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J Physiol. Apr 15, 2004; 556(Pt 2): 369–385.
Published online Jan 30, 2004. doi:  10.1113/jphysiol.2003.058420
PMCID: PMC1664938

Induction, modification and accumulation of HSP70s in the rat liver after acute exercise: early and late responses

Abstract

Liver cells synthesize HSP72, the cytosolic highly stress-inducible member of the 70 kDa family of heat-shock proteins (HSP70s), in response to acute exercise. This study was aimed at obtaining further insight into the physiological relevance of the hepatic stress response to exercise by investigating the induction and long-term maintenance of increased levels of HSP70s of the HSP and glucose-regulated protein (GRP) families, their post-translational modifications during or after exercise and the possible relation of HSP induction to oxidative stress. In a running rat model, acute exercise activated the synthesis and accumulation of HSP72, GRP75 and GRP78 in liver cells, pointing towards a multifactorial origin of this response. A peak HSP72 accumulation was observed shortly after exercise as a result of transcriptional activation. HSP72 was reduced shortly after exercise preceding the disappearance of its mRNA. Two further waves of HSP72 accumulation peaked 8 and 48 h after exercise without transcriptional activation. A transient increase in the proportion of acidic variants of HSP72 and HSP73 was also observed shortly after exercise as a result, at least in part, of protein phosphorylation. Free and protein-bound lipid peroxidation derivatives (TBARS) showed a tendency to increase in the early post-exercise and the free-to-protein-bound TBARS ratio decreased significantly after 2 h. During the early post-exercise period, protein-bound TBARS correlated positively with HSP72 and 73, but not with GRP75 or GRP78. Altogether, the reported results indicate that the early induction and post-translational modification of HSP70s in liver cells following exercise is a preliminary step of a series of long-lasting HSP70-related events, possibly designed to preserve liver cell homeostasis and to help provide a concerted response of the whole organism to physical stress.

The cellular stress response is an evolutionarily conserved defence mechanism characterized by a transcriptionally controlled induction of the synthesis and accumulation of heat-shock or stress proteins (HSPs) following exposure of cells to high temperatures or other environmental challenges (Welch, 1992; Morimoto, 1993). It is considered to be aimed at controlling the perturbation of homeostasis, protecting cells from injury, helping them to restore cellular function and enabling them to survive new stress situations (Gething & Sambrook, 1992). Underlying this protective effect is the ability of HSPs to act as molecular chaperones, interacting with denatured proteins and helping them to restore their native structure and their function (Buchner, 1996). The stress proteins of the family of 70 kDa (HSP70s) are amongst the most highly induced proteins of the cellular stress response in mammals (Welch, 1992). HSP72 and HSP73 are sensitive to a variety of stressors, including hyperthermia and ischaemia, while the glucose-regulated proteins, GRP75 and GRP78, are more affected by glucose deprivation or other treatments that perturb N-linked glycosylation of nascent polypetides (Pouyssegur et al. 1977; Munro & Pelham, 1986). All four HSP70s share the property of binding ATP and polypeptides while displaying different subcellular distribution and a different degree of constitutive expression (Welch & Feramisco, 1984; Beckmann et al. 1990; Sambrook, 1990). HSP72 (HSP70) and HSP73 (heat-shock cognate, HSC70) have been located within the cytoplasm and, following stress, in the nucleus. GRP78 (also known as BiP) is an endoplasmic reticulum-resident protein that binds different proteins crossing through this compartment, in addition to calcium ions (Sambrook, 1990; Villa et al. 1991). GRP75, also known as mitochondrial HSP70, is involved in the import of precursor proteins into the mitochondria, where it has been shown to interact functionally with HSP60 and other HSPs to facilitate protein folding and oligomerization of protein complexes (Mizzen et al. 1991). HSP73 is expressed constitutively to relatively high levels in different skeletal muscle types and other tissues and is usually only slightly inducible by stress, although its expression may increase considerably under the influence of proliferative stimuli both in fibroblasts and liver cells (Sorger & Pelham, 1987). In contrast, HSP72 usually displays low basal expression levels but is highly inducible in response to stress. Slow-twitch muscle fibres expressing type I myosin heavy chain show exceptionally high levels of HSP72 when compared with myocardium, fast-twitch skeletal muscle and liver (Locke et al. 1991; Hernando & Manso, 1997), suggesting that HSP72 performs a specific constitutive function in skeletal muscle that is mainly prevalent in slow-twitch fibres.

