Glycogenolytic Response to Glucagon of Cultured Fetal Hepatocytes

The glycogenolytic effect of glucagon has been studied in fetal hepatocytes cultured for 3 to 4 days in the presence of cortisol (10 muM). The hepatocytes, when transplanted from young fetuses (15-day-old), contain only minute amounts of glycogen, whereas when cultured 3 to 4 days in the presence of cortisol, they contain high levels of stored glycogen. Glucagon induced a rapid but partial mobilization of glycogen, which was maximal after 2 hours. The half-maximal response was observed with about 0.1 nM glucagon. The glycogenolytic effect of glucagon in fetal hepatocytes is probably mediated by cyclic adenosine 3':5'-monophosphate (cyclic AMP) as in adult liver. This effect was mimicked by cyclic AMP and N-6, O-2-dibutyryl cyclic AMP, (dibutyryl cyclic AMP), and potentiated by theophylline. Glucagon addition was followed by accumulation of cyclic AMP in the cells within 2 min. Glucagon produces a marked stimulation of the rate of glycogen breakdown and an inhibition of the rate of incorporation of [14-C] glucose into glycogen. The glycogeneolytic effect of a single addition of glucagon was reversed within 4 hours. A second addition of glucagon at this time was unable to induce a new glycogenolytic response. A resistance to glucagon stimulation appeared in the cells after a first exposure to the hormone. This refractoriness was also shown by the loss of glucagon-dependent cyclic AMP accumulation and was not linked to the release by the cells of a "hormone antagonist" into the medium. The hepatocytes resistant to the action of glucagon retained their response to cyclic AMP, dibutyryl cyclic AMP, and norepinephrine. Finally, glycogenolytic concentrations of cyclic AMP and of its dibutyryl derivative failed to induce a refractoriness to glucagon.

effect of glucagon has been studied in fetal hepatocytes cultured for 3 to 4 days in the presence of cortisol (10 PM). The hepatocytes, when transplanted from young fetuses (15-day-old), contain only minute amounts of glycogen, whereas when cultured 3 to 4 days in the presence of cortisol, they contain high levels of stored glycogen. Glucagon induced a rapid but partial mobilization of glycogen, which was maximal after 2 hours. The half-maximal response was observed with about 0.1 nM glucagon. The glycogenolytic effect of glucagon in fetal hepatocytes is probably mediated by cyclic adenosine 3': 5'-monophosphate (cyclic AMP) as in adult liver. This effect was mimicked by cyclic AMP and N6, 02-dibutyryl cyclic AMP, (dibutyryl cyclic AMP), and potentiated by theophylline. Glucagon addition was followed by accumulation of cyclic AMP in the cells within 2 min.
Glucagon produces a marked stimulation of the rate of glycogen breakdown and an inhibition of the rate of incorporation of [Wlglucose into glycogen. The glycogenolytic effect of a single addition of glucagon was reversed within 4 hours. A second addition of glucagon at this time was unable to induce a new glycogenolytic response. A resistance to glucagon stimulation appeared in the cells after a first exposure to the hormone. This refractoriness was also shown by the loss of glucagon-dependent cyclic AMP accumulation and was not linked to the release by the cells of a "hormone antagonist" into the medium. The hepatocytes resistant to the action of glucagon retained their response to cyclic AMP, dibutyryl cyclic AMP, and norepinephrine. Finally, glycogenolytic concentrations of cyc l!i c AMP and of its dibutyryl derivative failed to induce a refractoriness to glucagon.
Large stores of hepatic glycogen are built up during late gestation in the fetal rat under cortisol control (l-4). Immediately * This work was supported by grants from the Centre National de la Recherche Scientifique and from the Commissariat a l'Energie Atomique.
after birth, this glycogen is rapidly mobilized to supply the glucose needs of the newborn. Glucagon has been considered as a factor involved in this mobilization, both because premature depletion of glycogen is obtained after glucagon administration to the fetus (4-6), and because blood glucagon levels increase at birth (7,s).
