Glucagon degradation in isolated rat hepatocytes: Effect of ammonium chloride and chloroquine

Molecular and Cellular Endocrinology, 23 (1981) 311-320 Elsevier/North-Holland Scientific Publishers, Ltd.

311

GLUCAGON DEGRADATION IN ISOLATED RAT HEPATOCYTES: OF AMMONIUM CHLORIDE AND CHLOROQUINE Bertrand CANIVET, Phillip GORDEN, Jean-Louis CARPENTIER, Pierre FREYCHET

EFFECT

Lelio ORCI and

Institut National de la Santd et de la Recherche Mddicale (I.N.S.E.R.M., Groupe U 145), et Laboratoire de MPdecine Exp&imentale, Fact&P de Midecine, Nice (France) and Institut d’Histologie et d’Embryologie, Facultk de MPdecine, Geneva (Switzerland) Received 20 January 1981;revision received 21 May 1981; accepted 22 May 1981

It has recently been shown that, when [ ’ 2sIlglucagon is incubated with isolated rat hepatocytes at 37”, the radioIabelIed material is progressively internalized by the cell and is found to associate preferentially with lysosome4ke structures. To assess the role of this process in the degradation of the hormone, the degradation of [ ’ 25IJglucagon by isolated rat hepatocytes was examined both in incubation media and in ceil extracts, after exposure of the radiolabelled hormone to hepatocytes in the absence and in the presence of lysosomotropic agents NILtCl (8 mmoles/l) or chloroquine (10 rmoles/I); bacitracin (0.8 mg/ml, i.e. 0.6 mmoles/l) was present in aU experimental conditions to minimize extracellular degradation. Neither NHaCl nor chloroquine altered the time course and steady&ate binding of [ ’ 25Ilglucagon, or the degradation of the hormone in incubation media. However, both agents partially inhibited the degradation of cell-associated [ ’ 25Ilglucagon in steady-state conditions. In dissociation experiments, NHeCl, and even more so chloroquine, decreased the rate and the extent of release of radiolabelled material from the ceils. Moreover, after 60 min dissociation, the presence of either agent resulted in less degradation of both cell-associated [’ 25Ilghrcagon and that released into the medium. These results suggest that lysosomes are involved in the intraceilular degradation of glucagon. Keywords: isolated hepatocytes; tion.

glucagon; glucagon internalization;

glucagon degrada-

Glucagon interacts with specific binding sites on isolated rat-liver cells (Freychet et al., 1974; Sonne et al., 1978; Rouer et al., 1980). As shown for many peptide hormones, part of the cell-bound glucagon is internalized; morphological studies of isolated hepatocytes incubated with radiolabelled glucagon have shown that the translocated radioactivity preferentially associates with lysosome-like structures (Barazzone et al., 1980). Therefore, we investigated hepatocyte-associated glucagon degradation using the lysosomotropic agents NH4C1 and chloroquine. Ammonium chloride has been reported to inhibit insulin degradation in freshly isolated hepatocytes (Gorden et al., submitted) and in hepatoma cells (Terris et al., 1979) as well as EGF degradation by tibroblasts (Carpenter and Cohen, 1976) presumably by 0303-7207/81/0000-0000/$02.50

0 1981 Elsevier/North-Holland

Scientific Publishers, Ltd.

Bertrand Ganivet et al.

312

inhibiting lysosomal function (Seglen and Reith, 1976). Chloroquine inhibits intralysosomal degradation of low-density lipoproteins (Goldstein et al., 1975) and of epidermal growth factor (Carpenter and Cohen, 1976) in fibroblasts.