Acute exercise is known to induce HSP expression in different tissues, including skeletal muscle, heart, liver and brain and in leucocytes (see Locke, 1997; Fehrenbach & Niess, 1999, for a review). A variety of conditions that may be encountered during exercise, like hypoxia and metabolic stress (Iwaki K., 1993; Miller et al. 2002), hyperthermia (Cairo et al. 1985; Lindquist, 1986), oxidative (Storz et al. 1990) or mechanical (Knowlton et al. 1991) stress, changes in the intracellular calcium ion concentration and glucose deprivation (Welch et al. 1983; Lee, 2001), are known to induce the cellular stress response and could cause HSP induction following exercise. In addition, cytokines have also been suggested as possible regulators of HSPs (Fehrenbach & Niess, 1999). Because of its central role in locomotion, the cellular stress response to exercise has been studied with preferential attention in skeletal muscle (Locke et al. 1990; Ryan et al. 1991; Salo et al. 1991; Skidmore et al. 1995; Hernando & Manso, 1997; Fehrenbach & Niess, 1999; Febbraio et al. 2002b). An induction of the synthesis and accumulation of HSP72, GRP75, GRP78 and HSP60 was reported in rat muscle (Hernando & Manso, 1997) indicating that exercise activates the expression of heat-shock-inducible proteins (HSPs) as well as glucose-regulated proteins (GRPs). The nature of the exercise-activated signal responsible for HSP induction in skeletal muscle has been investigated in various reports (Skidmore et al. 1995; Hernando & Manso, 1997; Febbraio et al. 2002b; Mitchell et al. 2002; Khassaf et al. 2003). Since the accumulation of HSP72 and GRP75 (mtHSP70) in skeletal muscle occurred independently of core body temperature (Skidmore et al. 1995; Mitchell et al. 2002) and HSP72 induction was also observed following concentric (non-damaging) exercise (Khassaf et al. 2001), neither heat nor mechanical stress alone have been suggested to play a major role. Oxidative stress (Smolka et al. 2000; Khassaf et al. 2003) and reduced glycogen availability (Febbraio et al. 2002b) have been proposed more recently as main determinants of HSP72 induction following exercise in skeletal muscle.

Liver cells also induce HSP expression in response to acute exercise (Locke, 1990; Salo et al. 1991; Kregel et al. 1995). HSP72 expression seems to be a relevant mechanism of hepatic cell defence against stress as indicated by the observation that attenuation of HSP72 induction with increasing age correlates with reduced thermotolerance when compared with young counterparts (Hall et al. 2000). Thermal stress, however, does not appear to be a major determinant of the stress response to exercise in the liver since its response to hyperthermia alone was blunted with advancing age while exercise-induced hyperthermia elicited a robust HSP72-response even in older rats (Kregel & Moseley, 1996).

Given the essential roles played by liver cells during exercise, particularly in energy metabolism and protein secretion, we sought to investigate the hepatic stress response to exercise to get further insight into its physiological relevance. The first question we posed was whether this response also involved induction of GRPs, in addition to HSPs, as previously observed in skeletal muscle (Hernando & Manso, 1997), and how long liver cells maintained up-regulated HSP70 levels. A kinetic analysis of the induction of synthesis and accumulation of HSP70s at both the RNA and protein level during early and late post-exercise would answer these questions and therefore this was the first aim of this investigation. Due to the role of liver cells in maintaining glucose homeostasis during exercise (Issekutz et al. 1970; Kjaer, 1998; Bergeron et al. 2001; Miller et al. 2002) we hypothesized that exercise would induce hepatic HSP70s of the GRP-family (GRP75 and 78) in addition to HSP72. As suggested from the observation of increased levels of HSP72 in human sera following exercise, probably originating from the hepatosplanchnic tissue beds (Febbraio et al. 2002a), we also hypothesized that, in the running rat model, the accumulation of HSP72 post-exercise could be short-lived if the protein was released from hepatic cells into the circulation.

The presence of different charge variants of HSP72 and HSP73 in skeletal muscle fibres (Guerriero et al. 1989; Hernando & Manso, 1997) and their transitions shortly after exercise suggested a role for HSP72 phosphorylation as an early event of the stress response of skeletal muscle fibres to exercise (Hernando & Manso, 1997). The information relative to the presence, distribution and transitions of HSP70 variants in liver cells would contribute to understanding their possible relevance for synthesis, maturation, stabilization of increased protein levels or functioning of HSP70s. Therefore, the second aim of this investigation was the characterization of HSP70 variants in liver cells and the analysis of their transitions following exercise. We hypothesized that the molecular variants of HSP70s are the result of post-translational modifications inherent to the action mechanism of HSP70s that should be relevant to the hepatic stress response to exercise.

Oxidative stress, derived from an increased production of reactive oxygen species, has been implicated in the damaging effects of acute exercise in the liver (Davies et al. 1982; Jackson et al. 1985; Alessio & Goldfarb, 1988; Ji, 1999). A reduced blood supply (Granger & Korthuis, 1995) and/or an early decrease in hepatic reduced glutathione content (Lu et al. 1990), among other factors, could explain the early induction of oxidative stress (Di Meo & Venditti, 2001) and HSP70 expression during or following exercise in liver cells. Therefore, a third aim of this investigation was to assess oxidative stress indices during and after exercise and to test their possible relation to HSP70 expression.

