.G., Burnett, L.E., 2013. Effect of hypercapnic hypoxia and bacterial infection (Vibrio campbellii) on protein synthesis rates in the Pacific whiteleg shrimp, Litopenaeus vannamei. Am. J. Physiol. Regul. Integr. Comp. Physiol. 305, R1356–R1366. Hochachka, P.W., Lutz, P.L., 2001. Mechanism, origin, and evolution of anoxia tolerance in animals. Comp. Biochem. Physiol. B 130, 435–459. Hochachka, P.W., Buck, L.T., Doll, C.J., Land, S.C., 1996. Unifying theory of hypoxia tolerance: molecular/metabolic defense and rescue mechanisms for surviving oxygen lack. Proc. Natl. Acad. Sci. U. S. A. 93, 9493–9498. Hylland, P., Milton, S., Pek, M., Nilsson, G.E., Lutz, P.L., 1997. Brain Na+/K+-ATPase activity in two anoxia tolerant vertebrates: crucian carp and freshwater turtle. Neurosci. Lett. 235, 89–92. Jibb, L.A., Richards, J.G., 2008. AMP-activated protein kinase activity during metabolic rate depression in the hypoxic goldfish, Carassius auratus. J. Exp. Biol. 211, 3111–3122. Konovalova, S.A., Savina, M.V., Nikiforov, A.A., 2011a. Changes in intracellular Ca2+ concentration in hepatocytes of the Baltic lamprey (Lampetra fluviatilis L.) at the period of prespawning migration. J. Evol. Biochem. Physiol. 47, 204–206. Konovalova, S.A., Zubatkina, I.S., Savina, M.V., Nikiforov, A.A., 2011b. Intracellular pH in hepatocytes of the river lamprey Lampetra fluviatilis L. at the period of prespawning migration. J. Evol. Biochem. Physiol. 47, 389–391. Konovalova, S.A., Savina, M.V., Nikiforov, A.A., Puchkova, L.V., 2012. Mitochondrial and lysosomal pathways of lamprey (Lampetra fluviatilis L.) hepatocyte death. J. Evol. Biochem. Physiol. 48, 510–515. Krumschnabel, G., Schwarzbaum, P.J., Lisch, J., Biasi, C., Wieser, W., 2000. Oxygendependent energetics of anoxia-tolerant and anoxia-intolerant hepatocytes. J. Exp. Biol. 203, 951–959. Land, S.C., Hochachka, P.W., 1994. Protein turnover during metabolic arrest in turtle hepatocytes: role and energy dependence of proteolysis. Am. J. Phys. 266, C1028–C1036. Land, S.C., Buck, L.T., Hochachka, P.W., 1993. Response of protein synthesis to anoxia and recovery in anoxia-tolerant hepatocytes. Am. J. Phys. 265, R41–R48. Larsen, L.O., 1978. Subtotal hepatectomy in intact or hypophysectomized river lampreys (Lampetra fluviatilis L.): effects on regeneration, blood glucose regulation, and vitellogenesis. Gen. Comp. Endocrinol. 35, 197–204. Larsen, L.O., 1980. Physiology of adult lampreys, with special regard to natural starvation, reproduction, and death after spawning. Can. J. Fish. Aquat. Sci. 37, 1762–1779. Meijer, A.J., Williamson, J.R., 1974. Transfer of reducing equivalents across the mitochondrial membrane. I. Hydrogen transfer mechanisms involved in the reduction of pyruvate to lactate in isolated liver cells. Biochim. Biophys. Acta 333, 1–11. Mewes, K.R., Latz, M., Golla, H., Fisher, A., 2002. Vitellogenin from female and estradiolstimulated male river lampreys (Lampetra fluviatilis L.). J. Exp. Zool. 292, 52–72. Mommsen, T.P., Moon, T.W., Walsh, P.J., 1994. Hepatocytes: Isolation, Maintenance and Utilization. In: Hochachka, P.W., Mommsen, T.P. (Eds.), Biochemistry and Molecular Biology of Fishes 3. Elsevier Science, Amsterdam, pp. 355–373. Pakay, J.L., Withers, P.C., Hobbs, A.A., Guppy, M., 2002. In vivo downregulation of protein synthesis in the snail Helix apersa during estivation. Am. J. Physiol. Regul. Integr. Comp. Physiol. 283, R197–R204. Rognstad, R., Katz, J., 1972. Gluconeogenesis in the kidney cortex. Quantitative estimation of carbon flow. J. Biol. Chem. 247, 6047–6054. Rolfe, D.F., Brown, G.C., 1997. Cellular energy utilization and the molecular origin of standard metabolic rate in mammals. Physiol. Rev. 77, 731–756. Savina, M.V., Derkachev, E.F., 1983. Switch on and switch off phenomenon of liver gluconeogenic function in lamprey (Lampetra fluviatilis L.) under the influence of season and temperature. Comp. Biochem. Physiol. 75B, 531–539. Savina, M.V., Gamper, N.L., 1998. Respiration and adenine nucleotides of Baltic lamprey (Lampetra fluviatilis L.) hepatocytes during spawning migration. Comp. Biochem. Physiol. B 120, 375–383. Savina, M.V., Konovalova, S.A., Zubatkina, I.S., Nikiforov, A.A., 2011. Reversible metabolic depression in lamprey hepatocytes during prespawning migration: dynamics of mitochondrial membrane potential. Comp. Biochem. Physiol. B 160, 194–200. Shirokova, A.V., 2007. Apoptosis. Signaling pathways and cell ion and water balance. Cell Tissue Biol. 1, 215–224. Storey, K.B., Storey, J.M., 2004. Metabolic rate depression in animals: transcriptional and translational controls. Biol. Rev. Camb. Philos. Soc. 79, 207–233. Watford, M., Vinay, P., Lemieux, G., Gougoux, A., 1980. The regulation of glucose and pyruvate formation from glutamine and citric-acid-cycle intermediates in the kidney cortex of rats, dogs, rabbits and Guinea pigs. Biochem. J. 188, 741–748. Wieser, W., Krumschnabel, G., 2001. Hierarchies of ATP-consuming processes: direct compared with indirect measurements. Biochem. J. 355, 389–395. Wilkie, M.P., Pamenter, M.E., Alkabie, S., Carapic, D., Shin, D.S., Buck, L.T., 2008. Evidence of anoxia-induced channel arrest in the brain of the goldfich (Carassius auratus). Comp. Biochem. Physiol. C 148, 355–362. Zubatkina, I.S., Konovalova, S.A., Savina, M.V., Nikiforov, A.N., 2011. Membrane potential of mitochondria in hepatocytes of the river lamprey Lampetra fluviatilis at periods of metabolic depression and activity. J. Evol. Biochem. Physiol. 47, 97–99.