MATERIALS

AND METHODS

Hepatocyte isolation Hepatocytes were isolated from male Wistar rats (100-l 50 g) by collagenase dissociation of the liver as previously described (Le Cam et al., 1976; Fehlmann et al., 1979a). These cells have previously been shown to retain morphological integrity of their surface membrane structure and intracellular organelles (Le Cam et al., 1976; Fehlmann et al., 1979a), and to respond to various hormones in vitro (Fehlmann et al., 1979b; Canivet et al., 1980) including glucagon (Fehlmann et al., 1979b). Iodimtion of glucagon [“‘I]Glucagon was prepared at a specific activity of 90 &i/pg by a modification of the chloramine T method (Fouchereau-Peron et al., 1976). The labelled glucagon was purified by gel filtration on Sephadex G-50 at 4” before each experiment. Incubation procedure For binding experiments, hepatocytes were incubated in Krebs-Ringer bicarbonate buffer, pH 7.4, containing 10 mg bovine serum albumin (Fraction V) per ml and 0.8 mg bacitracin per ml (about 0.6 mmoles/l). The cell suspension (1 X lo6 cells/ml) was gassed with a mixture of 5% CO? and 95% OZ. Cells were incubated with [r2’I]glucagon (0.7 nmoles/l) for 60 min at 37’, or for 120 min at 20°, with or without NH4C1 (8 mmoles/l) or chloroquine (10 pmoles/l). After various times of incubation, 5009 aliquots of the cell suspension were collected and immediately centrifuged (10 set at 2 000 X g) in 1.5-ml tubes (Eppendorf) to separate the cell pellet and the supernatant: both were then submitted to a study of degradation of [1251]glucagon (see below). Identical incubations were carried out in the presence of 30 pmoles unlabelled glucagon per ml to determine the non-specific binding. Dissociation experiments were performed at 37” as follows. After [1251]glucagon had been incubated with hepatocytes for 30 min at 37’, as described above, cells were sedimented by centrifugation, washed twice in chilled buffer, and quickly resuspended in a lo-fold greater volume (compared with the initial incubation volume) of the corresponding media, i.e. with or without NH*Cl(8 mmoles/l) or chloroquine (10 pmoles/l). Aliquots were collected at various times of dissociation; cells were sedimented by centrifugation and the radioactivity remaining associated with the cell pellet was determined. .Both the cell pellet and the corresponding supernatant were then analyzed for degradation of [ 1251]glucagon, as described below.

Glucagon degradation in hepatocytes

313

Bacitracin (0.8 mg/ml) was present throughout association and dissociation studies to minimize glucagon degradation in incubation media; bacitracin decreases media degradation of glucagon with isolated hepatocytes (not shown) as well as with liver homogenates (Desbuquois and Laudat, 1974). Studies of / ’ 251/glucagon degradation

The supernatants collected at various times in association and dissociation experiments were diluted in Krebs-Ringer bicarbonate buffer to decrease the albumin concentration to 2-3 mg/ml. The integrity of [ ‘251]glucagon in these incubation media was estimated by precipitation by 5% trichloroacetic acid and adsorption to talc (Freychet et al., 1972). For the analysis of cell-associated radioactivity, cell pellets were extracted with a mixture of 0.1% Triton X-100, 3 M acetic acid, and 6 M urea (Terris and Steiner, 1975). The mixture was centrifuged in a Beckman microfuge at 12 000 Xg for 5 min: 93-99% of the total cellular radioactivity was recovered in the supernatant. The latter was applied to a Sephadex G-50 (fine) column (0.9 cm X 50 cm), which was equilibrated and eluted with 1 M acetic acid. The proportion of accessible (i.e., surface-bound) [‘251]glucagon during internalization was evaluated by a 6-min cold (4”) acid (pH 2.5) extraction of cell-associated radioactivity, according to Haigler et al. (1980). Chemicals

Porcine glucagon was purchased from Novo (Copenhagen, Denmark). Na’*‘I was purchased from the Commissariat a I’Energie Atomique (Saclay, France). Chloroquine, bacitracin, bovine serum albumin (Fraction V) were from Sigma. Other reagents were of the best grade commercially available.