Methods

Experimental animals and exercise protocol

Nineteen-week-old male Wistar rats served as experimental animals. The animals were bred and housed in the animal facilities of the Centre of Molecular Biology at the Autonomous University of Madrid, where they were housed in a temperature- (22–24°C) and humidity- (50–60%) controlled environment, with a 12 h photoperiod (8 a.m. to 8 p.m.), and provided with a standard laboratory diet (Panlab standard diet) and water ad libitum. Before exercising, rats were habituated to the motor-driven treadmill (Li 8706, Letica Scientific Instruments, Barcelona, Spain) by running at 10–20 m min−1 for 10 min a day during 5 days. After 3 days of total rest, they performed a single exercise bout consisting of 10 min of running at 15 m min−1 followed by 50 min at 27 m min−1, without incline. Two different experiments were performed, shown schematically in Fig. 1. After the exercise bout animals were returned to cages and were provided with water and food ad libitum. Immediately after exercise or at the indicated post-exercise periods, animals were anaesthetized either intraperitoneally with 0.2 ml per 100 g body weight of a mixture of 25 mg ml−1 ketamine, 2 mg ml−1 diazepan and 0.1 mg ml−1 atropine (first experiment) or with an isoflurane-based (induction: 2 l min−1 O2, 1 l min−1 N2O and 3.5% isoflurane for 2 min; maintenance: 0.8 l min−1 O2, 0.4 l min−1 N2O and 2.5% isoflurane) gaseous anaesthesia (second experiment) for sample collection. The experiments were approved by the Comité de Ética de la Investigación de la Universidad Autónoma de Madrid and the interventions were performed under the advice of specialized personnel following the recommendations included in the Guide for Care and Use of Laboratory Animals (US Department of Health and Human Services, NIH) and European laws and regulations on the protection of animals.

Figure 1
Experimental model and design used to investigate the induction of HSP70s during the hepatic response to acute exercise

Sample collection

After the quick dissection of various leg muscles, the liver was perfused with approximately 10 ml of cold saline and the right medial lobe extracted. Immediately after killing the animals by cervical dislocation, the myocardium was also obtained aseptically. A piece of the liver medial lobe was sliced for metabolic labelling. All organs were rapidly soaked in cold sterile saline, wiped free of water, weighed and frozen immediately in liquid N2 The remainder of the lobe was also frozen immediately in liquid N2 and stored at −70°C until used for measuring mRNA and protein expression levels as well as indices of lipid peroxidation.

Metabolic labelling and estimation of the rates of synthesis of stress proteins

The relative rate of synthesis of hepatic stress proteins was measured in 0.75 mm thick slices prepared immediately after extracting the liver by cutting a small piece consecutively in two perpendicular planes with a tissue chopper (The Mickle Laboratory Engineering Co. Ltd, Gomshall, Surrey, UK). Synthesis of stress proteins in liver slices was performed essentially as previously described for skeletal muscle (Hernando & Manso, 1997). Briefly, liver sections were equilibrated for 30 min in Dulbecco's modified Eagle's medium without methionine under cell culture conditions, which was changed twice, followed by incubation for 2 h in the same medium containing 35S-labelled methionine. The relative rates of synthesis of stress proteins were determined by fluorographic analysis of radiolabelled stress proteins separated from whole liver homogenates by two-dimensional electrophoresis. The incorporation of radioactivity into actin was used as an internal reference to compare rates of synthesis of HSPs in different samples. Since liver sections maintain their metabolic activity until frozen, synthesis rate data did not correspond temporarily with the HSP levels determined in samples frozen immediately after extraction. Thus, plotting of the former was shifted with respect to kill time. A horizontal discontinuous line indicates in the corresponding figure the time period of incubation with radioactive methionine. Under the experimental conditions used in this investigation, no appreciable induction of HSP synthesis merely due to tissue slicing was observed.

One- and two-dimensional electrophoresis of proteins, fluorography and immunoblotting procedures

Stress proteins were separated electrophoretically from whole liver homogenates prepared in 10 mm KCl, 10 mm Tris/HCl, pH 7.6, 180 mm 2-mercaptoethanol, 50% glycerol and 1 mm phenylmethylsulphonylfluoride. After diluting the homogenates to 1–2 mg ml−1 with 0.1 N NaOH, the protein content was determined by the Bradford assay using bovine serum albumin as a standard. For one-dimensional (1D) electrophoresis 80 µg protein was loaded per slot. This amount was reduced for the quantification of the more abundant HSPs. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate was performed using 12% acrylamide gels polymerized in the presence of 15% glycerol. Two-dimensional (2D) electrophoresis of HSPs was performed using a pH range of 4–6.5 in the first, isoelectric focusing dimension, as reported previously (Hernando & Manso, 1997). The radioactivity incorporated into HSPs was detected by fluorography (Chamberlain, 1979) using X-Omat AR film (Kodak). Proteins separated in polyacrylamide gels were transferred electrophoretically (Bio-Rad Trans-Blot Cell) to nitrocellulose membranes (0.2 µm, Schleicher and Schuell, Dassell, Germany). The membranes were stained with Ponceau S to visually control protein transfer and to mark the position of reference proteins, blocked with 5% non-fat dried milk in Tris-buffered saline, washed and processed for specific recognition of the different heat shock proteins using monoclonal antibodies. The peroxidase-conjugated secondary antibody was detected by enhanced chemiluminescence (ECL, Amersham) using CuriX RP2 film (Agfa). Quantification of radioautographs was performed by densitometry using a Laser densitometer (Molecular Dynamics, Image Quant Software v.3.0) or a Calibrated Imaging Densitometer GS-710 (Bio-Rad) with Quantity One v 4.1.1 software. The linear absorption range was assessed using increasing amounts of various stress proteins, including HSP72 and HSP73, and their corresponding antibodies. Various exposures of the same blot were usually made to guarantee that measurements were performed in the linear range.

