INTRODUCTION

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OVER THE LAST FEW YEARS, evidence has been gathered that shows that the liver may act as an afferent organ contributing to the metabolic and hormonal regulation of exercise (3, 21). The best evidence in favor of such a role by the liver during exercise comes from the demonstration that a selective hepatic vagotomy attenuates the exercise-induced pancreatic and norepinephrine responses in rats (21). However, the signal and the mechanism at the origin of this hepatic afferent information are still not fully understood. It has been suggested that substrates such as pyruvate have a hyperpolarizing effect on liver cells (7) and that the membrane potential of the hepatocytes determines the firing rate of afferent neurons (29).

The interest in studying hepatocyte cell volume changes during exercise stems from the recent reports that hepatic metabolism, not primarily serving cell volume regulation, appears to be regulated by a new parameter, i.e., cell volume (16, 17). It has been shown in isolated cell studies that pancreatic hormones such as insulin and glucagon can independently induce hepatocytes to swell or shrink, respectively (16). Physical exercise is a situation where the pancreatic hormone response is characterized by an increase in glucagon and a decrease in insulin. Both of these hormone responses would stimulate a shrinkage of the hepatocytes. It is possible that variations in hepatic cell volume could influence hepatic metabolism during exercise. It is also possible that a change in hepatic cell volume constitutes a signal at the origin of the hepatic afferent information transmitted through the hepatic vagus nerve. Supporting this view is the report that cell swelling hyperpolarizes the cell membrane (18). With this theory in mind, a first step toward the understanding of potential metabolic effects from changing in hepatocyte volume was to determine whether cell volume underwent changes subsequent to exercise. In addition, the relation between changes in hepatocyte volume and the reduction of liver glycogen stores during exercise was looked on by conducting the same experiments in both normally fed and semifasted animals. Because no in vivo models were available to directly measure the volume of the different liver compartments in exercising rats, we used an isolated perfused rat liver model and the multiple-indicator dilution curve technique described by Goresky (12) to achieve this first goal.

A second objective was to document the hepatic function in a perfused liver after an acute exercise. Some authors have reported an altered liver function in strenuous exercise in man (1, 9). A reduced extraction of propanolol (Pro) (1), increased liver injury (26), and the release of liver-specific enzymes (9) were observed. Some concerns have been, however, expressed relative to the inaccuracy of in vivo liver extraction of some compounds during exhaustive exercise due to caval reflux and catheter displacement in the hepatic vein occurring during high ventilation (2, 28). In the present study, we used a rat liver perfusion model to evaluate the effects of moderate-intensity exercise on hepatic function immediately after exercise.

	METHODS

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Animal care. Male Sprague-Dawley rats (Charles River, St. Constant, PQ) weighing 200-225 g were housed in individual cages and allowed pellet rat chow and tap water ad libitum for 15 days after they were received in our laboratory. Lights were on from 07:00 AM until 07:00 PM, and the room temperature was maintained at 20°C. Three days after their arrival, all rats underwent a habituation running protocol on a motor-driven rodent treadmill consisting of six sessions over 2 wk, beginning with 20 min/day at 15 m/min and progressively increasing in both speed and duration to 45 min/day at 30 m/min (0% grade), so that they were well accustomed to running and being handled. The experiments described in this report were conducted according to the directives of the Canadian Council on Animal Care.

Groups and exercise protocol. The night before experimentation, all rats were weighed and randomly assigned to either well-fed or semifasted groups. Semifasted rats received ~50% of their daily food intake (~ 11 g). On the day of the experiment, rats were again weighed and randomly divided into a rest and an exercise group. The experiments were run between 10:30 AM and 01:30 PM. The exercise test consisted of running on the treadmill at speeds and durations that totaled ~1,500 m (0% grade). The speeds and durations used were as follows: 5 min at 15 m/min, 5 min at 20 m/min, 40 min at 26 m/min, and 10 min at 28 m/min. Rested rats were killed at similar times as exercised rats. At the end of running or resting conditions, all rats were rapidly anesthetized with pentobarbital sodium (50 mg/kg ip). After complete anesthesia, the abdominal cavity was opened and the isolation of all vessels of the liver was rapidly started without closure of the blood circulation. Before the beginning of the perfusion, peripheral blood (~3 ml) was collected via the abdominal vena cava. After closure of the blood vessels and the cannulation of the bile duct, the portal vein was cannulated and the liver flushed with oxygenated Krebs-Henseleit buffer. Then the whole triangular lobe of the liver was cut, weighed, and then frozen. At the end of the 40-min perfusion, liver was flushed again with oxygenated Krebs-Henseleit buffer and the caudate lobe of the liver was taken, weighed, and then frozen. Pre- and postperfusion liver tissues were used to determine glycogen concentrations. This was done to verify whether the perfusion medium could affect glycogen content.