In a previous paper (9), an in vitro system has been described which permits the study of the development of glycogenesis and its regulation in hepatocytes. Primary cultures of hepatocytes were obtained from 15.day-old fetuses, i.e. 3 days before the stage where glycogen synthesis begins in viva. When these hepatocytes, which contain negligible or very minute quantities of glycogen, are grown in the presence of cortisol, glycogen is actively synthesized and accumulated within 3 to 4 days of culture (9). The purpose of the present work has been to study the response of the hepatocyte to glucagon at the level of its glycogen pool.
In the adult, in vitro studies performed with perfused liver (10) and with surviving isolated hepatocytes (11)(12)(13) have shown that the glycogenolytic effect of glucagon is linked to cyclic AMP' accumulation.
In the cultured hepatocytes, glucagon has a glycogenolytic action which is also mediated by cyclic AMP. In addition, this study has provided new information concerning the rapid reversal of the glycogenolytic response which becomes resistant to a second addition of glucagon. Some characteristics of this refractoriness which exists at the level of both cyclic AMP production and glycogen degradation (i.e. the physiological response) are also described in this paper.

EXPERIMEKTAL PROCEDURE
Culture Procedure-Primary cultures of hepatocytes were obtained from 15-day-old rat fetuses (Sprague-Dawley) by a method already described (9,14) with minor modifications. Dissociation of the cells was performed by trypsin treatment for 3 hours at 4", and then for 10 min at 37", followed by manual shaking in the presence of 0.16.mm diameter glass beads in a culture medium containing fetal calf serum. Hepatocytes were separated from hematopoietic cells as previously described (9,14), and grown for up to 4 days. The composition of the culture medium was as fol-  Fig. 2 shows that a decrease of glycogen radioactivity was obtained after addition of 10 nM glucagon. This decrease was cortsiderable after 1 hour, maximal after 3 hours, and leveled off after this, 40% of the stored glycogen being resistant to glucagon-dependettt degradation.
Similar results were obtained using the same experimental conditions with the esccption that glucagon addition was performed after transfer of the cells to an unlabeled medium. Under these conditions of labeling, a marked but again partial effect of glucagon on stored glycogen was demonstrated. On the contrary, when hepatocytes, after 4 days in the presence of cortisol, were incubated for only a 4-hour period with [%]glucase, 8Oo90 of the labeled glycogen was degraded (see Fig. 4); i.e. a larger proportiott than when glycogen was uniformly labeled. These results suggest that the last labeled glucosyl residues were the first to be removed by the actiott of glucagon. Thus, glucagottdependent glycogenolysis seems to concern preferentially newly synthesized glycogen. The glycogettolytic response to glucagon was obtained at, doses of glucagon which are in the range of the physiological blood concentrations in the adult and in the newborn rat (7,8). 1001 IM glucagon was added. The decrease in glycogen radioactivity was measured during a further 8-hour incubation period. Fig. 4A shows that glucagon induces a biphasic glycogenolytic response. A setnilogarithmic plot of these results (Fig. 4B) enabled the calculation of the half-life of glycogen degradation.
Immediately after glucagon addition, the half-life of glycogen was only 50 min, whereas 3 hours later, the rate of degradation was markedly reduced. However, the apparent half-life of glycogett (7 hours) during this second period still remained shorter than that measured in the absence of glucagon (12 to 14 hours). The biphasic glycogenolytic response to glucagon might be the result of uneven labeling of the glucosyl residues in which the last ones labeled were the first to be removed by the action of glucagott, as alread) suggested by the comparison of the results reported in Figs. 2 and 4. Attother possibility might be that some time after glucagott addition, the ratio betweett the rate of degradation and the rate of synthesis decreases markedly, thus resulting itt a slower apparent glycogen half-life.
The effect of glucagott on the rate of incorporation of [W]glucase into glycogen was measured using hepatocytes grown for 4 days in the presence of cortisol; [Vlglucose was thctt added with or without glucagon (10 11~). Glycogett labeling was then tneasured every hour durittg a further 8-hour incubation period. No attempt was made, for theoretical reasons (23), to estitnate the rate of glycogen synthesis immediately after glucagon addition, since the labeling time of glycogen (2 hours) is long with respect to the half-life of its degradation (50 mitt). However, 3 hours after glucagott additiott (Fig. 5), the rate of labeling appeared clearly reduced when cotnpared to assays performed in the absence of glucagon. After 4 to 5 hours, the rate of labeling accelerated and approached that measured in the absence of hormone.