Characterization of phosphorylated variants of cytosolic HSP70s by treatment with alkaline phosphatese and calcineurin

The presence of phosphorylated forms of the cytosolic HSP70s was assessed by analysing the transitions towards less acidic isoelectric points after incubating whole liver homogenates with alkaline phosphatase or calcineurin (exogenous dephosphorylation) or in the absence of added phosphatase (endogenous dephosphorylation) prior to separating the proteins by 2D electrophoresis. Alkaline phosphatase treatment was performed essentially as previously described (Gonzalez et al. 2002). For treatment with calcineurin, 0.2 ml samples of whole liver homogenate, at a protein concentration of 1.5 mg ml−1 in 20 mm Hepes/KOH, pH 7.4, 5 mm MgCl2, 1 mm CaCl2, were incubated at 36.5°C for 2 h with 1 unit each of bovine brain calcineurin (Sigma) and calmodulin, in the presence of a protease inhibitor mix. To confirm that the variant transitions observed upon incubation of liver homogenates, with or without added protein phosphatase should be effectively assigned to phosphatase activity, incubations were also performed in the presence of a phosphatase inhibitor mix containing 10 mm glycerol-2-phosphate, 1 mm sodium orthovanadate, 1 mm sodium pyrophosphate, 4 mm sodium tartrate and 5 mm NaF. After the incubations the protein was precipitated in cold acetone and the samples were prepared for the first, isoelectric focusing dimension of 2D electrophoresis.

Monoclonal antibodies

The following antibodies were used throughout the experiments, at the indicated dilution: monoclonal antibodies anti-HSP70 (mouse clone C92F3 A-5, StressGen SPA-810, at a 1 : 250 dilution; StressGen, Victoria, BC, Canada) and anti-HSC70 (rat clone 1B5, StressGen SPA-815; dilution 1 : 1000), for the recognition of the inducible and constitutively expressed cytosolic-nuclear HSP70, respectively, and anti heat-shock protein 70 (mouse clone BRM-22, Sigma, dilution 1 : 5000) for the simultaneous recognition of the molecular variants of both proteins in 2D-electrophoretograms. The monoclonal antibodies anti-GRP75 (mouse clone 30A5, StressGen SPA-825, dilution of 1 : 600–1000) and anti-GRP78 (mouse clone 10C3, StressGen SPA-827, dilution 1 : 250) were used to recognize the mitochondrial and endoplasmic reticulum lumenal members of HSP70s, respectively. Peroxidase-conjugated polyclonal goat antirabbit IgG (Transduction Laboratories, Canada), rabbit antimouse and rabbit antirat immunoglobulins (Dako, Denmark) were used as secondary antibodies, according to required specificity, at a 1 : 1500–2500 dilution.

RNA isolation and Northern and slot blot hybridization analysis

Total RNA was obtained from frozen liver samples (60 mg) using a commercial purification kit (Tripure Isolation Reagent, Roche) based on a previously described procedure (Chomczynski & Sacchi, 1987) according to the manufacturer's instructions. The amount of RNA extracted was typically around 4 µg per mg tissue, with 260–280 nm absorbance ratios from 1.8 to 1.9. For Northern blot analysis, 20 µg of formaldehyde-denatured RNA samples were separated by electrophoresis in 1% agarose gels containing 2.2 m formaldehyde. RNA was transferred to a nylon membrane (Hybond-N+, Amersham Pharmacia Biotech) by capillarity in 20 × SSC buffer (SSC is 0.15 m NaCl, 0.15 m sodium citrate, pH 7.0) and linked to the membrane by exposing it for 15 s to ultraviolet light in a Spectrolinker XL-100 UV-crosslinker (Spectronics Corp., Lincoln, NE). Once established by Northern blotting that the HSP72 cDNA-probe hybridized with a single mRNA band, the levels of HSP72 mRNA in liver samples were also determined by slot blot hybridization (White & Bancroft, 1982) using a Minifold II (Schleicher & Schuell). For this, 20 µg of total liver RNA, previously heated for 15 min at 68°C in SSC containing 50% formamide and 7% formaldehyde, were applied per sample on a positively charged nylon membrane and cross-linked by ultraviolet radiation.

Membranes were prehybridized in a hybridization-oven (Biometra, Germany) at 50°C for 6 h using a standard prehybridization buffer containing 50% deionized formamide, 2% blocking agent (Roche Diagnostics), 0.1%N-lauryl-sarcosine and 0.02% sodium lauryl sulphate in 5 × SSC. Hybridization was performed overnight under rotation at 44°C in the same buffer also containing 2.5 ng ml−1 of the digoxigenin (DIG)-labelled cDNA-probe. Membranes were washed and processed for the chemiluminiscent detection of the DIG-labelled probe using an alkaline phosphatase-based DIG-luminescent detection kit (Roche Diagnostics) following the manufacturer's instructions. Different exposures were made to guarantee measurements to be performed in the linear absorption range.

Preparation of a cDNA probe for HSP72 mRNA

A 220-bp region of maximal specificity within the coding sequence of the human HSP72 gene was identified by comparison with different HSP70 genes using the DNA-Star programme (DNASTAR Inc., Madison, WI, USA). The selected region of the human HSP72-gene, encompassing nucleotides 2082–2301, was 92% identical to the homologous rat sequence. This fragment was amplified by PCR from a human cDNA template (kindly provided by Dr J. M. Requena, Center of Molecular Biology, CSIC-UAM, Autonomous University of Madrid), using the oligonucleotides 5′-GGAATTCGCGAGAGGGTGTCAGCC-3′ and 5′-CCTTAAGCCCAATGTGTGGACGAG-3′ as primers. The single PCR-product of 220 bp was labelled with digoxigenin using the DIG random-primed DNA labelling kit (Roche Diagnostics), following the instructions provided by the manufacturer.