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Comparative Biochemistry and Physiology, Part A journal homepage: www.elsevier.com/locate/cbpa
ATP-consuming processes in hepatocytes of river lamprey Lampetra fluviatilis on the course of prespawning starvation Natalia I. Agalakova ⁎, Irina V. Brailovskaya, Svetlana A. Konovalova, Sergei M. Korotkov, Elena A. Lavrova, Anatolii A. Nikiforov Sechenov Institute of Evolutionary Physiology and Biochemistry, Russian Academy of Sciences, 44 Thorez av., Sankt-Petersburg 194223, Russia
a r t i c l e
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Article history: Received 23 November 2015 Received in revised form 5 July 2016 Accepted 5 July 2016 Available online 9 July 2016 Keywords: Lamprey Hepatocytes Prespawning starvation Metabolic depression Mitochondrial membrane potential Protein synthesis Na+-K+-pump Aminotransferases
a b s t r a c t The work was performed to establish which of the major ATP-consuming processes is the most important for surviving of hepatocytes of female lampreys on the course of prespawning starvation. The requirements of protein synthesis and Na+-K+-ATPase for ATP in the cells were monitored by the changes in mitochondrial membrane potential (MMP) in the presence of corresponding inhibitors from the peak of metabolic depression (January– February) to the time of recovery from it (March–April) and spawning (May). Integrity of lamprey liver cells was estimated by catalytic activities of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in blood plasma. In January–February, the share of ATP necessary for protein synthesis was 20–22%, whereas before spawning it decreased to 8–11%. Functioning of Na+-K+-pump required 22% of cellular ATP at the peak of metabolic depression, but 38% and 62% of ATP in March–April and May, respectively. Progression of prespawning period was accompanied by 3.75- and 1.6-fold rise of ALT and AST activities in blood plasma, respectively, whereas de Ritis coefficient decreased from 2.51 ± 0.34 to 0.81 ± 0.08, what indicates severe damage of hepatocyte membranes. Thus, the adaptive strategy of lamprey hepatocytes to develop metabolic depression under conditions of energy limitation is the selective production of proteins necessary for spawning, most probably vitellogenins. As spawning approaches, the maintenance of transmembrane ion gradients, membrane potential and cell volume to prevent premature cell death becomes the priority cell function. © 2016 Elsevier Inc. All rights reserved.
1. Introduction Many vertebrate species of different phylogenetic levels, which living cycle includes seasonal oxygen deprivation, hypothermia, hibernation, aestivation, anhydrobiosis, etc., are able to survive the natural phenomenon of reversible metabolic depression (Boutilier, 2001; Hochachka and Lutz, 2001; Storey and Storey, 2004). Although the defense strategies are divergent and broad array of adaptations were evolved by different species, a key mechanism of survival is an ability to fundamentally suppress ATP utilization and O2 demand to such hypometabolic state which balances the reduced capacity of cells to produce ATP. The intensity of ATP-demanding processes such as biosynthesis, protein turnover, membrane ion transport and secretion are reduced or almost suspended, leading to overall reduction in ATP consumption. Among the species able to reorganize their cellular metabolic rate, lamprey Lampetra fluviatilis is a unique example. Anadromous migration of these monocyclic animals is accompanied by prolonged
⁎ Corresponding author. E-mail address: [email protected] (N.I. Agalakova).
http://dx.doi.org/10.1016/j.cbpa.2016.07.002 1095-6433/© 2016 Elsevier Inc. All rights reserved.
(7–8 months) synchonia, genetically programmed and hormonally regulated behavior phenomenon of natural starvation (Larsen, 1980). The digestive system is atrophied, and the vitality of lampreys during all phases of prespawning period is maintained due to mobilization of glycogen, lipids and proteins mainly from muscle tissue (Emelyanova et al., 2004; Emel'yanova et al., 2007). However, the supply of hepatocytes with substrates is limited, because blood enters the liver through portal vein which undergoes degenerative changes. Hepatocyte metabolism is exclusively aerobic and based on oxidation of fatty acids, but the lipid drops accumulated in hepatocytes are not used until spring (Savina and Gamper, 1998; Gamper and Savina, 2000; Emel'yanova et al., 2007). As a result, to the middle of winter the hepatocytes develop deep metabolic depression, with endogenous respiration rate, oxidative phosphorylation and ATP content exhibiting considerable decline in comparison to values observed at the beginning of spawning migration (Savina and Gamper, 1998; Gamper and Savina, 2000; Gamper et al., 2001). When the spawning approaches, hepatocyte metabolism is activated again, the rates of endogenous respiration and ATP synthesis are restored and almost reach autumn values. In spite of metabolic depression, liver cells of female lampreys synthesize vitellogenins, precursors of the yolk proteins, which titer in the blood was confirmed to be constant throughout the spawning period (Mewes et al., 2002).
