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Endocrinology Vol. 138, No. 6 2601-2609
Copyright © 1997 by The Endocrine Society

ARTICLES

Interaction between Adrenaline and Epidermal Growth Factor in the Control of Liver Glycogenolysis in Mouse1

Montserrat Grau, Maria Soley and Ignasi Ramírez

Departament de Bioquímica i Biologia Molecular, Universitat de Barcelona, Barcelona, Spain

Address all correspondence and requests for reprints to: Dr. Ignasi Ramírez, Departament de Bioquímica i Biologia Molecular, Facultat de Biologia, Universitat de Barcelona, Avda Diagonal 645, 08071-Barcelona, Spain. E-mail: sunyer{at}porthos.bio.ub.es


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Epidermal growth factor (EGF) stimulates glycogenolysis in mouse liver, but the effect requires concentrations that are only achieved in plasma upon adrenergic stimulation of EGF release from submandibular salivary glands. Thus, we studied the interaction between adrenaline and EGF in liver glycogen metabolism, both in whole animals and in isolated hepatocytes. Adrenaline administered to anesthetized mice stimulated both the endocrine secretion of EGF from submandibular salivary glands and the degradation of glycogen in the liver. In sialoadenalectomized mice, adrenaline administration did not increase plasma EGF concentration. In these animals, the glycogenolytic response to adrenaline was enhanced. The sensitivity of hepatocytes to adrenaline was similar in cells from sialoadenalectomized and sham-operated mice. EGF, added to isolated hepatocytes, reduced the glycogenolytic effect of adrenaline (the maximal effect but not the ED50). Adrenaline stimulated glycogen degradation through both an {alpha}1-adrenergic mediated Ca2+ increase and a ß-adrenergic-mediated cAMP increase. EGF did not interfere with the rise of cytosolic Ca2+ but decreased the cAMP signal. EGF did not decrease the glycogenolytic effect of phenylephrine or VP (which increased cytosolic Ca2+ but not cAMP), but EGF decreased both the glycogenolytic effect and the cAMP signal generated by glucagon or forskolin. EGF did not interfere with the glycogenolytic effect of CPT-cAMP or bt2-cAMP. The effect of EGF on cAMP was blocked by 3-isobutyl-1-methylxanthine. These results demonstrate that the effect of EGF on the glycogenolytic action of adrenaline involves interference with the generation of the cAMP signal. We suggest that EGF induces such an effect through the activation of a phosphodiesterase.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THE LIVER is a target tissue for epidermal growth factor (EGF) action. It contains a large number of EGF receptors in both fetal and adult life (1, 2). The most studied effect of EGF in hepatocytes is the stimulation of DNA synthesis (3), and a role for EGF has been suggested in the early events of liver regeneration (4, 5, 6). Several metabolic pathways in the liver are affected by EGF. It rapidly increases gluconeogenesis in fasted rats, but the effect is transient (7, 8). Delayed effects are observed (9, 10, 11, 12), but they are secondary to the effect of EGF on cell redox state (8).

EGF stimulates glycogen degradation in both rat (13) and mouse (14) hepatocytes. This effect is mediated by the rise in cytosolic Ca2+ concentration (13, 14). Other studies report that EGF inhibits glycogen deposition in cultured hepatocytes (15) and counteracts the glycogenic effect of insulin (11, 16). In keeping with these results, EGF increases glycogen phosphorylase a in isolated hepatocytes (13, 17). In contrast, it was described that EGF, like insulin, inhibits phosphorylase activation brought about by phenylephrine (18). Recently, we have reported that the in vivo administration of EGF to mice rapidly decreases liver glycogen content and causes mild hyperglycemia (14). In contrast to catecholamines or glucagon, which increase glycogen breakdown and inhibit glycolysis, EGF does not affect glycolysis directly and, thus, a significant part of glucosyl residues ends in glycolysis (8, 14) and in the pentose-phosphate (19) pathways. The moderate glycogenolytic effect of EGF may provide glucose-6-phosphate for internal consumption and perhaps pentoses for nucleic acids synthesis. The metabolic effects of EGF in hepatocytes thus seem to be related to the mitogenic action of this factor.