Quantification of thiobarbituric acid reactive substances (TBARS) as indices of oxidative stress

Presence of malondialdehyde (MDA) and other lipid peroxidation products was assessed by the formation of pink-coloured adducts with thiobarbituric acid, with absorbance maxima at 532–534 nm. Determinations were performed for free- and protein-bound TBARS. Free TBARS were measured in the clear supernatants resulting from precipitating liver homogenates (prepared 1 : 10 w/v in 1.15% KCl at 4°C) in cold 6% TCA and washing with the same volume of cold 6% TCA. The washed protein sediment was used for determining protein-bound TBARS. The adducts were measured by a previously described method (Esterbauer & Cheeseman, 1990) using malondialdehyde, generated from a solution of malonaldehyde bis(dimethyl acetal) (Aldrich, Germany), as a standard. To increase the assay specificity, the MDA equivalents of TBARS were calculated from the absorbance at 532 nm, after subtracting that at 520 nm (Mihara & Uchiyama, 1978), using a molar extinction coefficient of epsilon= 153 mm−1 cm−1 at 532 nm for the MDA adduct.

Statistical analysis

Levels of stress proteins after a single exercise bout were expressed as a percentage of the means for the non-exercised group (arbitrarily assigned the 100% value). Groups of exercised and non-exercised animals were compared by means of a one-factor ANOVA using the statistical program SPSS 11.0. When the F-value indicated the existence of statistically significant differences among groups (P < 0.05), comparisons between individual groups were conducted using the Bonferroni post hoc test when the homogeneity of variance was accepted (Levene statistic), and a Dunnett T3 test when the homogeneity of variance was discarded. Statistical significance was accepted when P < 0.05. Correlations between variables were calculated using Pearson's correlation test (significance denoted by P < 0.05) using the same statistical programme.

Results

Induction kinetics of synthesis and accumulation of HSP70s in the rat liver immediately after a single exercise bout

Following 1 h of treadmill running, the hepatic rates of synthesis and the expression levels of HSP72, GRP75 and GRP78 increased. Animals anaesthetized and killed within the shortest possible time after exertion showed increased levels of HSP70s of the HSP and GRP families (Fig. 2). The constitutively expressed, cytosolic protein HSP73 only showed a trend towards increasing without changing in a statistically significant manner. HSP72 levels peaked 2 h post-exercise at a mean value of 5 times the control. Its abundance was reduced 4 h post-exercise. GRP75 and GRP78 maintained relatively constant, increased levels during the first 4 h post-exercise.

Figure 2
Post-exercise accumulation kinetics of HSP70s in the liver of sedentary rats

Consistent with their accumulation kinetics, the hepatic rates of synthesis of HSP70s increased immediately after exercising (Fig. 3) and at the next time point they were either below or essentially the same as before exercise. As shown for HSP72, the early induction of synthesis and accumulation of HSP70s following exercise was under transcriptional control (Fig. 4). HSP72 mRNA levels were very low or non-detectable in the liver of unstressed animals, reached relatively high values immediately after the exercise bout, with a maximum at 1 h post-exercise, and were reduced in most animals to practically undetectable levels at 4 h post-exercise.

Figure 3
Induction kinetics of synthesis of HSP70s in the liver following a single exercise bout
Figure 4
Induction of the HSP72 mRNA observed by Northern blot hybridization

Late response of the hepatic cytosolic HSP70s to a single exercise bout

Further insight into the hepatic HSP72 response to acute exercise was obtained from a second experiment. In this protocol, liver samples were obtained after 0.5 and 1 h of treadmill running, and longer post-exercise measurements were performed, covering 48 h. Gas anaesthesia was used to minimize the lapse between experimental time points and real times of sample collection. Analysis of the hepatic levels of HSP73 over the post-exercise period showed a trend towards increasing, but no statistically significant changes were observed either immediately after exercise or at later times (not shown). Both GRP75 and GRP 78 showed a tendency to decrease over the late post-exercise period (not shown). However, as shown in Fig. 5 for the accumulation kinetics of protein (Fig. 5A) and mRNA (Fig. 5B), HSP72 showed complex changes over the post-exercise period. The following conclusions were derived from the data presented in this figure. (1) After 30 min of treadmill running, HSP72 mRNA and protein levels were increased. (2) The maximal accumulation of HSP72 mRNA occurred 30 min after exercise and after this point, mRNA levels decreased along the next 3 h reaching non-detectable levels that persisted at least until 48 h post-exercise. (3) HSP72 protein showed a peak-accumulation 0.5 h post-exercise, coincident with that of its mRNA. HSP72 was reduced to control levels at 2 h post-exercise, thus preceding mRNA levels. (4) Two further waves of HSP72-accumulation, peaking around 8 h and 48 h post-exercise, were observed later in the absence of detectable HSP72 mRNA levels. The high standard deviation of the means observed in the peaks of both late waves of HSP72 accumulation indicated a considerable degree of inter individual variation indicative of either asynchrony or a highly variable response.