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Therefore, the strategy of lamprey hepatocytes to develop metabolic depression is not associated with unfavorable environmental factors (hypoxia or hypothermia), but serves to preserve energy resources for spawning. The goal of present work was to reveal the cellular processes being the most important for surviving of lamprey hepatocyte until spawning under conditions of energy limitation induced by prespawning starvation. For this purpose, we made an attempt to evaluate the sensitivity of major ATP-consuming processes to seasonal decrease in ATP production. In the cells of mammals and some other studied vertebrate species, major ATP-demanding processes under standard metabolic rate are protein synthesis (consuming 24–30% of ATP coupled to oxygen consumption) and activity of Na+-K+-ATPase (19–28%). The remaining is spent by Ca2+-ATPase activity, gluconeogenesis, protein degradation, ureagenesis, mitochondrial proton leak, mRNA synthesis, etc. (Buttgereit and Brand, 1995; Hochachka et al., 1996; Rolfe and Brown, 1997; Wieser and Krumschnabel, 2001). Biosynthesis of proteins and activity of sodium pump are also most sensitive to energy deprivation. Taking this into account, we monitored the requirement of protein synthesis and Na+-K+-pump in lamprey hepatocytes for ATP on the course of prespawning starvation, from the peak of metabolic depression (January–February) to the time of recovery from it (March–April) and spawning (May). We also evaluated the possible gluconeogenic capacity of liver cells at different energy states. Next, we measured the catalytic activities of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in lamprey blood plasma to monitor the integrity of liver cells at different seasons of prespawning period. The share of ATP necessary for the maintenance of examined cellular processes were evaluated by the rate of their inhibition measured as the changes in potential on inner mitochondrial membrane - MMP (Δψm). MMP is a key parameter controlling mitochondrial respiration and ATP synthesis, exerting a significant effect on the maximal ATPgenerating capacity. MMP accounts for the majority of the protonmotive driving force, which is in turn the central intermediate of aerobic energy production. Resting MMP reflects the balance between energy supply and ATP consumption. The study was based on two assumptions. First, acute inhibition of individual ATP-consuming processes causes a consequential drop in ATP demand and triggers a proportional drop in MMP due to decrease in the rate of electron transport by respiratory chain after administration of inhibitor. Second, an inhibition of one ATP-demanding process does not alter the rates of other processes. Since alterations of MMP values in lamprey hepatocytes on the course of prespawning starvation completely follows the dynamics of ATP production (Zubatkina et al., 2011; Savina et al., 2011), each of these parameters can be reliable indicator of cell energy state and enables studying the relationships between energy metabolism and activities of cellular processes requiring ATP. 2. Material and methods 2.1. Animals Lampreys (Lamperta fluviatilis L.) were caught in the Neva river in November–December after the beginning of their prespawning migration and maintained until May in the tanks with dechlorinated and permanently aerated water at 2–6 °C. The experiments were performed on females. 2.2. Solutions and chemicals The standard medium used for isolation and incubation of hepatocytes contained (mM): 137 NaCl, 5 KCl, 0.8 MgSO4, 0.44 KH2PO4, 0.33 Na2HPO4, 5 NaHCO3, 10 HEPES, 5 glucose, pH 7.6. All buffer salts, cycloheximide, emetine hydrochloride, sodium phenylpyruvate, alpha-cyano-4-hydroxycinnamate, ouabain, collagenase (type VIII), HEPES, FCCP (carbonyl cyanide-4-(trifluoromethoxy)
phenylhydrazone), DMSO (dimethyl sulfoxide) were purchased from Sigma (USA). FCCP was dissolved in ethanol, emetine and sodium phenylpyruvate - in distilled water. Tetramethyl rhodamine methyl ester (TMRM) was from Fluka. Stock solution of TMRM was prepared on DMSO, aliquoted and stored at −20 °C in the dark.
2.3. Preparation of hepatocytes Hepatocytes were isolated by a modified collagenase method (Mommsen et al., 1994). The liver was perfused for 2–3 min in situ with the standard medium through the ventral hepatic vein, then carefully minced with a razor blade and incubated for 1 h at room temperature in the same medium supplemented with 0.1% collagenase type VIII. After incubation, the digested tissue was passed through nylon mesh and the cells were washed three times by centrifugation in the standard medium supplemented with 1.5 mM CaCl2 and 0.5% bovine serum albumin (BSA). The final cell pellet was suspended in the same medium at concentration of 4–6 millions of cells per ml and stored on ice. Cell viability of hepatocytes, evaluated by Trypan blue, was 90–95%.