The plasma EGF concentration in the adult mouse is close to 0.1 nM (20, 21). Because the metabolic effects of EGF in hepatocytes have an ED50 of 1–5 nM (13, 14, 18, 22), it is conceivable that EGF does not influence liver metabolic function in resting conditions. Rather, some of these effects may be exerted by transforming growth factor-{alpha} (TGF-{alpha}), which is produced locally in some circumstances (23). Indeed, an effect of TGF-{alpha} on glycogen metabolism similar to that of EGF was reported by Peak and Agius (24).

In mouse, the submandibular salivary glands contain a large amount of EGF (25). These glands are sensitive to catecholamines, which induce salivation and the consequent depletion of EGF stores (21, 26, 27). Some of the EGF is routed to plasma (21, 27), where EGF concentration can reach 100 nM (26, 27). Therefore, the liver may become sensitive to circulating EGF when it receives simultaneous adrenergic stimulation. Both EGF and catecholamines bring about glycogenolysis in the liver, although the effect of catecholamines is stronger (13, 14).

To determine the metabolic significance of the secretion of EGF from submandibular glands to the circulation, we studied the role of EGF, released from these glands upon adrenaline administration, in hepatic glycogen metabolism in mice. Because EGF interfered with the glycogenolytic effect of adrenaline, we checked this effect in isolated hepatocytes, where we also studied the mechanism of the interference. Our results indicate that EGF interfered with the cAMP component of adrenaline signaling but not with the Ca2+ component.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals
Adult (2-month-old) Swiss-CD1 mice were obtained from Interfauna (Barcelona, Spain). All animals were male, fed ad libitum, and maintained under a constant 12-h light, 12-h dark cycle (lights on at 0800 h) and controlled conditions of humidity (between 70–80%) and temperature (22 ± 1 C). The experimental procedures were approved by the Committee on Animal Care of the University of Barcelona.

Sialoadenalectomy
In diethyl ether-anesthetized mice, a small incision was made to expose the submandibular salivary glands, which were then ligated and excised. In control (sham-operated) animals, the glands were exposed, and a ligature was passed but not tied. The wound was stitched and disinfected. Sham-operated and sialoadenalectomized animals were fasted for 24 h to allow similar conditions for recovery. Two weeks later, the mice had recovered completely, and the experiment was started. In some experiments, hepatic DNA was determined as described (28).

Experiments in whole animals
Sham-operated or sialoadenalectomized mice were anesthetized (sodium pentobarbital, 60 mg Kg-1) before receiving an iv (0.37 mg Kg-1) plus an ip (1.25 mg Kg-1) injection of adrenaline (Sigma, St. Louis, MO). Control animals received identical volumes of saline. Ten minutes later, blood was collected into heparinized syringes from the inferior vena cava. The liver and both submandibular glands were then immediately excised and frozen in liquid N2. A sample of the liver was digested in 3% HClO4, and the supernatant was used to determine glycogen (29). Another sample was homogenized in 10 vol of buffer (40 mM glycerol 2-phosphate (pH 6.8)/40 mM ß-mercaptoethanol/10 mM NaF/0.1% albumin), and glycogen phosphorylase a was determined, as glucose-1-phosphate release from glycogen at 30 C (30). One unit of enzyme activity was defined as the amount of enzyme that catalyzed the release of 1 µmol glucose-1-phosphate per min. Blood plasma was processed, as indicated in Ref. 21, for EGF quantification. Submandibular salivary glands were homogenized in 10 ml PBS; after centrifugation (100,000 x g for 60 min at 4 C), the supernatant was stored at -40 C.