Figure 5
Accumulation of HSP72 protein and mRNA in the liver of sedentary rats immediately after a single exercise bout and along an extended time period

Changes in hepatic lipid peroxidation indices immediately following exercise and at different time points along the post-exercise period

Induction of oxidative stress in liver cells following a single exercise bout was tested by measuring the formation of adducts with thiobarbituric acid in both the 6% cold TCA-extractable (free TBARS) and in the TCA-precipitable (protein-bound TBARS) material of the same homogenate. During the early post-exercise period both free and protein-bound TBARS showed a trend towards increasing though differences did not reach statistical significance (Fig. 6). The free-to-protein-bound TBARS ratio, however, was lower than in controls after 2 and 4 h. Considering paired data from the first four post-exercise hours, the levels of protein-bound TBARS were found to correlate directly with accumulated HSP72 (0.642, P < 0.001) and HSP73 (0.638, P < 0.001), but not with GRP75 or GRP78. No correlation was observed between free TBARS and HSPs or GRPs.

Figure 6
Effect of acute exercise on the levels of free and protein-bound TBARS in the rat liver

Free TBARS did not increase along the late post-exercise period. However, mean levels of protein-bound TBARS were found to increase in a statistically significant manner 48 h post-exercise (not shown), coincidently with a burst of the mean accumulated HSP72. However, animal by animal lipid peroxidation indices did not correlate with HSP72 expression levels in either peak of late HSP72-accummulation, suggesting that in the late post-exercise these events were not related to each other.

Transitions among charge variants of the hepatic cytosolic HSP70s following a single exercise bout

Using a monoclonal antibody that simultaneously recognized both proteins, at least three isoelectric point variants of each of the cytosolic HSP70s were detected in immunoblots of whole liver homogenates separated by two-dimensional electrophoresis. In non-stressed animals the hepatic HSP72 levels were very low and usually only the major variant was detected. Following induction, however, the pattern of variants of HSP72 was similar to that of HSP73, with a major variant (denoted 1) flanked by one more acidic (denoted 2) and another less acidic (denoted 3) (Fig. 7A). Variants 2 and 3 of HSP73 displayed a separate behaviour following exercise. While the relative amount (referred to variant 1) of HSP733 did not change during the early post-exercise period, HSP732 increased transitorily shortly after exercise (Fig. 7B). HSP722 also showed a transient increase during the early post-exercise, when the major variant also increased. A peak in the HSP722-to-HSP721 ratio shortly after exercise (Fig. 7C), indicated that the generation of the acidic variant was an early response of hepatic cells to exercise stress.

Figure 7
Presence and distribution of charge variants of the cytosolic HSP70s in the liver of rats under unstressed conditions and following a single exercise bout

Analysis of the abundance of the different variants of the hepatic cytosolic HSP70 in the peaks of late HSP72 accumulation (referred to the major variant of HSP73, the level of which did not change along the post-exercise period) and of their percent distribution within each protein (Fig. 8), indicated that: (1) the proportion of HSP732 was very low at 8 h post-exercise but was present at 48 h at about the same proportion than immediately after exercise; (2) HSP733 was absent in both peaks of late accumulation of HSP72; and (3) The amounts of HSP721 and HSP722 increased in the peaks of late HSP72 accumulation; HSP721 reached its maximum 8 h post-exercise while HSP722 was maximal at 48 h. In the late post-exercise period the amount of immunodetectable HSP72 surpassed that of its constitutively expressed counterpart.

Figure 8
Relative abundance and proportions of variants of rat hepatic cytosolic HSP70s in the peaks of the waves of HSP72 accumulation in early and late post-exercise

Molecular mechanism of generation of variants of the cytosolic HSP70s

To establish whether the generation and transitions among molecular variants of the cytosolic HSP70s involved phosphorylation/dephosphorylation processes, whole liver homogenates displaying high HSP72 levels (48 h post-exercise) were incubated with alkaline phosphatase or calcineurin and the distribution of variants in treated and non-treated samples was analysed following separation by 2D electrophoresis (Fig. 9). Both cytosolic HSP70s showed the same distribution of variants following treatment with either type of protein phosphatase, though the effect was slightly more pronounced on HSP72 than on HSP73. In both proteins, the proportion of the major variant did not change appreciably with phosphatase treatment, while the most acidic variant diminished in the same proportion as the less acidic one increased (Fig. 9). Incubation of samples at 36.5°C without added phosphatase (endogenous dephosphorylation) resulted in essentially the same transitions as following its addition (not shown). Using whole liver homogenates from animals killed 8 h post-exercise, also displaying high HSP72 levels but with a reduced proportion of variant 2 (Fig. 10), it was confirmed that the increase in the abundance of the less acidic variant of HSP72 and HSP73, observed upon incubation with or without added alkaline phosphatase or calcineurin, was effectively dependent on phosphatase activity. Addition of phosphatase together with a phosphatase inhibitor mix (10 mm glycerol-2-phosphate, 1 mm sodium orthovanadate, 1 mm sodium pyrophosphate, 4 mm sodium tartrate and 5 mm NaF) prevented the transition towards the less acidic variant (Fig. 10). However, contrary to expectation, the relatively small fraction of the most acidic variant present in these liver samples increased following incubation with either phosphatase, and this effect was not prevented by adding the phosphatase inhibitor mix.