Isolated perfused rat liver. The liver was perfused for 40 min through the portal vein in a closed circuit using a perfusion apparatus (Mx/Ambex Two/ten; Mx International, Aurora, CO). The perfusion medium (250 ml total volume) consisted of Krebs-Henseleit buffer, pH 7.4, containing 20% prewashed bovine red blood cells (RBCs) (vol/vol), 20 g/l albumin (wt/vol), and 1 g/l dextrose (wt/vol). The perfusate was saturated by equilibration with 95% O₂-5% CO₂, and the temperature was kept at 37°C. Liver temperature was also kept at 37°C using a heating lamp. The perfusion flow was measured volumetrically and fixed at 20 ml/min (~7 ml · min¹ · 100 g body wt¹ for all groups). The measurements of taurocholate (TC) and Pro clearance assessed the hepatocyte cell function. Throughout each perfusion period, a loading dose of unlabeled TC and Pro (mixed with tracer doses of [¹⁴C]TC and [³H]Pro) was added to the reservoir to attain theoretical plasma concentrations of 11.5 ng/ml and 100 ng/ml of TC and Pro, respectively, followed by a continuous infusion to maintain these levels. Viability of the perfused liver was first assessed subjectively on the basis of gross appearance and, thereafter, objectively on the basis of lactate dehydrogenase (LDH) release, O₂ consumption, bile production, and intrahepatic resistance (perfusion pressure) (4, 11).

Assessment of TC and Pro clearance. After 20 min of equilibration, perfusate plasma samples were obtained at several time points (20, 24, 29, 35, and 40 min). [¹⁴C]TC and [³H]Pro plasma levels were determined in duplicate using a beta-counter (Beckmann, Montreal, Canada) (30). TC and Pro elimination were measured by their hepatic clearance (Cl) using the following formula where Q is the perfusate flow, E is the extraction, and C_in and C_out are [¹⁴C]TC or [³H]Pro plasma levels at inflow and outflow, respectively. The hepatic Pro clearance and extraction were calculated similarly as for TC. [³H]Pro and Pro perfusate plasma levels were measured in duplicate using a specific high-pressure liquid chromatographic method as previously reported (8).

Multiple-indicator dilution curve. At the end of the liver function study, the hepatic venous outflow was diverted to avoid recirculating radioactive materials; then the multiple-indicator dilution curves were obtained after a 0.1-ml injection of radioactive tracer mixture consisting of the following substances: ⁵¹Cr-labeled RBCs (10⁵ dpm), ⁹⁹Tc-labeled albumin (2 × 10⁵ dpm), [¹⁴C]sucrose (2 × 10⁵ dpm), and [³H]water (3 × 10⁵ dpm) in a solution of Krebs-Henseleit buffer adjusted to a hematocrit matched to that of the perfusate containing albumin (20 g/l). The total hepatic venous outflow was collected in serial tubes at the rate of 1 tube/s for 90 s. An aliquot from each tube was used for the determination of gamma- and beta-activity and processed as previously described (31). The background contamination by [¹⁴C]TC and [³H]Pro contained in the perfusate was quantitatively negligible compared with the levels of [¹⁴C]sucrose and [³H]H₂O, respectively (<0.01% of the peak value of labeled sucrose and water curves). Outflow activity (disintegrations per minute per milliliter) was divided by the total amount injected (disintegrations per minute) to yield a normalized outflow fraction per milliliter of blood, thus providing the basis for comparison of each tracer.