Both of these results (Figs. 4 and 5) show, therefore, that after addition of glucagon, the rate of glycogen degradation was in- creased, whereas the rate of incorporation of [Wlglucose into glycogen was reduced, and that these changes were reversed after 4 hours. The glycogen content of the cells was also measured during the time course of the glycogenolytic effect of glucagon. The result represented in Fig. 1 shows that 4 hours after glucagon addition, glycogen content was reduced by SO'%. After a further 4 hours of incubation (results not shown), the glycogen content increased to 80% of the initial value. Thus, the reversal of the glycogenolytic effect of glucagon was also observed at the level of the glycogen content. come resistant to a second stimulation by the hormone. To confirm this point, experiments were designed (Fig. 6) whereby the effect of glucagon was estimated by measuring the inhibition of incorporation of [14C]glucose into glycogen. At Day 4 of the culture, [14C]glucose was introduced, and the glycogen radioactivity determined 4 hours later. This labeling was performed during the period either from 0 to 4 hours or from 4 to 8 hours after a first addition of 10 nM glucagon; a second addition of 10 nM glucagon was then performed in some cases, 4 hours after the initial one. The control experiment illustrated in Fig. 6A shows that in the absence of glucagon, the rate of glycogen labeling was identical during the two successive 4-hour periods. A single addition of 10 nM glucagon performed at zero time (Fig. 6B) or after 4 hours (Fig. 6C) was followed within 4 hours by a drastic inhibition of the glycogen labeling. Fig. 6B shows that 4 hours after a single glucagon addition, the rate of 14C incorporation returned to normal, as expected from the results shown in Fig. 5. Fig. 6D shows that this restoration of high rates of glycogen synthesis was unaffected by a second addition of 10 nM glucagon. One possible explanation for these results is that the second dose of glucagon is rapidly inactivated when added to "conditioned" medium, i.e. to a medium where the hepatocytes have been cultured for 4 hours in the presence of the hormone. To test this hypothesis, the second glucagon addition was performed after removal of the conditioned medium and its replacement by a 'Lnonconditioned" medium. A nonconditioned medium is one in which hepatocytes have been cultured in the absence of glucagon. Table I shows that in the presence of either conditioned or nonconditioned medium, a second addition of glucagon was ineffective. The same result was obtained when the cells were washed three times with fresh medium before replacement of the medium. Finally, supramaxi-ma1 concentrations of glucagon (1 mM) failed to suppress this resistance to glucagon action. These results show that this refractoriness does not depend on the inactivation of the second dose of hormone by the conditioned medium. In addition, the fact that glucagon remains ineffective when added a second time together with nonconditioned medium suggests that the resistance to glucagon action is not linked to a modification appearing in the medium during the first incubation period performed in the presence of the first dose of glucagon. This modification might have resulted from the release by the cells of a diffusible inhibitory factor. If such an inhibitory factor were released by the cells, it should be present in the conditioned medium. Therefore, the   (Fig. 7A).
In addition, 0.5 mM dibutyryl CAMP produced a marked glycogenolytic effect. Fig. 8 shows the time course of the decay of glycogen radioactivity in the presence of 0.5 mM dibutyryl CAMP, which was similar to that observed with glucagon ( Fig. 2)  on hepatocytes previously exposed to glucagon The experimental protocol is as described in Fig. 6  AMP levels was obtained after addition of 10 nM glucagon plus 1 mM caffeine in hepatocytes which have been exposed to glucagon 4 hours before. It should be noted that the basal level of cyclic AMP in the resistant hepatocyte was higher (4.fold) than that in fresh cells.