RESULTS To study cell-related degradation it was first necessary to establish that neither NH4Cl nor chloroquine affected degradation in the incubation media (Table 1). The rate or extent of [ ‘*‘I ]gl ucagon binding at both 20 and 37’ (Fig. 1) was unaffected by these agents. When extracts of cell-associated radioactivity were applied to a Sephadex G-50 column, 2 main peaks of radioactivity were observed in the elution profile (Fig. 2; Table 2): a major peak eluted in the position of intact [‘*‘I]glucagon, and a minor peak eluted slightly beyond the iodide marker, presumably in the position of iodotyrosyl peptides that loosely adsorb to the column. A small (~2.5%) fraction of the total radioactivity recovered from the column eluted in the void volume (Fig. 2). At early times of association (5 min at 20”; 2 min at 37’), Sephadex elution profiles of the radioactivity in cell extracts were similar in the absence or presence of NI&Cl or chloroquine, as shown by the percentages of radioactivity recovered in peak II

314

Bertrand Canivet et al.

Table 1 Analysis of media radioactivity of [ r2sI]glucagon

incubated with isolated hepatocytes

Hepatocytes were incubated with [ ras~]glucagon for various times, temperatures and conditions of binding media. At the times indicated, cells were collected by centrifugation, and the integrity of [rssI]glucagon in the supernatant was estimated by talc adsorption and by 5% trichloroacetic acid (TCA) precipitation. Values represent the percentages of radioactivity precipitated by TCA or adsorbed to talc. Incubation conditions

Time of incubation Temperature

Talc TCA Talc TCA

20”

Talc TCA Talc TCA

31”

mm

5 120 2 60

Buffer a

Buffer a +NH4 Cl

Buffer a +chloroquine

94 89 84 60

95 87 82 66

95 89 81 62

92 82 66 50

93 85 60 48

91 84 70 50

a Buffer refers to Krebs-Ringer bicarbonate medium containing 0.8 mg bacitracin per ml (see Materials and Methods).

37’

Fig. 1. Time course of specific binding of [ ’ 25Ilglucagon to isolated hepatocytes in KrebsRinger bicarbonate buffer (see Materials and Methods) containing 0.8 mg bacitracin per ml (9); in the same buffer plus NH4Cl at 8 mmoles/l (A); and in the same buffer plus chloroquine at 10 nmoles/l (0). Specific binding was determined as total binding minus non-specific binding; nonspecific binding did not exceed 16% of total binding. Each point represents the mean of quadruplicate determinations.

Glucagon degradation in hepatocytes

315

‘*%GLUCAGON

‘251-GLUCAGON 125 I ’

I

“\ (I)

I (II)

j25’ cm,

37” 2 min

FRACTION

I

1

I

0

25

50

NUMBER

( 1 ml

: vol)

Fig. 2. Sephadex G-50 fntration elution profile of extract of cell-associated radioactivity. Hepatocytes were incubated with [ 1z s I]glucagon in Krebs-Ringer bicarbonate buffer containing 0.8 mg bacitracin per ml. After 2-min or 60-min incubation at 37”, cells were collected by centrifugation, and the cell pellets were extracted as described in Materials and Methods. The supematants of the cell extracts were then applied to a Sephadex G-50 (fme) column (0.9 cm x 50cm)which was equilibrated and eluted in acetic acid (1 mole/l). The peak elution volume of the void (Vo), [ ’ 25Ilglucagon and lzsI are shown at the top. The slashes designate the fractions pooled for peaks I (void), II (intact [ rZ51]glucagon) and III (degraded products). The percentages of radioactivity recovered in peaks II and III for various times, temperatures and conditions of binding media are presented in Table 2.

(intact glucagon) and peak III (peptide fragments) (Table 2). By contrast, after 120 min at 20”, and even more so after 60 min at 37’, the presence of NH&l or chloroquine in incubation media resulted in a marked inhibition of degradation of cellassociated [‘251]glucagon (Table 2). In dissociation experiments, the presence of lysosomotropic agents (throughout the association and dissociation study) resulted in a lower extent of release of radioactivity from hepatocytes. Thus, after 90 min of dissociation at 37’ in the absence of these agents, 75% of the cell-associated radioactivity was released. In contrast, in the presence of NH&l or chloroquine, only 64 and 54% were released, respectively (Fig. 3). In the absence of lysosomotropic agents, the radioactivity released in the medium was evidently largely degraded, because about 80% of the radioactivity

316

Bertrand Canivet et al.

