Direct Evidence of a Glucagon-Dependent Regulation of the Concentration of Glucagon Receptors in the...

Eur. J. Riochem. 121, 671 -677 (1982) 0 FEBS 1982

Direct Evidence of a Glucagon-Dependent Regulation of the Concentration of Glucagon Receptors in the Liver

Angel SANTOS and Enrique BLAZQUEZ

Departamento de Bioquimica, Facultad de Medicina de la Universidad Complutense, Madrid, and Departamento Intcrfacultativo de Fisiologia, Universidad de Oviedo

(Received June 29/September 15, 1981)

Chronic treatment with exogenous glucagon in the rat results in a reduced concentration of glucagon receptors in the liver. To determine if this is a direct effect of the hormone on its own receptors, the following experimental model in vilro was designed. Cultured rat hepatocytes were exposed to glucagon at 37 "C, washed and incubated with mono[1251]iodoglucagon (1251-glucagon), resulting in reduced binding of '251-glucagon. The magnitude of the diminution in binding was dependent on the concentration of glucagon present as well as on the duration of the exposure. Analysis of the data indicated that the decrease in binding of '251-glucagon was due to a loss of receptor numbers per cell rather than any change in receptor affinity for the hormone. Degradation of '251-glucagon during the incubation of cells in the presence or absence of glucagon was studied and did not account for the differences observed in binding. The binding of mono['2sI]iodoinsulin of these cells was not affected by glucagon, suggesting specificity of the phenomenon. Although the cells incubated with glucagon incorporate less ~-['~C]valine into hepatocyte proteins, this effect was smaller than the reduction in glucagon receptors. Exposure of the cells to cycloheximide (1 pg/ml) produced a progressive loss of glucagon receptors, and this effect was additive to the glucagon-induced receptor loss. This suggests that cycloheximide inhibited receptor synthesis while glucagon accelerated receptor loss. Glucagon-dependent reduction of glucagon receptors closely correlated with the decreased glucagon-stimulated production of CAMP. Furthermore, epinephrine (0.5 pM) stimulation of cyclic AMP production was similar in hepatocytes previously incubated with or without glucagon, indicating a specific role for glucagon in modifying target-cell sensitivity.

A hormone-dependent receptor regulation has been described for catecholamines, steroids and peptide hormones, which directly modify target-cell response to hormones [l - 31.

In relation to glucagon, there is contradictory evidence about its role in the regulation of its own receptors. In fact, the hyperglucagonemia of fasted [4] and developing rats [5] and animals under liver regeneration [6] is associated with a decrease in glucagon-binding sites in the liver and a proportional reduction of glucagon-stimulated adenylate cyclase activity. However, under other pathophysiological situations, high levels of circulating glucagon have been described, either with increase or reduction in the binding of glucagon to liver membranes and with variable modifications of the glucagon- stimulated activity of adenylate cyclase [7 - 101.

In a previous work [ I l l we have reported that in rats chronically treated with a long-acting glucagon preparation, the glucagon receptor is functionally normal, though present in decreased concentration. Also, when isolated hepatocytes of non-treated animals were cultured in the presence of glucagon, a diminished binding of this hormone to its receptor was observed. Reproducibility of the findings in vivo to

systems in vitro seems to be important in order to demonstrate if the effect of glucagon on its receptors is direct or mediated by other factors. Also, the possible mechanism involved in the reduction of glucagon receptors by the homologous hormone could be elucidated by using isolated hepatocytes maintained in culture. In fact, the amount of a given membrane protein seems to be a consequence of an equilibrium between synthesis and degradation. For com- parative purposes, the turnover of membrane proteins [12] has been applied to the dynamics of insulin and growth hormone receptors. In cultured human lymphocytes, the reduction of binding sites for insulin and growth hormone induced by the homologous hormones has been related more to an increase in the rate of degradation of their receptors than to a decrease in biosynthesis [3 - 131.

In this paper we report the effect of glucagon on the regulation of its own receptors and on target-cell sensitivity as seen in studies with hepatocytes incubated in vitro. Special attention has been focused on the specificity and of mechanism involved in the process.

Abbreviations. '251-glucagon, mono['251]iodoglucagon; '251-insul~n, m~no['~~I]iodoinsulin.

Enzymes. Adenylate cyclase (EC 4.6.1 . I ) ; Closrridium hislolyticum collagenase (EC 3.4.24.3); cyclic AMP phosphodiesterase (EC 3.1.4.17); glucose-6-phosphate dehydrogenase (EC 1.1.1.49); hexokinase (EC 2.7.1.1); 5'-nucleotidase (EC 3.1.3.5); peroxidase (EC 1.11.1.7).

MATERIALS AND METHODS

Experimental Animals

Male rats of the Wistar strain, weighing 120- 150 g were housed in an animal room under constant conditions of

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lighting and temperature. Animals were fed on a standard diet.