Quantification of EGF
EGF was determined by enzyme-linked immunosorbent assay (ELISA) in blood plasma and in submandibular glands, as described (21). In brief, 1 ng EGF (in 0.1 ml PBS) was adsorbed (overnight at 4 C in a humidified chamber) onto polystyrene ELISA plates and fixed by adding 0.1 ml of 25%-isopropanol in 10%-acetic acid for 15 min at room temperature. Plates were then rinsed (three times) with PBS at room temperature, and the remaining adsorbent sites were blocked with 5% defatted milk powder in PBS (30 min at 37 C in a humidified chamber). The plates were then rinsed three times with MTP (0.5% defatted milk powder, 0.1% Tween-20 in PBS) and incubated with 0.05 ml of the primary antibody (rabbit antiserum anti-mEGF diluted 1:1250 in MTP) and 0.05 ml of sample (or standards ranging from 6.6 pM to 66.6 nM EGF in MTP) for 4 h at 37 C in a humidified chamber. After rinsing three times with MTP, the plates were incubated with 0.1 ml of the secondary antibody (goat-IgG antirabbit IgG/peroxidase conjugate diluted 1:10,000 in MTP) for 90 min at 37 C in a humidified chamber. The plates were finally rinsed three times with MTP and developed with 0.1 ml OPD solution (0.4 mg/ml o-phenylendiamine, 60 ppm H2O2 in 0.15 M citrate buffer, pH 5.0) for 20 min at room temperature, and the reaction was stopped with 0.05 ml of 2.5 M HCl. Absorbance was measured at 492 nm. The mean intraassay variation coefficient was 1.6% and the mean interassay variation coefficient was 4.4%. Our ELISA procedure does not cross-react with mouse nerve growth factor, rat or human EGF, or rat recombinant TGF-{alpha} (21).

Experiments in isolated hepatocytes
Hepatocytes were isolated from the liver of adult male mice, as described (14). Initial cell viability measured by the trypan blue exclusion test was over 90% and decreased less than 10% during the incubations (up to 60 min). Hormones did not affect this decrease. Isolated hepatocytes were then incubated (2 x 106cells/ml; final vol, 2 ml) in a 20 mM-HEPES (pH 7.4)-containing buffer supplemented with 1%-albumin (11) but without glucose (buffer A). At the end of the incubation, a sample of the suspension was placed into enough ice-cold HClO4 to give a final concentration of 3%. After neutralization, glucose (31) [and in some experiments, lactate (32) and pyruvate (33)] concentrations were determined. Glycogen was determined in HClO4 extracts, as indicated in Ref. 29. To determine glycogen phosphorylase a activity, a sample was taken at the indicated times and centrifuged (30 sec at 10,000 x g, 4 C). The medium was discarded, and the cells were immediately frozen in liquid N2. Cell pellets were processed as indicated in Ref. 13. Glycogen phosphorylase a activity was determined as indicated above.

Determination of cAMP
At indicated times (usually 5 min after hormone additions), a sample (1 ml) of the hepatocyte suspension was obtained and deproteinized as above. cAMP was determined in neutralized supernatans with a radiochemical-binding assay kit (TRK-432, from Amersham International, UK), following manufacturer’s instructions.

Determination of cytosolic free Ca2+
Cytosolic free Ca2+ was measured in fura-2/AM-loaded hepatocytes. Isolated hepatocytes (3 x 106 cells/ml) were incubated in buffer A supplemented with 5.5 mM-glucose, amino acids, and vitamins (composition given as buffer D in 34 for 45 min at 37 C in the presence of 5 µM-fura-2/AM. The cells were then rinsed three times in fresh fura-2/AM-free medium and further incubated for 15 min to allow deesterification of the dye. Cells were rinsed twice in fresh medium and then used to monitor fluorescence in a Shimadzu RF5001PC spectrofluorimeter (excitation at 340 and 380 nm and emission at 505 nm). At the end of each experiment, 0.06 ml of Triton X-100 and 0.1 ml of 100 mM-EGTA were sequentially added. Cytosolic Ca2+ was quantified as in Ref. 35.

The chemicals used were obtained as indicated elsewhere (8, 13, 21, 36). Statistical comparisons were made by ANOVA. When significant F values were obtained, multiple comparisons were made using InStat (ISI Software, Philadelphia, PA).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Effect of EGF on adrenaline action in whole animals and in isolated cells
To determine the role of endogenous EGF (released to bloodstream from submandibular salivary glands) in liver glycogen metabolism, we studied the response of sham-operated and sialoadenalectomized adult male mice to adrenaline. This experiment was performed 2 weeks after surgery. At that time, both groups of animals had equivalent growth pattern, with similar intake of chow (6.7 ± 0.2 and 7.0 ± 0.5 g/animal/day) and water (8.9 ± 1.0 and 6.7 ± 0.8 ml/animal/day). They also had similar body weight (47.3 ± 0.7 and 47.2 ± 0.9 g), liver weight (2.25 ± 0.11 and 2.18 ± 0.04 g), and liver DNA (2.59 ± 0.08 and 2.48 ± 0.06 mg/g), as well as similar plasma glucose (9.80 ± 0.34 and 9.82 ± 0.39 mM for sham-operated and sialoadenalectomized animals in each pair of data).