2.4. Determination of mitochondrial membrane potential Mitochondrial membrane potential was determined by flow cytometry using fluorescent dye tetramethyl rhodamine methyl ester (TMRM). TMRM is a lipophilic cationic compound accumulating in the mitochondrial membranes space in inverse proportion to mitochondrial membrane potential according to the Nernst equation. The hepatocytes were incubated for 30 min in the standard medium with tested inhibitors at room temperature and then loaded for 30 min with TMRM (500 nM) in the dark. After loading the cells were washed twice by centrifugation for 3 min at 1000g with fresh medium. Because TMRM is rapidly lost from the cells in the absence of external probe, the hepatocytes were placed in incubation medium containing 50 nM TMRM to allow continuous reequilibration of fluorophore across the plasma membrane and immediately subjected for flow cytometry. An analysis of orange TMRM fluorescence was performed on EPICS-XL (Beckman Coulter, USA) at wavelength of 575 ± 15 nm. The intensity of TMRM fluorescence was set on logarithmic scale. The data were first plotted using SSC (side scatter) vs FSC (forward scatter) to reveal cell size and complexity, and the gate was applied to isolate the population of cells with normal volume and remove the debris along the axes. Then the data were re-plotted using FSC and TMRM and the gate was used to remove dying cells with low TMRM fluorescence. Finally, the data were collected and gated so that only hepatocytes with high TMRM fluorescence were used for analysis. 30,000 events were collected for each sample. Mean fluorescence intensity in arbitrary units obtained for control samples (without inhibitors) in each experiment were taken as 100%. The fluorescence of samples in the presence of inhibitors was expressed as the percentage of control. According to the Nernst equation, intracellular distribution of cationic mitochondrial probe (TMRM) reflects the differences in the transmembrane potential across both the plasma membrane and inner mitochondrial membrane, i.e., besides mitochondria, TMRM is accumulated in the cytoplasm as well. The specificity of TMRM fluorescence for MMP was assessed using protonophore FCCP. FCCP completely depolarized the mitochondria, thus causing a collapse in MMP and, respectively, loss of TMRM signal. After determination of TMRM fluorescence, 40 μM FCCP was added to each sample, and the fluorescence was determined again in 10 min. TMRM fluorescence in the presence of FCCP (negative control) was quite similar in all samples and did not exceed 14–18 arbitrary units, whereas without FCCP it was 200– 300 units. For analysis, TMRM signal in the presence of FCCP was subtracted from the total TMRM signal in the sample.
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2.5. Determination of ALT and AST activity The activities of ALT and AST in lamprey blood plasma were determined by enzymatic reactions using commercial colorimetric assay kits (Sigma). 2.6. Statistics The obtained results were processed by SigmaPlot software package version 11.0 (Jandel Scientific). The statistical differences were assessed using one-way ANOVA for many groups followed by Tukey's test. The level of significance was established at p ≤ 0.05. The data are presented as arithmetic means ± SE. 3. Results 3.1. Effects of protein synthesis inhibitors on MMP in lamprey hepatocytes on the course of prespawning starvation The metabolic cost of protein synthesis in lamprey hepatocytes on the course of prespawning starvation was evaluated using two distinct inhibitors of protein synthesis, cycloheximide (CHX) and emetine (EME), at the doses of 10-fold concentration range to reveal the minimal saturating concentration. Treatment of the cells with 1 μM CHX at all three seasons examined caused similar ~7% decrease in the intensity of TMRM fluorescence (Fig. 1). However, inhibitory potential of higher CHX concentrations depended on the time of prespawning starvation. During January– February the effect of CHX was clearly concentration-dependent, with 10 and 100 μM showing ~18 and 20% decrease, respectively, being statistically significant in comparison to 1 μM. At other time points, March– April and May, the decline in MMP induced by 10 and 100 μM was not statistically different from that caused by 1 μM, overall not exceeding 8%. Different inhibition profile of MMP was observed in lamprey hepatocytes exposed to the same range of EME concentrations (Fig. 2). In January–February, EME produced the similar dose-dependent decline in MMP as CHX, with 100 μM causing ~22% suppression. However, no effect on MMP was observed for all three EME doses in March–April. In
Fig. 1. Effect of protein synthesis inhibitor cycloheximide (CHX) on mitochondrial membrane potential (MMP) in lamprey hepatocytes on the course of prespawning starvation. Isolated lamprey hepatocytes were incubated for 30 min with 1, 10 and 100 μM of cycloheximide (CHX) at room temperature, then loaded for 30 min with 500 nM of tetramethyl rhodamine methyl ester (TMRM) in the dark, and flow cytometry analysis of orange TMRM fluorescence was performed in FL2. The data are calculated as percentages to corresponding controls taken as 100%. Presented are arithmetic means with standard errors. Number of animals: January–February – 12, March–April – 10, May – 7. *p b 0.001, #p b 0.05 in comparison to corresponding control.
Fig. 2. Seasonal dynamics of MMP suppression by protein synthesis blocker emetine (EME). Lamprey hepatocytes were incubated for 30 min with 1, 10 and 100 μM of emetine (EME) at room temperature, loaded for 30 min with 500 nM of TMRM and subjected for flow cytometry analysis. The data are calculated as percentages to corresponding controls taken as 100%. Shown values are arithmetic means ± SE (number of animals is indicated on Fig. 1). *p b 0.001, #p b 0.05 in comparison to corresponding control.
May, 1 and 10 μM EME did not affect MMP, whereas 100 μM induced ~11% decrease. 3.2. Seasonal dynamics of MMP inhibition associated with Na+-K+-ATPase activity The portion of ATP production necessary for the maintenance of Na+-K+-ATPase activity was examined using different concentrations of specific sodium pump inhibitor, ouabain. Previous experiments have shown that 2 mM ouabain inhibited 96% of Na+-K+-pump activity, measured as Rb+ influx to the cells in the medium where K+ was replaced by Rb+ (Gamper et al., 2001). A clear seasonal dynamics was revealed for Na+-K+-pump-dependent MMP changes (Fig. 3). During January–February, incubation of hepatocytes with 2 mM ouabain decreased the intensity of TMRM fluorescence by 22% in average. In March–April, MMP sensitive to highest of applied ouabain dose
Fig. 3. Effect of Na+-K+-pump inhibitor ouabain on MMP in lamprey hepatocytes at different seasons of prespawning period. Experimental procedure and number of animals as on Fig. 1. Presented values are arithmetic means ± SE. *p b 0.001 in comparison to corresponding control.