DISCUSSION
In cultured fetal hepatocytes, addition of 10 nM glucagon produced a rapid mobilization of glycogen, which concerned 60% of the glycogen pool ( Figs. 1 and 2). Norepinephrine (10 IBM) was also found to have a marked glycogenolytic effect (Table IV). In explants from fetuses at term, after preincubation where spontaneous depletion of glycogen occurs, glycogenolytic effects of epinephrine (24) and glucagon (25) have also been observed. It appears, therefore, that both glucagon and catecholamines are likely glycogenolytic agents for the fetal liver. Addition of glucagon was followed within 2 min by the accumulation of cyclic AMP (Fig. 7A). In addition, the glycogenolytic effect of glucagon was mimicked by cyclic AMP and its dibutyryl derivative, and potentiated by theophylline. In adult in vitro systems, perfused liver (lo), and surviving isolated hepatocytes (11)(12)(13)) glycogenolysis was always accompanied by a great increase in cyclic AMP levels. It may be postulated that in cultured fetal hepatocytes as in the adult systems, glucagon acts according to the scheme of Sutherland. The fixation of the hormone to a specific membrane site is accompanied by stimulation of a plasma membrane adenylate cyclase system (26)(27)(28). The cyclic AlIP produced activates in turn a protein kinase (29,30), which regulates via phosphorylation the activity of two key enzymes for glycogen metabolism: glycogcn phosphorylase and glycogen synthetase (31). This assumption implies the presence of all of these steps in 15.day-old hepatocytes grown for 4 days in the presence of cortisol. Adenylate cyclase activity has been shown to be present and to respond to glucagon and epinephrine 4 days before birth (32)(33)(34)(35). At earlier stages (15 to 16 days of gestation), adenylate cyclase activity was also present, but, according to some authors, it responded to glucagon and epinephrine (36)) and according to others, it did not (35). It should be pointed that the adenylate cyclase thus detected may belong at least partly to the hematopoietic cells, which represent, at these stages, 607C of the liver population (37,38). The present work shows that glucagon stimulates the accumulation of cyclic AMP in 15.day-old hepatocytes grown for 4 days in the presence of cortisol. Plas and Nunez2 also suggest that this system is not induced by cortisol during the culture period, and that it is present before significant amounts of glycogen are stored.
In the adult liver, cyclic AMP produced after glucagon administration is responsible for both the activation of glycogen phosphorylase (39) and the inactivation of glycogen synthetase (39,40). From in viva studies, it. has been postulated that the regulation by glucagon of these enzymatic activities appears around the time of birth (41). In explant liver cultures from term fetuses, glucagon was found to activate glycogen phosphorylase by sorne authors (42), but no effect was found by others (25). In cultured fetal hepatocytes, it is clear that cyclic AhIP activates the glycogcn degradation system (Fig. 5). In addition, this effect of glucagon was rapidly reversed (after 4 hours), suggesting a reversibility of the activation state of glycogen phosphorylase, probably via the action of phosphatases (43).
In fact, it was found that the reversal of the response to glucagon was not modified by a second addition of glucagon (Figs. 4 to 6). This second addition of glucagon was also ineffective after transfer of the hepatocytes to a fresh medium with or without repetitive washing of the cells (Table I). Consequently, the loss of the glycogenolytic response depends neither on a fast inactivation of the hormone in the conditioned medium, nor on the occupation of the receptor sites by inactivated glucagon. This latter possi-bility is unlikely because it has been shown that the binding of or& through the cell membrane in explants (51). In cultured glucagon and t.he inactivation of the hormone are clearly inde-fetal hepatocytcs, release of all antagonist similar to that found pendent (27,44). These results support the hypothesis that the in isolated adipose ctlls was not observed (Table II). If such an cells really become resistant to a second addition of glucagon antagonist is produced in fetal hcpatocytes, it must be unable to after a first exposure and response to the hormone. It should be diffuse out of the cultured hepatic cells, and must exert its effect emphasized that this resistance is expressed at the level of glyco-intracellularly. gen metabolism, i.e. the physiological response. IIowever, it Another question is whether the onset of this autoresistant cannot be excluded that the receptor sites are occupied by irproperty induced by glucagon is mediated by cyclic AAlP, as is reversibly bound native glucagon or by some contaminant present in the glucagon solution.