Ten minutes after adrenaline administration to sham-operated mice, the EGF content of submandibular glands decreased (Table 1Go). In contrast, EGF concentration in plasma increased. In sialoadenalectomized animals, adrenaline administration did not result in any significant change in plasma EGF levels. Similar results were described previously by us (21) and others (26, 27). Adrenaline had a potent glycogenolytic effect, as shown by the increased glycogen phosphorylase a activity and the decreased glycogen content found in the liver of these animals (Table 1Go). In sialoadenalectomized animals (in which the administration of adrenaline is not followed by an increase in plasma EGF concentration), the glycogenolytic response to adrenaline was enhanced. This was observed in both glycogen phosphorylase a activation and glycogen depletion (Table 1Go). Both animal groups had similar values in basal conditions (saline administration).


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  		Table 1. Sialoadenalectomy enhances the glycogenolytic effect of adrenaline in whole animals

 
Next, we determined whether the differential response to adrenaline in sham-operated and sialoadenalectomized animals was attributable to a change in the sensitivity of liver cells to adrenaline. Thus, we compared the dose-dependent increase in glycogenolysis in hepatocytes isolated from sham-operated or sialoadenalectomized mice. The sensitivity (ED50 49.4 ± 2.9 and 50.9 ± 3.8 nM in cells from sham-operated and sialoadenalectomized animals, respectively) and the maximal response (4.02-fold ± 0.39 and 4.29-fold ± 0.32 increase in cells from sham-operated and sialoadenalectomized animals, respectively) of hepatocytes to adrenaline were similar in both groups (differences were nonsignificant) (Fig. 1Go). Therefore, the enhanced glycogenolytic response of sialoadenalectomized mice to adrenaline was not caused by a change in the sensitivity of hepatocytes to the catecholamine.



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  		Figure 1. Sialoadenalectomy does not affect the sensitivity of isolated hepatocytes to the glycogenolytic action of adrenaline. Two weeks after sialoadenalectomy (o) or sham-operation (control animals) (•), hepatocytes were isolated and incubated in a glucose-free medium and in the presence of increasing concentrations of adrenaline. After 20 min of incubation, samples were taken to determine glucose output (as glycogenolysis index). The basal (no adrenaline added) output was 374 ± 22 and 391 ± 23 nmol glucose/106 cells for control and sialoadenalectomized animals, respectively (nonsignificant differences). The results, expressed as the relative (to the basal value) effect, are the mean ± SE of four different experiments. The curves were adjusted to sigmoids and compared by ANOVA. There were nonsignificant differences between the curves (Fisher value = 0.50, with 4 and 76 degrees of freedom).

 
The next question was whether EGF interfered with adrenaline action in isolated hepatocytes. We incubated the cells with increasing concentrations of adrenaline, either in the absence or in the presence of 100 nM EGF. The results are shown in Fig. 2Go. At concentrations of adrenaline below 10 nM, the cells that were incubated in the presence of EGF degraded more glycogen than did control cells (Fig. 2aGo). This is in keeping with our previous studies (13, 14). However, as we increased adrenaline concentration, the cells incubated in the presence of EGF responded less than control cells, and thus, at saturating concentrations of adrenaline (1 µM or higher), the cells incubated in the presence of EGF degraded less glycogen than control cells. This is more clearly shown in panel b, which presents the relative effect of adrenaline in control and EGF-exposed cells. EGF decreased the maximal glycogenolytic response to adrenaline and did not affect the ED50 (46 and 67 nM in the absence or in the presence of EGF, respectively). Six experiments like that shown in Fig. 2aGo were performed. In summary, EGF decreased the maximal relative effect of adrenaline from 2.60-fold ± 0.16 increase to 1.84 ± 0.06 (Fig. 2cGo).



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  		Figure 2. EGF decreases the glycogenolytic response of isolated hepatocytes to adrenaline. Isolated hepatocytes from nonoperated mice were incubated with increasing concentrations of adrenaline and in the presence (o) or in the absence (•) of 100 nM EGF. Panel A, Glycogen degraded in 20 min of incubation. The data corresponds to a representative experiment. Panel B, The same data as in panel A but expressed as the relative effect (to the corresponding no-adrenaline value) of each adrenaline concentration. Panel C, Relative effect of 10 µM adrenaline, in the absence and in the presence of 100 nM EGF. The bars correspond to the mean ± SE of six different experiments. **, P < 0.01.