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Fig. 4. Seasonal dynamics of MMP suppression in the presence of sodium phenylpyruvate (SPP). Experimental procedure and number of animals as on Fig. 1. Average values ± SE are shown. *p b 0.001, #p b 0.05 in comparison to corresponding control.
accounted for 38% in average, while in May it increased to 62% of the total MMP value.
3.3. Effect of sodium phenylpyruvate and alpha-cyano-4-hydroxycinnamate on MMP in lamprey hepatocytes at different seasons of prespawning starvation To ascertain a possible share of gluconeogenesis in ATP turnover in lamprey hepatocytes, we used two chemicals known to impair glucose synthesis - sodium phenylpyruvate (SPP), an inhibitor of pyruvate carboxylase (Meijer and Williamson, 1974), and alpha-cyano-4hydroxycinnamate (CHC), blocker of the mitochondrial pyruvate transporter (Halestrap and Denton, 1975). Although 1 and 10 mM SPP decreased the intensity of TMRM fluorescence in comparison to control, no evident differences were revealed between the values of SPPsensitive MMP at three stages of prespawning starvation (Fig. 4). Overall, the effect of 10 mM SPP did not exceed 10%. An addition of increasing CHC concentrations diminished MMP in a dose-dependent manner
Fig. 6. Catalytic activity of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in lamprey blood plasma at different seasons of prespawning period. A – seasonal dynamics of ALT, B – seasonal dynamics of AST, C - de Ritis coefficient.
(~15% by highest concentration) as well, however, the inhibitory effect of CHC on MMP was equal at all three stages examined (Fig. 5). 3.4. ALT and AST activities in lamprey blood at different seasons of prespawning period The integrity of lamprey liver cells at the different seasons of prespawning period was monitored by estimating the catalytic activity of alanine aminotransferase (ALT, EC 2.6.1.2) and aspartate aminotransferase (AST, EC 2.6.1.1) in blood plasma. The prolonged starvation of animals was accompanied by progressive rise of both ALT and AST activities. Fig. 6 and Table 1 demonstrate that catalytic activity of ALT exhibited 3.75-fold increase from November to May, whereas that of AST showed only 1.6-fold rise. Accordingly, de Ritis coefficient diminishes in N 3 times. Table 1 Averaged catalytic activity of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in lamprey blood plasma at the different seasons of prespawning period. Enzyme activity is expressed as units/l (mean ± SEM).
Fig. 5. Effect of alpha-cyano-4-hydroxycinnamate (CHC) on MMP in lamprey hepatocytes on the course of prespawning starvation. Experimental procedure and number of animals as on Fig. 1. Presented values are arithmetic means ± SE. *p b 0.001, #p b 0.05 in comparison to corresponding control.
Month
ALT
AST
de Ritis coefficient (AST/ALT)
November–December (n = 8) February–March (n = 7) May (n = 5)
16 ± 2 34 ± 5⁎ 60 ± 4⁎
37 ± 3 45 ± 3⁎ 48 ± 3⁎
2.51 ± 0.34 1.43 ± 0.19 0.81 ± 0.08⁎
n – number of experiments. ⁎ The differences are statistically significant (p b 0.05) in comparison to October values.
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4. Discussion 4.1. Protein synthesis Protein synthesis was established to be the ATP consumer most sensitive to energy deprivation. Thus, the protein synthesis is suppressed under anoxia in hepatocytes of western painted turtle Chrysemys picta bellii (Land et al., 1993; Land and Hochachka, 1994), goldfish Carassius auratus and trout Oncorhynchus mykiss (Krumschnabel et al., 2000), under hypoxia in the liver of goldfish Carassius auratus (Jibb and Richards, 2008) and under hypoxia/hypercapnia in hepatopancreas of Pacific whiteleg shrimp Litopenaeus vannamei (Hardy et al., 2013). Aestivation reduces the rate of protein synthesis in liver of Australian desert frog Neobatrachus centralis (Fuery et al., 1998) and hepatopancreas of snail Helix apersa (Pakay et al., 2002), daily torpor is accompanied by protein synthesis suppression in the liver of Djungarian hamster (Phodopus sungorus) (Berriel Diaz et al., 2004). The situation in lamprey hepatocytes is different. Thus, in January– February, in spite of deep metabolic depression, protein synthesis remains one of the prominent ATP-utilizing processes accounting for consumption of 20–22% total energy produced in the cells (Figs. 1–2). This proportion is comparable to that reported for vertebrate cells under standard metabolic rate (Buttgereit and Brand, 1995; Hochachka et al., 1996; Rolfe and Brown, 1997). An ability of female lamprey hepatocytes to maintain the energy-costly process of protein synthesis is most probably associated with the production of vitellogenins (VTGs), hepatic phospho-glyco-lipoprotein precursors of the yolk proteins secreted to bloodstream and transported to developing oocytes. VTGs are the only proteins crucially required to be synthesized at this period of lamprey life. Recently, Mewes et al. (2002) have shown that VTG titer in the blood of female lampreys Lampetra fluviatilis is high throughout an entire prespawning period, which confirms that the deposition of VTG into maturing oocytes occurs directly before spawning. In contrast, in March–April and May, when energy metabolism is markedly activated, but the maturation of oocytes is terminated, the portion of ATP necessary for protein synthesis is reduced to 7–8% (Figs. 1–2). Thus, the adaptive strategy of lamprey hepatocytes to develop metabolic depression is not only to survive until spawning but also allow the selective production of proteins necessary for oocyte maturation. 