The dilution curves were corrected for catheter distortion and delay as previously described by Goresky and Silverman (14). To calculate the sinusoidal volume, data were analyzed according to Goresky et al.'s flow-limited model (12). This model states that the labeled RBCs delineate the vascular space, whereas the interstitial space indicator (high-molecular weight or labeled albumin, and low-molecular weight or labeled sucrose), as well as the whole organ tracer (labeled water), undergo flow-limited distribution from the sinusoidal into the Disse's space and hepatic cellular space, respectively, in a manner characterized by a delayed wave type of behavior. Therefore, the dilution curve of each diffusible substance can be superimposed on that of the RBCs if every point is corrected by a constant factor to its corresponding point on the RBC curve using the following equation in which C_RBC and C_dif are the concentration values for RBC and diffusible substance at corresponding arbitrary points in time of t(RBC) and t'(dif), respectively; t_o is the large vessel transit time delay, and y is the ratio of extravascular to vascular volume of distribution of diffusible substance. The values of t_o and y were determined as those yielding the least sum of square of deviations between the RBC curves and the diffusible substance curves when the latter is transformed linearly to be superimposed on the first. The optimization procedure was guided by a least-square minimization algorithm programmed in Turbo Pascal and Lotus 123 on a Hewlett-Packard computer (Vectra 286 series, Palo Alto, CA). Results of the nonlinear fitting procedure were examined by visual inspection of the fitting curve (25) and by measuring the coefficients of variation (13) and determination (10) of the fit. Once the y and t_o were obtained, the vascular (sinusoidal) volume (V_sin) was calculated as in which t_RBC is the corrected mean transit time of RBC calculated according to Meier and Zierler (24). Extravascular volume (EV) accessible to albumin and sucrose (EV_Suc) was calculated as a model-independent parameter according to the transit time method of Chinard et al. (5) as where t_dif is the corrected mean transit time of diffusible substances (albumin and sucrose). Extravascular water space was calculated using the same formula with the exception that, in that case, Q equals water flow (Q_w), which must be previously calculated as in which 0.7 and 0.93 represent the proportion of water (ml/ml) in RBC and plasma, respectively (6). The hepatic cellular water space (V_cell) is consequently Analytic methods. Peripheral blood was collected (<1.5 min) into three 1-ml syringes with 15% EDTA and immediately separated into two fractions. One fraction of blood (500 µl) was preserved in 50 µl Trasylol and centrifuged at 4°C, and the plasma was used for glucagon determination. The remainder of the blood was also centrifuged (Eppendorf centrifuge, no. 5415), and the plasma was stored (70°C) for subsequent glucose, insulin, and free fatty acids (FFA) determinations.

Plasma glucose concentrations were determined by the use of a glucose analyzer (Yellow Springs Instruments 2300, Yellow Springs, OH). Insulin and glucagon concentrations were determined by commercially available radioimmunoassay kits (Radioassay System Laboratory; ICN Biomedicals, Costa Mesa, CA; distributed by Immunocorp, Montreal, Quebec). Free fatty acids were assessed enzymatically with the use of reagent kits from Boehringer Mannheim Laboratories (distributed by Immunocorp). The liver was precisely weighed with an electronic balance (Mettler AE 100). Liver glycogen concentration was determined by use of the phenol-sulfuric acid reaction (23). The nonperfused areas were finally assessed histologically by the use of Trypan blue (200 mg% Krebs-Henseleit buffer, Aldrich 30, 264-3). All data are reported as means ± SE. Statistical analyses were performed using a two-way analysis of variance with exercise and dietary state as factors. Scheffé's post hoc test was used in the case of a significant (P < 0.05) F ratio.

	RESULTS

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As expected, the mean body weights of the semifasted rats were significantly (P < 0.01) lower than those of the fed rats (Table 1). There was, however, no difference in body weights between the exercised and the rested groups in both fed and semifasted groups. Because the liver-to-body weight ratio was significantly (P < 0.01) decreased with exercise, whereas the rat body weight remained unchanged (Table 1), it was decided to express all parameters related to hepatic measurements per one hundred grams of body weight instead of per gram of liver weight.