 
As in glycogen degradation, the maximal relative effect of adrenaline on glucose output was decreased by EGF (4.25-fold ± 0.26 and 2.14-fold ± 0.24 increase, P < 0.001, in the absence and in the presence of EGF, respectively; data from eight experiments). Adrenaline not only has a glycogenolytic effect, but it also inhibits glycolysis. The quantification of lactate and pyruvate showed that EGF decreased the effect of adrenaline on glycolysis: the percentage of inhibition of glycolysis was 78 ± 3% and 58 ± 6% in the absence and in the presence of EGF, respectively (P < 0.01).

Involvement of {alpha}1- and ß-adrenergic receptors in the glycogenolytic effect of adrenaline
In hepatocytes isolated from adult male rats, the glycogenolytic effect of adrenaline is mediated predominantly by {alpha}1-adrenergic receptors (37, 38). These receptors induce the rise of glycogen phosphorylase activity, mediated primarily by an increase of cytosolic Ca2+. Here we show (Fig. 3Go) that adrenaline, phenylephrine, and isoproterenol increased glycogen degradation in a dose-dependent manner, although the effect of phenylephrine or isoproterenol was less potent than that of adrenaline. Looking at intracellular signals, we observed that only adrenaline increased both cAMP and Ca2+. Phenylephrine increased Ca2+, but not cAMP, and isoproterenol increased cAMP but not Ca2+. In hepatocytes isolated from male rats, the increase in cAMP, observed upon stimulation with adrenaline, involves both ß2- and {alpha}1-adrenergic receptors (38). In hepatocytes isolated from male mice, adrenaline increased cAMP through ß-adrenergic receptors, and {alpha}1-adrenergic receptors were not involved. This conclusion is based on the finding that the increase in cAMP concentration induced by 1 µM adrenaline (from a basal level of 6.79 ± 0.13 pmol/106 cells to 20.12 ± 1.12) was blocked by 10 µM propranolol (7.54 ± 0.14 pmol/106 cells) but not by 10 µM prazosin (18.37 ± 0.45 pmol/106 cells). Neither propranolol nor prazosin modified basal cAMP concentration (data not shown).



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  		Figure 3. Effect of {alpha}1- and ß-adrenergic agonists on glycogen degradation and on intracellular second messengers in mouse hepatocytes. Isolated hepatocytes were incubated in the presence of increasing concentrations of adrenaline (ADR), phenylephrine (PHE), or isoproterenol (ISO). At 5 and 20 min of incubation, a sample was taken to determine, respectively, cAMP and glycogen (glycogen was determined also at zero time). Cytosolic Ca2+ was determined in fura-2-loaded hepatocytes. The peak Ca2+ concentration is plotted against agonist concentration.

 
To determine whether both adrenergic receptor types were involved in the glycogenolytic response of mouse hepatocytes to adrenaline, we incubated the cells with {alpha}1- and ß-adrenergic agonists and antagonists. The effect of adrenaline on glycogen degradation was moderately decreased by the ß-antagonist propranolol and markedly decreased by the {alpha}1-antagonist prazosin (Fig. 4Go). On the other hand, the addition of 10 µM phenylephrine plus 10 µM isoproterenol stimulated glycogenolysis to a level similar to that induced by 1 µM adrenaline. Individually, these agonists had a lower effect than 1 µM adrenaline. Therefore, for a maximal effect of adrenaline on glycogen degradation in mouse hepatocytes, not only a Ca2+ (mediated by {alpha}1-adrenergic receptors), but also a cAMP (mediated by ß-adrenergic receptors) signal is required.



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  		Figure 4. Both {alpha}1- and ß-adrenergic receptors are involved in the glycogenolytic effect of adrenaline in mouse hepatocytes. Isolated hepatocytes were incubated for 30 min in the presence of the indicated agonists and/or antagonists. At the beginning and at the end of the incubation, samples were taken to determine the glycogen content of the cells. The results are the mean ± SE of triplicate values from a representative experiment. **, P < 0.01; ***, P < 0.001; C, control; A, 1 µM adrenaline; P, 10 µM phenylephrine; I, 10 µM isoproterenol; Pro, 10 µM propranolol; Pra, 10 µM prazosin.