4.2. Sodium pump Suppression of the Na+-K+-ATPase activity is another adaptive mechanism aimed to survive the periods of energy limitation by diminishing ATP demands. Active ion pumping is reduced in response to oxygen deficit in the brain cells of goldfish (Carassius auratus) (Wilkie et al., 2008) and freshwater turtles (Trachemys scripta) (Hylland et al., 1997), in hepatocytes of trout Oncorhynchus mykiss (Krumschnabel et al., 2000). Hibernation was shown to be the cause of Na+ pump suppression in the red blood cells of black bear (Chauhan et al., 2002) and muscles of frogs Rana temporaria (Boutilier, 2001). In contrast, Na+-K+-ATPase activity was stable in anoxic brain of crucian carp Carassius carassius (Hylland et al., 1997) and goldfish hepatocytes (Krumschnabel et al., 2000). Another example of surviving is Na+-K+-ATPase in hepatocytes of western painted turtle Chrysemys picta bellii, which in response to anoxic incubation reduced ATP demand by 75%, but in percentage terms this suppression is less than for overall ATP turnover, as a result, Na+ pump becomes the dominant energy sink of the cell consuming 75% of cellular ATP (Buck and Hochachka, 1993). In lamprey hepatocytes even at the depth of metabolic depression (January–February) the functioning of Na+-K+-pump requires 22% of total cellular ATP (Fig. 3), being within the range described for the standard metabolic rate. This indicates that Na-K-pump energy supply during metabolic depression is kept at appropriate level. The stability of active cation transport was confirmed earlier by work of Gamper et al. (2001), reporting that Na+-K+-pump in lamprey hepatocytes is capable of
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successful functioning within the wide range of cellular ATP (2.5– 1 nmol per 10−6 cells) and is suppressed only when ATP content drops below 0.8 nmol per 10−6 cells. However, the recovery of lamprey hepatocytes from metabolic depression (March–April) is accompanied by dramatic increase in ATP portion necessary for activity of Na+-K+-ATPase (38%) (Fig. 3). Before spawning (May), the greatest part of ATP produced in lamprey hepatocytes (62%) is spent for transport of Na+ and K+ across plasma membrane, most probably associated with the necessity to maintain ion balance of the cells. Earlier, metabolic depression of lamprey hepatocytes has been shown to be accompanied by a moderate increase in cytosolic Ca2+ content (Konovalova et al., 2011a) and decrease in intracellular pH (Konovalova et al., 2011b), which are considered to give these cells an opportunity of surviving in low-energy state for a few months. However, such ion changes, when reach the threshold concentrations, inevitably lead to disturbances in cellular ion and water balance and induce cell death (Shirokova, 2007). Indeed, the number of lamprey hepatocytes undergoing both apoptosis and necrosis increases following progression of prespawning starvation (Konovalova et al., 2012). Thus, enhanced activity of Na+-K+-ATPase can indicate a compensatory response to reestablish transmembrane ion gradients, membrane potential and cell volume, being the priority cell function associated with the necessity to prevent their premature death. Indirect evidence supporting degenerative damage of lamprey hepatocyte membranes following progression of prespawning starvation is the alterations in ALT and AST activities in blood plasma (Fig. 6, Table 1). These enzymes are released from the cells at a constant rate, and their levels in the blood reflect the equilibrium between the normal level of hepatocytes programmed death (apoptosis) and the clearance of enzymes from plasma. In mammals, altered activity of these two aminotransferases is known to be a sensitive marker of liver inflammation or disease, whereas de Ritis ratio (de Ritis coefficient AST/ALT) gives a possibility to estimate the character and degree of hepatocellular injury (Botros and Sikaris, 2013). ALT resides predominantly in the liver, with negligible quantities found in other organs, thus being a more specific indicator of hepatocellular damage. ALT is a cytoplasmic enzyme, and an increased ALT activity is observed not only upon damage of hepatocytes membrane but also due to increase in its permeability. AST presents in both hepatocyte cytoplasm and mitochondria, therefore, its increase always indicates severe cell damage. In our study, elevation of ALT and AST catalytic activities alongside with progressive decrease in de Ritis coefficient confirm the damage of hepatocytes associated with development of apoptosis and necrosis as the spawning approaches. 