The experimental groups of rats included a group of animals injected subcutaneously with 40 pg/lOO g body weight of long-acting glucagon (protamine-zinc-glucagon, Novo Industries AS . , Copenhagen), every 8 h for a period of 4 days, and a control group ofrats which were injected subcutaneously with a placebo (protamine-zinc).

Isolation of Liver Membranes and Hepatocytes

Partially purified liver plasma membranes were prepared by the method of Neville [14] as modified by Pohl et al. [I 51, and stored in liquid nitrogen. To provide an index of membrane purification, binding of '251-glucagon to its liver receptors and 5'-nucleotidase activity [16] were determined at the different steps in the purification procedure. Membrane protein was determined by the method of Lowry et al. [17].

Isolated hepatocytes were prepared from normal and glucagon-treated rats. Animals were anaesthetized with ether, the abdomen opened through a wide incision and the portal vein exposed and cannulated. The liver was then perfused with a 120 ml of calcium and magnesium-free Krebs-Ringer bicarbonate buffer pH 7.4 for 4 min. Another 150 ml of perfusion media with 40 mg of collagenase type I (Sigma Chemi- cal Co., St Louis, USA) were infused into the liver for 5 min. At the end of the perfusion period, the liver was removed, minced with scissors and incubated with Krebs-Ringer bicarbonate buffer pH 7.4 (NaCl 120 mM, KCI 4.8 mM, CaClz 1.3mM,KH~P041.2mM,MgS0~1.2mM,NaHC0~24mM), 1 % bovine serum albumin at room temperature with slow stirring for 5 min. The digested liver was filtered, and the cells spun down at 50 x g for 2 min. All procedures with isolated cells and liver membranes were carried out in plastic labora- tory ware. Cell number was determined by counting in a Neubauer chamber.

Test of Hepatocyle Viability

Biochemical and morphological approaches were used in order to test cell viability. Trypan blue exclusion was determined as judged by the ability of hepatocytes to exclude 0.5 % (w/v) of the stain in the absence of bovine serum albumin for 2 min. Ultrastructure of the isolated hepatocytes was studied with an electron microscope [HI. The adenosine tri- phosphate (ATP) content of the cells was determined by the method of Williamson and Corkey 1191. Binding of glucagon to their hepatocyte receptors and cyclic AMP production in the absence or presence of different glucagon concentrations, were also determined.

Iodination of Insulin and Glucagon

M~no[~~~I] iodoglucagon (1251-glucagon) was obtained according to the procedure of Nottey and Rosselin [20] with specific activities of 450- 500 Ci/g. Trichloroacetic acid pre- cipitability or talc adsorption of the iodinated hormone was 80- 85 %. Immediately after iodination, the reaction mixture was applied to DEAE-cellulose column (0.9 x 30 cm What-

man 52) equilibrated with 0.05 M Tris/HCl, 7 M urea pH 9.3 at 4 "C. After the passage of 80 ml of the same buffer, a linear gradient of NaCl from 0-0.2 M was passed. Monoiodinated hormone obtained after column chromatography was dialyzed against 0.3 M phosphate buffer pH 7.5 and immediately afterwards, stored at - 20 "C.

Using a similar procedure [21], porcine insulin mono- component (Novo Research Institute, Denmark) was iodinated with Nalz5I (Radiochemical Center, Amersham) to yield specific activities of 240 - 300 Ci/g. Mono ['251]iodoinsulin (1251-insulin) was obtained after the iodinated mixture was chromatographed on DEAE-cellulose column (0.9 x 30 cm, Whatman 52) with 0.05 M Tris/HCl pH 9.3 and a linear gradient of NaCl (0-0.1 M).

Measurement of Hormone Binding to Isolated Hepatocytes and Liver Membranes

Isolated hepatocytes were incubated with Krebs-Ringer Tris pH 7.4 (NaCI 120 mM, KCI 4.8 mM, CaClz 1.3 mM, KH2P04 1.2 mM, MgS04 1.2 mM, Tris 24 mM), 1 bovine serum albumin and iodinated hormones (50 pM) at the times and temperature stated in the figure legends. At the end of the incubation periods, the samples were diluted rapidly in Krebs-Ringer bicarbonate buffer pH 7.4 with 2 ml of ice- chilled solution of 1 % bovine serum albumin and immediately filtered on oxoid filters (0.45 pm) soaked in 10 % bovine serum albumin for 30min prior to use. To account for non- specific adsorption of the peptides to the cells, the binding of radioactive hormones was determined in the presence of 1 pM of unlabelled hormones, a concentration which would saturate specific hormone-binding sites. The difference between cell-bound radioactivity in the presence of an excess of unlabelled hormones was considered to represent binding of labelled hormones to specific binding sites. Radioactivity was determined in a well-type scintillation counter. The same experimental procedure was followed in the binding studies with purified liver membranes, except that Krebs-Ringer phosphate buffer pH 7.5 (NaCl 118 mM, KC1 5 mM, MgS04 1.2mM, KH2P04 10mM) was used as the incubation medium. Hormone degradation was studied after the preincubation of isolated hepatocytes (1.5 x lo6 cells) in the absence or presence of glucagon (28 nM) for 6 h at 37°C. Immediately after, cells were washed and incubated with Krebs-Ringer bicarbonate pH 7.4 containing 1 % bovine serum albumin and '251-glucagon (0.1 nM) for 15 min, 30 rnin and 60 rnin at 20 "C. The supernatant then was removed and the percentage of the radioactivity that bound to fresh liver membranes during a subsequent incubation period for 90 rnin at 20 "C, was determined. Tubes incubated under identical conditions without cells in the first incubation served as con- trols. The amount of degraded hormone was calculated by subtracting the sum of the hepatocyte-bound glucagon and of the biologically active glucagon in the supernatant (as measured by the ability of '251-glucagon to bind to liver membranes) from the total amount of glucagon originally present in the incubation mixture.