 
EGF interferes with the cAMP- but not with the Ca2+-component of the glycogenolytic effect of adrenaline
To study whether EGF interfered with the Ca2+ component of the glycogenolytic effect of adrenaline, we studied first the effect of EGF on the Ca2+ signal generated by adrenaline, phenylephrine, or VP, another Ca2+-mobilizing hormone. In this experiment we used saturating concentrations of these hormones, as determined in preliminary dose-response studies (data not shown). In agreement with previous reports (13, 14, 39, 40), EGF increased cytosolic free Ca2+ concentration (Fig. 5Go). Adrenaline increased cytosolic Ca2+ concentration to a higher level than phenylephrine or VP (Fig. 5Go). EGF addition not only did not decrease, but actually increased the Ca2+ peak achieved by a maximal concentration of phenylephrine or VP. EGF had no effect on a maximal concentration of adrenaline. However, with lower adrenaline concentrations (which increased cytosolic Ca2+ to a level similar to that observed with phenylephrine or VP), EGF increased the Ca2+ peak (data not shown).



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  		Figure 5. EGF does not interfere with the Ca2+ signal generated by adrenaline, phenylephrine, or VP. Fura-2-loaded hepatocytes were monitored continuously in a Shimadzu RF5001PC spectrofluorimeter. When indicated (arrow), adrenaline (Adr, 10 µM final concentration), phenylephrine (Phe, 10 µM final concentration), or VP (30 nM final concentration) were added either alone (continuous line) or in combination with EGF [100 nM final concentration (dotted line)]. The graphs correspond to representative experiments.

 
When we looked at the effect of EGF on the glycogenolytic response of hepatocyte to these hormones, we observed that EGF decreased the glycogenolytic effect of adrenaline but not that of phenylephrine or VP (Table 2Go). The glycogenolytic response to both phenylephrine and VP actually was increased. Therefore, the interference was only observed with adrenaline, which increased not only cytosolic free Ca2+, but also cAMP (Fig. 3Go and Table 2Go). Phenylephrine did not affect, and VP decreased basal cAMP concentration (Table 2Go).


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  		Table 2. EGF does not interfere with the glycogenolytic response of hepatocytes to phenylephrine or vasopressin (which do not increase cAMP concentration)

 
We had previously observed that in mouse (14), but not in rat (13) hepatocytes, EGF decreased basal cAMP concentration. In Table 2Go, we show that EGF also decreased cAMP concentration in cells exposed to phenylephrine but not to VP. This may be because VP, but not phenylephrine, decreased the basal cAMP level. EGF decreased cAMP concentration in cells incubated with adrenaline (Table 2Go). Glucagon has a potent effect on glycogen degradation in the liver, caused by an increase in cAMP, and Bosch et al. (18) had shown that EGF could decrease both the cAMP signal and the phosphorylase activation induced by submaximal doses of glucagon. Therefore, we compared the effect of EGF on cAMP concentration raised by increasing concentrations of adrenaline and glucagon (Fig. 6Go). EGF decreased the maximal effect of both adrenaline and glucagon on cAMP.



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  		Figure 6. EGF interferes with the cAMP signal generated by adrenaline or glucagon. Isolated hepatocytes were incubated in the absence (•) or in the presence (o) of 100 nM EGF with the indicated concentrations of adrenaline (panel a) or glucagon (panel b). After 5 min, a sample was obtained to determine cAMP concentration in the cells. The results are the mean ± SE of triplicate values from a representative experiment.

 
In the next experiment, we studied the effect of EGF on the glycogenolytic response of hepatocytes to glucagon, forskolin [which directly activates adenylate cyclase (41)], 8-(4-chlorophenylthio) cAMP, and dibutyryl cAMP. Each agent was tested at a near half-maximal dose, as determined in dose-response experiments (data not shown). The results are shown in Fig. 7Go. EGF decreased the glycogenolytic effect of glucagon or forskolin but not that of 8-(4-chlorophenylthio) cAMP or dibutyryl cAMP. In these experiments, we also determined the effect of EGF on cAMP. We observed a potent effect of EGF on both glucagon- and forskolin-stimulated cells (cAMP concentration expressed as pmol/106 cells: 23.2 ± 2.2 and 6.9 ± 1.0 for glucagon and glucagon plus EGF, respectively, P < 0.01; and 29.9 ± 1.2 and 15.6 ± 0.3 for forskolin and forskolin plus EGF, respectively, P < 0.01).