4.3. Influence of gluconeogenesis inhibitors Gluconeogenesis, the metabolic pathway generating glucose from non-carbohydrate carbon substrates such as pyruvate, lactate, glycerol, and glucogenic amino acids, is a major mechanisms to keep normal blood glucose levels during periods of fasting or starvation. In this study, sodium phenylpyruvate (SPP) and alpha-cyano-4hydroxycinnamate (CHC), the inhibitors of gluconeogenic pathway from pyruvate, diminished MMP by 10–15% during the period of energy limitation (Figs. 4–5). Unexpectedly, after the recovery of hepatocytes from metabolic depression, when intracellular ATP content rises to the levels able to support gluconeogenesis, the degree of MMP inhibition by SPP and CHC remains the same. From one hand, it would indicate that the rate of gluconeogenesis in lamprey hepatocytes is stable during an entire period of natural starvation, with the share of ATP supply close to that revealed in vertebrate cells under standard metabolic rate. On the other hand, previous studies established that lamprey liver is not a “glucostat”. Thus, gluconeogenesis in lamprey hepatocytes during winter months of prespawning starvation was shown to be depressed, since at existing ATP concentrations the carboxylation of pyruvate is not occurred, hence, the synthesis of glucose from amino acids is not possible (Savina and Derkachev, 1983). Moreover, liver metabolism is
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suppressed to such negligible level that hepatectomized animals do not differ from the sham operated ones for several weeks, but the level of glucose in blood are maintained within normal range for prolonged time (Larsen, 1978). All this indicate that the effects of SPP and CHC cannot be linked with gluconeogenesis. Most probably, this portion of cellular ATP is consumed for the maintenance of redox-potential of the cytoplasm, which occurs due to functioning of “basal” or “futile” processes implicated in the transfer of reducing equivalents across mitochondrial membrane. The existence of three such cycles, associated with recirculation of malate and pyruvate across the mitochondrial membrane, was described in the rat hepatocytes (Meijer and Williamson, 1974) and rat kidney proximal tubules (Rognstad and Katz, 1972; Watford et al., 1980). All these cycles include pyruvate carboxylase and pyruvate transporter, so they can be suppressed in the presence of SPP and CHC. The physiological role of such cycles is not known, apparently they may have a role in metabolic regulation or may be used to maintain thermal homeostasis. In conclusion, the results of present work clearly show that the pattern of ATP utilization by the major cellular processes in hepatocytes of lampreys Lampetra fluviatilis on the course of prespawning starvation differs from that described for the cells of other vertebrates under hypoxia or anoxia (Hochachka et al., 1996; Rolfe and Brown, 1997). First, although the total absolute rates of ATP-demanding processes in lamprey hepatocytes might be quantitatively decreased at the peak of metabolic depression, their percentage proportions are unaltered and comparable to that revealed in vertebrate cells under standard metabolic rate, reflecting an ability of these cells to suppress ATP-consuming processes in coordination with reduction of total ATP production. Second, the development of metabolic depression is the adaptive strategy of lamprey hepatocytes in order not only to survive until spawning, but provide the selective production of proteins necessary for oocyte maturation. Acknowledgements The work was financially supported by Russian Fund for Basic Research (project 13-04-00011). References Berriel Diaz, M., Lange, M., Heldmaier, G., Klingenspor, M., 2004. Depression of transcription and translation during daily torpor in the Djungarian hamster (Phodopus sungorus). J. Comp. Physiol. B. 174, 495–502. Botros, M., Sikaris, K.A., 2013. The de ritis ratio: the test of time. Clin. Biochem. Rev. 34, 117–130. Boutilier, R.G., 2001. Mechanisms of cell survival in hypoxia and hypothermia. J. Exp. Biol. 204, 3171–3181. Buck, L.T., Hochachka, P.W., 1993. Anoxic suppression of Na+-K+-ATPase and constant membrane potential in hepatocytes: support for channel arrest. Am. J. Phys. 265, R1020–R1025. Buttgereit, F., Brand, M.D., 1995. A hierarchy of ATP-consuming processes in mammalian cells. Biochem. J. 312, 163–167. Chauhan, V.P., Tsiouris, J.A., Chauhan, A., Sheikh, A.M., Brown, W.T., Vaughan, M., 2002. Increased oxidative stress and decreased activities of Ca2+/Mg2+-ATPase and Na+/K+ATPase in the red blood cells of the hibernating black bear. Life Sci. 71, 153–161. Emelyanova, L.V., Koroleva, E.M., Savina, M.V., 2004. Glucose and free amino acids in the blood of lampreys (Lampetra fluviatilis L.) and frogs (Rana temporaria L.) under prolonged starvation. Comp. Biochem. Physiol. A 138, 527–532. Emel'yanova, L.V., Savina, M.V., Belyaeva, E.A., Brailovskaya, I.V., 2007. Peculiarities of functioning of liver mitochondria of the river lamprey Lampetra fluviatilis and the common frog Rana temporaria at periods of suppression and activation of energy metabolism. J. Evol. Biochem. Physiol. 43, 564–572. Fuery, C.J., Withers, P.C., Hobbs, A.A., Guppy, M., 1998. The role of protein synthesis during metabolic depression in the Australian desert frog Neobatrachus centralis. Comp. Biochem. Physiol. A Mol. Integr. Physiol. 119, 469–476. Gamper, N.L., Savina, M.V., 2000. Reversible metabolic depression in hepatocytes of lamprey (Lampetra fluviatilis) during pre-spawning: regulation by substrate availability. Comp. Biochem. Physiol. B 127, 147–154.