Table 2 Analysis of extracts of cell-associated radioactivity hepatocytes

of [ 12si]glucagon

incubated with isolated

Hepatocytes were incubated with [ i2si]glucagon for various times, temperatures and conditions of binding media. At the times indicated, cells were collected by centrifugation, and the cell pellets were extracted as described in Materials and Methods. The supernatants of the cell extracts were then applied to a Sephadex G-50 column as detailed in Fig. 2. Peaks II and III correspond to the elution of intact [ 1asI]glucagon and of degraded products, respectively (see Fig. 2). Values represent the percentages of total radioactivity recovered from the column. Incubation conditions

Time (mini

Buffer a +NH&l ichloroquine

5

Buffer a +NIQC!l +chloroquine

120

Buffer a +Nl$CI +chloroqume

2

Buffer a +NH4Cl +chIoroquine

60

a Buffer refers to Krebs-Ringer Materials and Methods).

Temperature

Peak II

20*

87.0 87.1 88.5

6.5 5.9 5.9

77.9 86.3 81.5

13.6 8.7 8.3

83.6 84.6 86.7

8.9 8.1 8.4

69.4 82.9 84.1

20.2 9.4 9.3

37O

bicarbonate medium cont~~g

Peak III

0.8 mg bacitracin per ml (see

released was accounted for by degraded [’ 251]glucagon after 90 min of dissociation at 37’. The presence of NH&l or chloroquine reduced this to about 68 and 60%, respectively (Fig.4). A greater proportion of intact [‘251]glucagon was released into the medium at all time points of dissociation when the lysosomotropic ,agents were present (Fig. 4). When cell extracts obtained at various times of dissociation fo~o~ng a 30-min a~ociation were analyzed by gel fdtration (Table 3), after 5 min dissociation the proportion of intact [ 1251]glucagon in the cell extract (which eluted from the column as peak II) was similar whether or not the lysosomotropic agents were present. However, after 60 min dissociation the presence of NH&l or chloroquine resulted in less degradation of cell-associated [r2’I]glucagon (Table 3). When hepatocytes were treated for 6 mm with acetic acid at 4” according to Haigler et al. (1980), only 21% of the cell-associated radioactivity was recovered from hepatocytes after 60 mm of dissociation, whereas 70% of the radioactivity was released from the cells at the end of the 30-min association period (at 37°C).

Glucagon degradation in hepatocytes

317

L

‘

,

I

0

30

60

90

TIME, min Fig. 3. Tie course of ~c~tion of [ t251Jglucagon from hepatocytes at 37”. After a 30-m&1 association period, dissociation was performed as described in Materials and Methods. Throughout association and dissociation, cells were incubated in Kmbs-Ringer bicarbonate buffer containing 0.8 mg bacitracin per ml (u) or in the same buffer supplemented with NH,+Cl at 8 mmoles/l (A) or with chloroquine at 10 rtmoles/l (a). Results are expressed as percentage of initial binding (measured at the end of the association period), Each point is the mean of quadruplicate determinations. ...

Table 3 Analysis of extracts of cellassociated isolated hepatocytes at 37”

radioactivity during dissociation of [ 1asI]ghrcagon from

Experimental conditions were as described in Table 3. At the times indicated, cells were collected by centrifugation, and the cell pellets were extracted as described in Materials and Methods. The supernatants of the cell extracts were then applied to a Sephadex G-50 column as detailed in Fig. 2. Peaks II and III correspond to the elution of intact [r asI]glucagonand of degraded products, respectively (see Fig. 2). Values represent the percentages of total radioactivity recovered from the column. Incubation conditions

Buffer +I@%&1 +chloroquine Buffer +hIII$c1 ~hloroqu~e

Time (mm)

Peak II

Peak III

5

81.7 82.4 81.6

9.6 8.1 8.1

60

61.0 76.4 75.3

18.0 10.0 11.0

Bertrand Canivet et al.