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  		Figure 7. EGF decreases the glycogenolytic response of hepatocytes to glucagon or foskolin, but not that to cAMP analogs. Isolated hepatocytes were incubated in the absence (white bars) or in the presence (dotted bars) of 100 nM EGF with the indicated glycogenolytic agents (FORSK, forskolin). At zero time and at 30 min of incubation, a sample was obtained to determine glycogen. The results are the mean ± SE of triplicate values from a representative experiment. Statistical comparisons vs. no-EGF value: *, P < 0.05, **, P < 0.01.

 
The effect of EGF on the glycogenolytic action of glucagon (and also on that of forskolin) disappeared at saturating concentrations of this hormone (data not shown). This can be understood, in the light of the relationship between cAMP rise and glycogenolytic response to glucagon: glycogen degradation reaches maximal rate at cAMP concentrations between 25 and 50 pmol/106 cells in mouse hepatocytes (14). Saturating concentrations of glucagon, even in the presence of EGF, increased cAMP concentration over these values (see Fig. 6Go), and thus, glycogenolysis was insensitive to the effect of EGF on cAMP.

Hepatocytes express a variety of phosphodiesterase isoforms, most of which are sensitive to inhibition by 3-isobutyl-1-methylxanthine (42). In Table 3Go, we show that both EGF and insulin decreased basal, and adrenaline- or glucagon-increased cAMP levels. In the presence of 3-isobutyl-1-methylxanthine, the effect of adrenaline or glucagon on cAMP was enhanced. This inhibitor abolished the effect of both insulin and EGF on cAMP (either basal or hormone-stimulated levels). Dimethyl sulfoxide, used as solvent, did not affect basal cAMP levels or the response to hormones (data not shown).


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  		Table 3. 3-Isobutyl-1-methylxanthine blocks EGF action on cyclic AMP levels

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In search of the significance of EGF effects on liver glycogen metabolism, we found that adrenaline stimulates the release of EGF, which in turn, interferes with the glycogenolytic effect of adrenaline in the liver. This was observed in whole animals (the glycogenolytic effect of adrenaline was lower in control than in sialoadenalectomized animals, which cannot be attributed to any difference in the sensitivity of liver cells to adrenaline between animal groups) and in isolated hepatocytes. We previously found that EGF interferes with another important metabolic effect of catecholamines: the stimulation of lipolysis in adipocytes (36, 43). This effect of EGF is a consequence of the interference with the generation of a cAMP signal by ß-adrenergic agonists in these cells.

Our results indicate that the glycogenolytic effect of adrenaline involves both {alpha}1- and ß-adrenergic receptors in male mice hepatocytes. Indeed, the {alpha}1-receptor-mediated increase in cytosolic Ca2+ seems to be the major component, considering that the {alpha}1-blocker prazosin produced a stronger effect than the ß-blocker propranolol. However, the ß-receptor-mediated increase in cAMP was also involved, because propranolol reduced the effect of adrenaline. Although in male rats, the increase in cAMP induced by adrenaline involves both {alpha}1- and ß-receptors (38), our results indicate that in male mice, only ß-receptors are involved. This conclusion is based on the finding that propranolol blocked the cAMP rise induced by adrenaline; further, the selective stimulation of {alpha}1-adrenergic receptors with phenylephrine did not result in any increase in cAMP concentration.

We conclude that EGF does not interfere with the Ca2+-mediated increase in glycogen degradation. First, EGF did not decrease the Ca2+ signal induced by adrenaline; and second, when glycogen breakdown was prompted by a Ca2+ increase without any rise in cAMP (as was that induced by phenylephrine or VP), it was enhanced by EGF. The effect was not additive to that of EGF itself, but this can be attributed to the nonadditive enhancement of the Ca2+ signal observed in these conditions. These later results suggest that EGF does not affect the Ca2+-mediated glycogenolysis down-stream of the Ca2+ signal. The conclusion is partly in contrast to Bosch et al. (18), who found that EGF did not affect the dose-dependent increase in glycogen phosphorylase activity induced by VP and that EGF decreased both the Ca2+ signal and the increase in phosphorylase activity induced by phenylephrine. We have no clear explanation for the discrepancies, but it should be noted that those authors used rat, instead of mice, hepatocytes in their studies. In addition, the monitoring of cytosolic Ca2+ was performed with a different fluorescent dye (Quin-2, in the early studies of Bosch et al.), which can give artefactual results because of the Ca2+ buffering capacity of this dye (35).