Gamper, N.L., Savina, M.V., Brailovskaya, I.V., Vereninov, A.A., 2001. Respiration rates, ATP content and ionic regulation in hepatocytes of starving lamprey during the prespawning period of their life cycle. J. Fish Biol. 58, 230–239. Halestrap, A.P., Denton, R.M., 1975. The specificity and metabolic implications of the inhibition of pyruvate transport in isolated mitochondria and intact tissue preparations by alpha-cyano-4-hydroxycinnamate and related compounds. Biochem. J. 148, 97–106. Hardy, K.M., Burnett, K.G., Burnett, L.E., 2013. Effect of hypercapnic hypoxia and bacterial infection (Vibrio campbellii) on protein synthesis rates in the Pacific whiteleg shrimp, Litopenaeus vannamei. Am. J. Physiol. Regul. Integr. Comp. Physiol. 305, R1356–R1366. Hochachka, P.W., Lutz, P.L., 2001. Mechanism, origin, and evolution of anoxia tolerance in animals. Comp. Biochem. Physiol. B 130, 435–459. Hochachka, P.W., Buck, L.T., Doll, C.J., Land, S.C., 1996. Unifying theory of hypoxia tolerance: molecular/metabolic defense and rescue mechanisms for surviving oxygen lack. Proc. Natl. Acad. Sci. U. S. A. 93, 9493–9498. Hylland, P., Milton, S., Pek, M., Nilsson, G.E., Lutz, P.L., 1997. Brain Na+/K+-ATPase activity in two anoxia tolerant vertebrates: crucian carp and freshwater turtle. Neurosci. Lett. 235, 89–92. Jibb, L.A., Richards, J.G., 2008. AMP-activated protein kinase activity during metabolic rate depression in the hypoxic goldfish, Carassius auratus. J. Exp. Biol. 211, 3111–3122. Konovalova, S.A., Savina, M.V., Nikiforov, A.A., 2011a. Changes in intracellular Ca2+ concentration in hepatocytes of the Baltic lamprey (Lampetra fluviatilis L.) at the period of prespawning migration. J. Evol. Biochem. Physiol. 47, 204–206. Konovalova, S.A., Zubatkina, I.S., Savina, M.V., Nikiforov, A.A., 2011b. Intracellular pH in hepatocytes of the river lamprey Lampetra fluviatilis L. at the period of prespawning migration. J. Evol. Biochem. Physiol. 47, 389–391. Konovalova, S.A., Savina, M.V., Nikiforov, A.A., Puchkova, L.V., 2012. Mitochondrial and lysosomal pathways of lamprey (Lampetra fluviatilis L.) hepatocyte death. J. Evol. Biochem. Physiol. 48, 510–515. Krumschnabel, G., Schwarzbaum, P.J., Lisch, J., Biasi, C., Wieser, W., 2000. Oxygendependent energetics of anoxia-tolerant and anoxia-intolerant hepatocytes. J. Exp. Biol. 203, 951–959. Land, S.C., Hochachka, P.W., 1994. Protein turnover during metabolic arrest in turtle hepatocytes: role and energy dependence of proteolysis. Am. J. Phys. 266, C1028–C1036. Land, S.C., Buck, L.T., Hochachka, P.W., 1993. Response of protein synthesis to anoxia and recovery in anoxia-tolerant hepatocytes. Am. J. Phys. 265, R41–R48. Larsen, L.O., 1978. Subtotal hepatectomy in intact or hypophysectomized river lampreys (Lampetra fluviatilis L.): effects on regeneration, blood glucose regulation, and vitellogenesis. Gen. Comp. Endocrinol. 35, 197–204. Larsen, L.O., 1980. Physiology of adult lampreys, with special regard to natural starvation, reproduction, and death after spawning. Can. J. Fish. Aquat. Sci. 37, 1762–1779. Meijer, A.J., Williamson, J.R., 1974. Transfer of reducing equivalents across the mitochondrial membrane. I. Hydrogen transfer mechanisms involved in the reduction of pyruvate to lactate in isolated liver cells. Biochim. Biophys. Acta 333, 1–11. Mewes, K.R., Latz, M., Golla, H., Fisher, A., 2002. Vitellogenin from female and estradiolstimulated male river lampreys (Lampetra fluviatilis L.). J. Exp. Zool. 292, 52–72. Mommsen, T.P., Moon, T.W., Walsh, P.J., 1994. Hepatocytes: Isolation, Maintenance and Utilization. In: Hochachka, P.W., Mommsen, T.P. (Eds.), Biochemistry and Molecular Biology of Fishes 3. Elsevier Science, Amsterdam, pp. 355–373. Pakay, J.L., Withers, P.C., Hobbs, A.A., Guppy, M., 2002. In vivo downregulation of protein synthesis in the snail Helix apersa during estivation. Am. J. Physiol. Regul. Integr. Comp. Physiol. 283, R197–R204. Rognstad, R., Katz, J., 1972. Gluconeogenesis in the kidney cortex. Quantitative estimation of carbon flow. J. Biol. Chem. 247, 6047–6054. Rolfe, D.F., Brown, G.C., 1997. Cellular energy utilization and the molecular origin of standard metabolic rate in mammals. Physiol. Rev. 77, 731–756. Savina, M.V., Derkachev, E.F., 1983. Switch on and switch off phenomenon of liver gluconeogenic function in lamprey (Lampetra fluviatilis L.) under the influence of season and temperature. Comp. Biochem. Physiol. 75B, 531–539. Savina, M.V., Gamper, N.L., 1998. Respiration and adenine nucleotides of Baltic lamprey (Lampetra fluviatilis L.) hepatocytes during spawning migration. Comp. Biochem. Physiol. B 120, 375–383. Savina, M.V., Konovalova, S.A., Zubatkina, I.S., Nikiforov, A.A., 2011. Reversible metabolic depression in lamprey hepatocytes during prespawning migration: dynamics of mitochondrial membrane potential. Comp. Biochem. Physiol. B 160, 194–200. Shirokova, A.V., 2007. Apoptosis. Signaling pathways and cell ion and water balance. Cell Tissue Biol. 1, 215–224. Storey, K.B., Storey, J.M., 2004. Metabolic rate depression in animals: transcriptional and translational controls. Biol. Rev. Camb. Philos. Soc. 79, 207–233. Watford, M., Vinay, P., Lemieux, G., Gougoux, A., 1980. The regulation of glucose and pyruvate formation from glutamine and citric-acid-cycle intermediates in the kidney cortex of rats, dogs, rabbits and Guinea pigs. Biochem. J. 188, 741–748. Wieser, W., Krumschnabel, G., 2001. Hierarchies of ATP-consuming processes: direct compared with indirect measurements. Biochem. J. 355, 389–395. Wilkie, M.P., Pamenter, M.E., Alkabie, S., Carapic, D., Shin, D.S., Buck, L.T., 2008. Evidence of anoxia-induced channel arrest in the brain of the goldfich (Carassius auratus). Comp. Biochem. Physiol. C 148, 355–362. Zubatkina, I.S., Konovalova, S.A., Savina, M.V., Nikiforov, A.N., 2011. Membrane potential of mitochondria in hepatocytes of the river lamprey Lampetra fluviatilis at periods of metabolic depression and activity. J. Evol. Biochem. Physiol. 47, 97–99.