6 Ip

0

TIME of

DlSSOClAflON

30

60

90

(mid

Fig. 4. Analysis of radioactivity released during dissociation of [ ‘251]glucagon from isolated hepatocytes at 37”. After a 30-min association of [ ’ 2’ I]ghmagon with hepatocytes at 37”) dissociation was performed, at 37”, as described in Materials and Methods. Throughout association and dissociation, cells were incubated in Krebs-Ringer bicarbonate buffer containing 0.8 mg bacitracin per ml (a) or in the same medium supplemented with NH& at 8 mmoleslf (A) or with chloroquine at 10 pmoles/l (*). The integrity of [ I25 Ifghrcagon releasedfrom the cells was analyzed by 5% tricbloroacetic acid (TCA) precipitation and by talc adsorption.Values represent the percentages of radioactivity precipitated by TCA or adsorbed to talc.

DISCUSSION It has previously been shown that insulin degradation in the medium is largely independent of its binding to receptor sites (Freychet et al., 1972; Le Cam et al., 1975). However, other studies have shown that a more specific degradation occurs as a result of the insulin-receptor interaction in hepatocytes (Terris and Steiner, 197.5). Furthermore, recent studies have shown that, after binding to its cell surface receptors, insulin is internalized in hepatocytes (Bergeron et al., 1977, 1979; Gorden et al., 1978, 1980; Carpentier et al., 1979a,b). Although the internalization of glucagon has not been so extensively documented as that of insulin, a similar pattern of time- and temperature-dependent intracellular penetration and preferential association to lysosomal structures has been observed (Barazzone et al., 1980). The present work was carried out to examine the intracellu~r degradation of glucagon in hepatocytes, and to see whether this process is related to lysosomal activity. In all experiments, bacitracin was present in incubation media to minimize the extracellular degradation of the hormone (Desbuquois and Laudat, 1974). We found that neither WC1 nor chloroquine exhibited any protective effect against the degradation of glucagon in the ~cubation media. The specific binding of [r 251]-

Glucagon degradation in hepatocytes

319

glucagon was not influenced by either agent. By contrast, the degradation of cellassociated glucagon was partially inhibited by both agents, but only at relatively late times (120 min at 20°, or 60 min at 37”) of association of the hormone to hepatocytes. This observation is in agreement with morphological observations because internalization is timedependent (Barazzone et al., 1980). This approach, however, does not distinguish between the degradation of surface-bound radioactivity and that of translocated material. To get more insight into the degradation process occurring inside the cell, dissociation studies were performed. Dissociation of cell-associated [ “‘1 ] p eptide is thought to affect first the membrane-bound fraction and subsequently the internalized material; this was demonstrated, for insulin, by autoradiographic studies during dissociation (Gorden et al., submitted). The present study has shown that, as the release of hepatocyte-associated [12sI]glucagon progresses, a greater amount of radiolabelled material remains assqciated with the cells incubated in the presence of NH&l and chloroquine. Gel-filtration analysis of cell extracts after 60 min dissociation showed a greater proportion of intact glucagon in the presence of NH4C1 or chloroquine. At that time, most of the cell-associated radioactivity could be accounted for by intracellular radioactivity; this is strongly suggested by our observation that 79% of cell-associated radioactivity could not be released from hepatocytes by acetic acid treatment. Released radioactivity was also degraded to a lesser extent when lysosomotropic agents were present. Therefore, we conclude that glucagon is degraded within the hepatocyte, in agreement with a recent report by Rouer et al. (1980). Furthermore, we found that lysosomotropic agents such as NH&l and chloroquine could inhibit, partly at least, this degradation whereas neither agent affected the binding or extracellular degradation of the hormone. These results suggest that the lysosome is involved in glucagon intracellular degradation and support the notion that receptor-linked degradation is mediated by receptor-linked internalization. A similar process appears to be involved in many diverse ligands that bind to cell surfaces (Gorden et al., 1980; Ascoli and Puett, 1978).

ACKNOWLEDGEMENTS We thank A. Kowalski for technical assistance, G. Visciano for illustration work, and L. Capolongo for typing the manuscript. This work was supported in part by research funds from the University of Nice, from Fondation pour la Recherche MBdicale, and from Comite Doyen Jean LBpine (City of Nice).

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