Food restriction (one-half fast) the night before experimentation resulted in significantly (P < 0.01) lower levels of liver glycogen, plasma glucose, and insulin concentrations (Fig. 1, A, B, and D). As expected, exercise resulted in a significant (P < 0.05) decrease in plasma glucose, insulin, and liver glycogen levels in the fed state while significantly (P < 0.05) increasing glucagon and plasma FFA concentrations (the latter in semifasted rats only; Fig. 1, C and D). A small but significant (P < 0.05) difference in liver glycogen in pre- vs. postinfusion period was found only in the postexercise fed situation (Fig. 1A).

View larger version (23K): [in this window] [in a new window]   Fig. 1.   Liver glycogen (pre- and postperfusion), peripheral plasma glucose, free fatty acids, insulin, and glucagon concentrations at rest and postexercise. Values are means ± SE; n = 5 or 6 and 6-8 rats at each point in fed and semifasted groups, respectively. * Significantly different from corresponding resting values, P < 0.05. + Significant difference at P < 0.001 between semifasted and fed state. § Significant difference at P < 0.05 between preperfusion and postperfusion.

Liver viability parameters such as hepatic oxygen consumption, bile secretion, intrahepatic resistance, and LDH release, measured between the 20th and 40th min of the perfusion, were within normal values in all groups and were not significantly (P > 0.05) affected by fasting or exercise (Fig. 2). The only exception to these observations was the LDH release, which was found to be significantly (P < 0.01) more elevated in semifasted than in fed rats (Fig. 2D). Pro and TC hepatic clearance was not significantly (P > 0.05) changed with exercise either in fed or semifasted rats (Fig. 3). However, the intracellular water space of hepatocytes was significantly (P < 0.01) decreased following exercise (~15%; Fig. 4A). The exercise-stimulated decrease in liver cell volume was observed in fed as well as in semifasted rats, even though in the latter, cell volume was significantly (P < 0.01) lower than in the former. Sinusoidal space as well as albumin and sucrose interstitial spaces were not significantly (P > 0.05) changed in all experimental conditions.

View larger version (21K): [in this window] [in a new window]   Fig. 2.   Liver O2 consumption, bile secretion, intrahepatic resistance, and lactate dehydrogenase (LDH) release at rest and postexercise. Values are means ± SE; n = 5 or 6 and 6-8 rats at each point in fed and semifasted groups, respectively. BW, body wt. + Significant difference at P < 0.005 between semi-fasted and fed state.

View larger version (18K): [in this window] [in a new window]   Fig. 3.   Liver taurocholate (TC) and propanolol (Pro) clearance at rest and postexercise. Values are means ± SE; n = 5 or 6 and 6-8 rats at each point in fed and semifasted groups, respectively.

View larger version (21K): [in this window] [in a new window]   Fig. 4.   Hepatocyte cellular water space, sinusoidal space, and sucrose and albumin interstitial spaces at rest and postexercise. Values are means ± SE; n = 5 or 6 and 6-8 rats at each point in fed and semifasted groups, respectively. * Significantly different from corresponding resting values, P < 0.005. + Significantly different at P < 0.005 between semifasted and fed state.

	DISCUSSION

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The present experiment was designed to ascertain the decrease of liver cell volume induced by a 60-min exercise bout of moderate intensity. The present data are the first to our knowledge to confirm the hypothesis of an exercise-induced hepatic cell shrinkage. The hypothesis that physical exercise could potentially reduce the intracellular water content of liver has previously been made (22). Lehmann et al. (22) suggested that the average 7.6% plasma volume increase with a mean 3.3-kg body mass decrease following an ultratriathlon (23 h) in man was due to multiple factors, such as the decrease in intracellular water of muscles and liver. Other researchers have reported an exercise-induced 21% loss of hepatic water using gravimetric measurements (20). In the present report, we show an exercise-induced 15% decrease of the hepatocellular hydration level using a specific and reliable technique in isolated perfused rat liver. With this method we can confirm a selective hepatocyte water loss during exercise without any significant changes in interstitial and sinusoidal spaces.