Figure 9
Identification of phophorylated variants of the cytosolic HSP70s by treatment with alkaline phosphatase or calcineurin
Figure 10
Dependence on phosphatase activity of the increases in the proportions of variants 2 and 3 observed after incubating whole liver homogenates with a low proportion of variant 2 with protein phosphatase

Discussion

The biological functions currently assigned to HSPs in preserving cell homeostasis during and following stress, together with previous reports on their induction in liver cells in response to a variety of stressors (Locke, 1990; Salo et al. 1991; Schiaffonati et al. 1994; Kregel et al. 1995; Kregel & Moseley, 1996; Hall et al. 2000; Bergeron et al. 2001), suggested that the transcriptionally controlled up-regulation of HSP70s could be a relevant mechanism of hepatic cell defence to exercise stress in sedentary individuals, a notion that is supported by the data reported in this paper. In addition, it has also been observed that the hepatic stress response to acute exercise did not culminate with the transient accumulation, post-translational modification and, possibly, release of HSP72 in the early post-exercise, since liver cells maintained a close, discontinuous relationship to HSP72 during at least 48 h post-exercise.

Induction of the glucose regulated proteins GRP75 and GRP78, in addition to HSP72, was the first evidence that the stress response of liver cells to exercise may have a multifactorial origin. The observation that during the early post-exercise the levels of protein-bound TBARS correlated with HSP72 and 73 but not with GRPs adds further support to this hypothesis. In addition, GRPs and HSP72 showed subtle differences in their post-exercise accumulation kinetics. GRPs maintained their levels over time during the early post-exercise period with a tendency to lower in the late post-exercise, while the accumulation of HSP72 in the early post-exercise was transient. A short-lived up-regulation of HSP72 was contrary to expected and seemed inappropriate for a protein thought to be involved as a molecular chaperone in protecting cells from stress and conferring on them increased stress tolerance when present at increased levels in the intracellular milieu. Since the time course of induction of protein synthesis was similar for all four proteins, the short-lived accumulation of HSP72 was puzzling. An interpretation based on a rapid degradation of the protein was consistent with the very low stationary level of HSP72 normally found in liver, but was difficult to reconcile with its putative intracellular function as a molecular chaperone. The observation that the hepatic levels of HSP72 declined faster than those of its mRNA (Fig. 5) suggested that the protein was released from liver cells, in agreement with the observation of increased arterial HSP72 in humans following exercise, probably originating from hepatosplanchnic viscera (Febbraio et al. 2002a). This interpretation opens new insights into the possible physiological role of HSP72 in the response of the whole organism to exercise stress, particularly in the light of evidence suggesting that HSP72 freed from cells might have important extracellular functions acting as a signalling molecule (Guzhova et al. 1998; Asea et al. 2000b; Asea et al. 2002). Further support for the notion that HSP72 freed from liver cells could have the relevant signalling role is provided by the recent proposal in epithelial cells in culture of a mechanism explaining the targeting of HSP72 to the extracellular space, by means of sphingolipid–cholesterol-rich structures known as lipid rafts (Broquet et al. 2003).

The hepatic stress response to exercise involved the transient generation of an acidic variant of both cytosolic HSP70s (Fig. 7). This post-translational modification has been previously observed in skeletal muscle fibres and characterized as a phosphorylation process suggested to play a role in stabilizing newly synthesized proteins in active muscle (Hernando & Manso, 1997). In liver cells, however, HSP72 levels declined shortly after its post-exercise rise indicating that the transient generation of acidic variants was not related to stabilization of the intracellular protein levels. Whether this post-translational modification plays a role in the stress regulated association/dissociation of HSP72 with and from the lipoprotein structures suggested to be involved in the stress regulated release of HSP72 from epithelial cells in culture (Broquet et al. 2003) is an interesting matter for future research. Concerning the generation of charge variants of the cytosolic HSP70s, the reported results point towards protein phosphorylation as the major, if not only, mechanism accounting for this diversity. The reduction in the proportion of the most acidic variant (variant 2), concomitantly with an increase in the proportion of the less acidic one (variant 3) after phosphatase treatment of liver samples expressing a high level of HSP72 and a high proportion of variant 2 (1 h and 48 h post-exercise) is evidence that phosphorylation is involved in the generation of the most acidic of these variants. The reason why phosphatase effects were incomplete is unknown. Inaccessibility of the phosphoryl groups under the in vitro conditions used could be a reason. We interpret the results of phosphatase treatment as a net result, probably involving two successive dephosphorylation steps with variant 1 as an intermediate. Since dephosphorylation assays were performed in whole liver homogenates, where endogenous enzymes are still active, the level of protein-bound phosphoryl groups at the end of the treatment will depend on a dephosphorylation and back-phosphorylation cycle that will operate as long as endogenous ATP remains available. This is the possible reason why after incubating with protein phosphatase, only part of variant 2 disappeared. It could also explain why in samples with a low proportion of variant 2 (8 h post-exercise) phosphatase treatment resulted in an increased, rather than a reduced, proportion of this variant. Consistent with this hypothesis is also the observation that, while the increase in the proportion of variant 3 observed following phosphatase treatment was effectively dependent on phosphatase activity (as demonstrated by the absence of phosphatase effect when inhibitors of this activity were added simultaneously), the increase in the proportion of variant 2 observed in samples expressing a low percentage of this variant was phosphatase-independent (but probably the result of a phosphorylation process catalysed by endogenous kinases). The reported results do not allow the exclusion of part of variant 1 being the result of post-translational modifications other than protein phosphorylation as, for example, oxidative processes. A further possibility is that variant 2 represents an intermediate state of the cycle of ATP binding, hydrolysis, ADP–ATP exchange and release regulating the interaction of HSP70s with denatured proteins during chaperone activity.