The results reported here suggest that the basis of the interaction between adrenaline and EGF in glycogen metabolism is the ability of EGF to interfere with the rise of cAMP induced by adrenaline. Because EGF also interfered with the cAMP signal prompted by glucagon or forskolin, we may conclude that this effect is not the consequence of any direct action of the EGF-signaling system on ß-adrenergic receptors. Rather, it seems to be the consequence of an action on some phosphodiesterase, because the effect of EGF on cAMP was blocked by 3-isobutyl-1-methylxanthine. Because EGF did not interfere with the glycogenolytic effect of either CPT-cAMP or bt2-cAMP, we may further conclude that EGF does not affect the cAMP-stimulated glycogenolysis down-stream of the cAMP signal.

EGF modulates the action of several hormones through an effect on the cAMP generating system (see Ref. 44 for recent review). In some cells, like A-431 human epidermoid carcinoma cells, in which EGF inhibits cAMP accumulation induced by isoproterenol (45) or by bradykinin (46), EGF seems to induce the phosphorylation of the Gs {alpha}-subunit in tyrosine residues, leading to reduced guanosine nucleotide exchange (46). In others, like gastric mucosa parietal cells, in which EGF inhibits glucagon-like peptide-1-induced acid production, the effect seems to involve the activation of a Gi protein by EGF (47). A somewhat different effect of EGF on Gi protein function was described by Tebar et al. (36, 43) in rat adipocytes. In this system, EGF seemed to increase the sensitivity of Gs-stimulated adenylate cyclase to the inhibitory effect of Gi proteins. Our studies in mouse hepatocytes do not rule out an effect of EGF at the G protein level, but (as discussed above) they strongly suggest that a phosphodiesterase is the main target of EGF action on the cAMP-generating system in this particular cell type.

The interaction between EGF signaling and cAMP is complex. We report here that EGF interferes with the generation of the cAMP signal induced by adrenaline, glucagon, or forskolin in rat hepatocytes. On the other hand, cAMP-raising agents, including ß-agonists, decrease the activation of mitogen-activated protein kinases by EGF and other mitogens (48, 49, 50, 51, 52). This effect was also observed in cultured rat hepatocytes (53).

In conclusion, EGF induces two opposite effects on liver glycogen metabolism: 1) When added alone to hepatocyte suspensions, or injected to intact mice, EGF moderately increases glycogen breakdown, an effect that seems to be mediated by the increase in cytosolic Ca2+ concentration (13, 14); 2) When added together with adrenaline, EGF decreases the glycogenolytic response of hepatocytes. This effect is mediated by the interference with the generation of cAMP, but not Ca2+, signal. We discussed elsewhere (13, 14) (also see the Introduction) the relevance of the effect of EGF, when added alone, on glycogen metabolism. The physiological significance of the interaction between adrenaline and EGF is still a matter of speculation. We suggest that the effect on glycogen metabolism reported here may be one among the consequences of such interaction (perhaps not the most relevant) because first, it is inconceivable that adrenaline might induce the release of EGF from submandibulary gland with the only purpose being to reduce its own metabolic effect; and second, because EGF interferes only with the minor component of the glycogenolytic effect of adrenaline, and the extent of the interference is quite moderate. Rather, we believe that the interaction between EGF and adrenaline will be understood by looking at cellular effects of cAMP other than the metabolic ones and taking into consideration the complexity of the interaction between EGF and cAMP. Studies are in progress in our laboratory to shed new light on this interaction.


   Acknowledgments
 
We thank Robin Rycroft for editorial help.


   Footnotes
 
1 This work was supported by Grants PB91–0251 and PB94–0863 from Dirección General de Investigación Científica y Técnica (Ministerio de Educación y Ciencia, Spain). Back

Received December 2, 1996.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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