As expected, the 60-min exercise period resulted in a decrease in blood glucose and insulin and in an increase in glucagon concentrations in all groups. Because liver glycogen levels were already decreased in semifasted rats, liver glycogen concentrations were decreased significantly only in the fed rats following exercise. Consequently, the amount of liver glycogen used during exercise was ~15 times higher in the fed than in the semifasted animals. Interestingly, the different amount of liver glycogen used during exercise did not lead to a different decrease in hepatocellular volume, the reduction being ~15% in both groups. This observation suggests that the reduction in hepatocyte volume following exercise is not entirely due to the loss in glycogen and to the water that is associated with it (27). In search of an alternative explanation for the exercise-induced decrease in liver cell volume, one has to consider the decrease in insulin and the increase in glucagon levels during exercise. In perfused liver, insulin, by acting on different transport systems, leads to cellular accumulation of K⁺, Na⁺, and Cl and, consequently, to cell swelling (16, 19). Glucagon, on the other hand, is known to decrease cellular K⁺ in isolated perfused rat liver, resulting in cell shrinkage (15). During exercise, with insulin concentration decreasing and glucagon concentration increasing, both of these stimuli should lead to a shrinkage of liver cells. It is therefore possible that the typical pancreatic hormone response to exercise, by acting on different transport systems in the liver, leads to cell shrinkage. Overall, the present results indicate that the present 15% decrease in hepatocyte water space following exercise cannot solely be attributed to a loss of glycogen. This observation suggests that liver cell shrinkage during exercise is induced by specific mechanisms and is not only a consequence of an increased glycogen breakdown.

The physiological relevance of the liver cell shrinkage during exercise could be related to specific hepatic functions or to a hepatic afferent contribution to the overall metabolism. After 60 min of exercise, Kasperek et al. (20) reported an increased osmotic fragility in liver cell lysosomes, which was associated with the exercise increase in the rate of protein degradation via the autophagolysosomal system. These authors (20) mention that, although the signal that causes autophagy is not known with certainty, the typical hormonal status of exercise could induce autophagy. It is interesting to relate these speculations with the concept put forward by Lehmann et al. (22) that a protein-catabolic signal might be triggered by a decrease in cellular hydration of liver cells at the background of the particular hormonal pattern during prolonged exercise. A decrease in hepatocellular hydration state has also been reported to stimulate glycogenolysis (17), which would be particularly relevant to exercise energy metabolism. Whether the variation of cellular volume of hepatocyte could represent an afferent signal perceived or transmitted through hepatic nervous pathways, contributing to hormonal and other changes that occur during exercise, remains hypothetical. It is interesting to note, however, that cell swelling hyperpolarizes the cell membrane (18), providing a potential link with the activity of afferent neurons (29).

A second objective of the present study was to document how acute physical exercise can affect liver function. The hepatic clearance of TC and Pro were unchanged in all rats following exercise. This observation indicates that an acute 60-min bout of exercise at moderate intensity is not detrimental to the liver function in rats. Even though LDH release was found to be slightly but significantly higher in semifasted compared with fed rats, there were no effects of exercise on LDH release either in fed or in semifasted rats. This indicates that hepatocyte lysis was not changed by the present exercise protocol.

Other viability parameters, such as the hepatic oxygen consumption and intrahepatic resistance, were all within normal values whether in rested or exercised rats. Finally, the bile secretion values remained normal throughout the perfusion in all groups and do not support the reported reduction in bile flow or cholestasis with exhaustive exercise in rats (32). Overall, our results indicate a normal hepatic functionality following exercise under the present experimental conditions.

In summary, acute physical exercise in rats results in a shrinkage of the liver cells, as assessed in situ under basal unstimulated conditions. Exercised livers displayed a ~15% decrease in the hepatocellular hydration level compared with rest condition. These results confirm the hypothesis that there is an exercise-induced hepatocyte shrinkage and show that it cannot be solely linked to a decrease in the hepatic glycogen level. The hepatic function, as assessed by the clearance of TC and Pro, is not altered following a moderate exercise, which suggests an absence of postexercise hepatic function deterioration.

Perspectives