We conclude in this paper that the transcriptionally controlled induction of HSP72 in liver cells shortly after exercise is only a prelude to of a series of long-lasting HSP72-related events occurring along the late post-exercise. The existence of two late waves of HSP72 accumulation that took place in the absence of transcriptional activation is another intriguing finding of this investigation. Whether these waves are a consequence of the inability of liver cells of sedentary animals to cope with the stressful conditions generated during the acute bout of exercise by merely inducing the stress response, or whether they represent the landmarks of a cross-talk between liver and other tissues involving HSP72 as a signalling molecule, cannot be answered at present. The existence of defined waves of HSP72 accumulation over a long period of time following exercise is difficult to reconcile with a single synthetic event. Activation of HSP72 gene expression is known to occur rapidly and transiently, preceded by the activation of the DNA-binding activity of the heat-shock factor. The absence of detectable levels of the HSP72 mRNA during the late post-exercise indicates that the secondary waves of protein accumulation were not the result of consecutive events of transcriptional activation. They would be better interpreted as the result of release and uptake of the protein out of and into hepatic cells, either as a free entity or bound to invading cells or circulating proteins. The observation of an association of HSP72 with lipid rafts and their release from epithelial cells through a pathway independent of the classical secretory route (Broquet et al. 2003) suggests the involvement of lipoprotein structures in HSP72 movements out from and into liver cells, a hypothesis that constitutes an interesting matter for future research.

Finally, concerning the possible involvement of oxidative stress in signalling the hepatic stress response to exercise, the correlation of protein-bound TBARS with HSP72/73 observed during the early post-exercise is evidence in favour of this hypothesis. Since free TBARS, i.e. malondialdehyde and other lipid peroxidation products, did not correlate with cytosolic HSP70s, the possibility of oxidative stress signalling HSP expression in hepatic cells appears to depend on protein modification. Aldehydes produced by peroxidation of lipids are known to react with proteins and other biomolecules generating stable adducts (Esterbauer et al. 1991; Uchida & Stadtman, 1992; Uchida et al. 1994). Protein-bound TBARS, representing a fraction of modified, unfolded proteins, would be able to induce the cellular stress response (Kelley & Schlesinger, 1978). As proposed by the HSP70 hypothesis of autoregulation of HSP genes, under unstressed conditions the heat-shock transcription factor HSF1 would be mainly present in the cytosol bound to HSP70s. Following stress, a rise of unfolded proteins would lead to disruption of HSP70–HSF1 complexes, because of the engagement of HSP70s in chaperone activity. HSF1, freed from its association with HSP70s, would increase its oligomerization rate and DNA-binding activity, and activation of HSP gene transcription would ensue. This model would explain why the level of cytosolic HSP70s correlated with oxidatively modified proteins, simply considering that damaged proteins are used for titration of HSP70s. Induction of HSP70 expression following small changes of oxidatively modified proteins would indicate a high degree of sensitivity that is consistent with the relevance of this cellular response. Even though no statistically significant increase in either free or protein-bound TBARS was observed in this study, the change in their ratio in the early post-exercise is an evidence of the dynamics of generation of lipid-peroxidation derivatives and of their transformation into stable protein-bound adducts, to be finally chaperoned and eventually degraded.

In conclusion, the results reported in this paper provide evidence that the hepatic stress response to acute exercise involves the synthesis and accumulation of HSP70s of the HSP and GRP families and suggest a multifactorial origin for its induction. Oxidatively modified proteins have been found to be related to HSP72/73, but not to GRP78/75. The stress response of liver cells to acute exercise did not culminate with the transient accumulation, modification and, possibly, release of HSP72 early after exercise. Liver cells maintained a close, discontinuous relationship to HSP72 during at least 48 h post-exercise, with two later waves of HSP72 accumulation taking place in the absence of transcriptional activation. The data suggest a role for hepatic HSP72 as a stress signal, possibly contributing to provide a concerted response of the whole organism to exercise stress, in agreement with the notion that HSP72 might be secreted from cells to exert relevant physiological functions (Hightower & Guidon, 1989; Asea et al. 2000b; Guzhova et al. 2001; Dybdahl et al. 2002; Broquet et al. 2003).

Acknowledgments

The authors are indebted to M. Fernández-Algar, V. Blanca and N. Zamarreño for efficient technical assistance, to M. J. Pérez-Lorenzo for performing part of the experiments leading to Figs 810, to J. Palacín and M. L. Vega for assisting with animal care and to E. Prentice for revising the English manuscript. This work was supported by grants PB98-0084 and BFI2002-02419 from the CICYT and 01/UNI32/00 from the Consejo Superior de Deportes. B G was financed in part by the Autonomous University of Madrid. The Centre of Molecular Biology ‘Severo Ochoa’ is the recipient of an Institutional Grant from the Fundación Areces.

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