The loss of response to glucagon implies a blocking of some step in the glucagori-dependent glycogenolytic pathway, which may be located before or after cyclic A;\II' production.
Escape from blocking was obtained with maximal glycogenolytic COIIcentrations of cyclic A?*11 and dibutyryl cA?*Il' (Table IV). Therefore, refractoriness does not occur, at least qualitatively, at the protein kinase activation step or later, but probably at the level of cyclic ,411I' production.
This conclusion was cow firmed by the fact that in the resistant hepatocytes, 110 cyclic A;1IP production was observed after a second glucagon addition. A similar loss of hormonal response at the level of cyclic AMP production has been shown in several in vitro systems after a first exposure to a hormone (45551). For example, with adipose tissue, both in isolated cells (46,47,51) and in tissue explants (51), a lack of response to a second epinephrine or ACTH stimulation has been described. However, a relationship between the refractoriness in the production of cyclic AX' and the lipolytic response to epinephrine and ACTH was not demoilstrated (51). On the other hand, in the present work, the fact that the glycogenolytic response to glucagon was rapidly reversed permitted the demonstration of refractoriness both for the production of cyclic AMP and for the physiological response. A glycogenolytic response to norepinephrine was observed even while the cells exhibited refractoriness to glucagon (Table IV). If the glycogenolytic effect of norepinephrine is mediated by cyclic AXU' (10,52)) this result again suggests that the refractoriness to glucagon is not located at a step in the glycogenolytic response involving activation by cyclic AJvIP. In addition, refractoriness to glucagon seems specific, since norepinephrine was able to induce a glycogenolytic response in the hepatocytes re sistant to glucagon. In adipose tissue, no specificity for the onset of refractoriness at the level of cyclic AMP production by epinephrine and ACTH was found (47, 51). A('TH was unable to enhance the production of cyclic AlzlP after prior exposure to epinephrine, and \-ice versa (47, 51). On the contrary, in other in vitro systems, such as brain slices for epinephrine and histamine (45), ovarian follicles for LH and I-'GE2 (49), and macrophages for epinephrine and PGEz (50), specificity was found for the hormone-induced refractoriness at the level of cyclic AMP release.
In isolated adipose cells, the refractoriness was shown to be linked to a factor released by the cells into the medium after the first hormonal exposure (46,51). This factor was called a "hormone antagonist" by Ho and Sutherland (46). This antagonist, released by isolated adipose cells after a first exposure to epinephrine and then partially purified, was found to inhibit the release of cyclic AI&U' by epinephrine, but also by ACTH and glucagon in fresh cells (46). On the contrary, in adipose tissue explants (51), release of such an antagonist was not observed. In addition, the "antagonist" produced by isolated adipose cells after a first exposure to epinephrine was unable to transfer the refractoriness to adipose explants (51). These contradictory findings could be explained by the nondiffusibility of the antag-the glycogenolytic response. Experiments summarized in Table  III show clearly that glycogenolytic concentrations of cyclic A1IP and dibutyryl cAMI' were unable to induce the resistant property to glucagon at the level of glycogenolytic response. This observation suggests that the appearance of refractoriness occurs via a mechanism where cyclic AlI is not involved. On the contrary, in isolated adipose cells, the production of the antagonist by cpinephrine seems to be mediated by cyclic AAIl' (46). 1)ata on the dose response of glucagon with regard to the refractoriness phenomenon will be necessary to know if the refractoriness is related to an interaction between glucagon and its receptor. Finally, the fact that this hormonal autoregulation was shown to exist at the level of the glycogenolytic response rnight have a physiological significance by providing an additional regulatory mechanism for glycogenolysis.
However, such a refractoriness toward glucagon has not been described in adult perfused liver (lo), where continuous presence of this hormone was accompanied by a continuous production of cyclic AMP. It is not known at the present time if this refractoriness is specific to the fetal hepatocyte. In any way, this mechanism may be one factor cow tributing to the maintenance of high stores of hepatic glycogen before birth in spite of the presence in fetal blood, during late gestation, of pancreatic glucagon levels similar to those of adult rats (7,53).