Preincubation of Hepatocytes

Isolated hepatocytes from control or glucagon-treated rats, were incubated at 37°C in a 199 medium (Gibco Bio- Cult) supplemented with 1 % bovine serum albumin, penicillin and streptomycin (2000 U and 2 mg/ml, respectively) for 0-6 h in the presence or absence of glucagon and/or cycloheximide.

Washing Procedure before '251-glucagon Binding

After preincubation, aliquots of cell suspensions were transferred to plastic centrifuge tubes containing 2 ml of

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Krebs-Ringer bicarbonate and spun at 60 x g for 2 min. The Statistical Analysis cells wcre &mediately rcsuspended in 3 ml of Krebs-Ringer bicarbonate, 1 '%, bovine serum albumin and incubated at 37°C for 30 min. This incubation time was expected to be long enough to dissociate all the glucagon molecules bound to its receptors during the preincubation period. In order to demonstrate this hypothesis, aliquots of hepatocytes were incubated at 37 "C for 10 min, with 0.10 pM glucagon, and it was found using the above procedure, that the hormone dissociates immediately afterwards. No differences in 1251- glucagon binding to cells previously incubated with or without glucagon (0.1 pM) were found. Similar results were obtained when hepatocytes were incubated with unlabelled glucagon for longer periods of time.

25t-glucagon Binding to Washed Cells

For binding studies, '251-glucagon was incubated with washed cells ( 5 x 10') in a total volume of 0.5 ml at 20°C in the presence (0.3 - 1000 ng/ml) or absence of glucagon. After the incubation periods, the cells were separated from the assay buffer by microfiltration. The radioactivity bound to cells in the presence of 1 pM glucagon was considered non-specific binding and subtracted from total binding.

Stimulation of Cyclic A M P Production in Rat Hepatocytes by Glucagon and Epinephrine

Isolated hepatocytes (2 x lo6 cells/ml) were incubated with Krebs-Ringer bicarbonate pH 7.4, containing 4 % bovine serum albumin, 30 mM alanine, 2 mM theophylline and glucagon (from 0-1 pM) or epinephrine ( S O pM). Cell suspension were gassed with 95 % oxygen, 5 carbon dioxide, and incubated with gentle shaking at 37 "C for 2.5 min. Frac- tion V bovine albumin powder (Sigma Chem. Co., St Louis, USA) was defatted prior to use by the procedure of Guillory and Racker [22]. Cyclic AMP accumulation by hepatocytes was increased by glucagon with a maximal effect at 2.5 min, the values decreasing almost to basal levels by 20min. At 60 min and 120 rnin of incubation, cyclic AMP values were indistinguishable from basal concentrations. Therefore, 2.5- rnin incubation periods were selected to study the glucagon- stimulated production of cyclic AMP. At the end of the incubation period, cell suspensions were treated with trichloroacetic acid (5 final concentration), centrifuged and the supernatant washed with ether saturated with water. In aliquots of the aqueous phase, cyclic AMP was assayed by radioimmunoassay as described by Steiner et al. [23].

Incorporation qf L - ( ' ~ C ] Valine into Heputocyte Proteins

Isolated hepatocytes (1 x lo6 cells/ml) were incubated at 37 "C with 199 medium (Gibco Bio-Cult, USA) and L-['~C]- valine (sp. act. 2 Ci/mol; 25 mg/l). At 15 min, 30 min, 60 min and 120 min, 0.5-ml aliquots were taken from the incubation medium, mixed with 1 ml of Krebs-Ringer bicarbonate pH 7.4 and centrifuged at SO x g for 5 min. The resulting pellets then were resuspended with 0.5 ml of distilled water and 0.5 ml of 5 % trichloroacetic acid, and immediately filtered on Whatman GF/C filters. Radioactivity present in the dried filters was determined in a Tricarb liquid-scintillation spectrophotometer, using a mixture of toluene, PPO and POPOP (4 g/l, 0.2 g/I respectively) as scintillation liquid.

Results have been expressed as means & S.E.M.; for statistical comparisons Student's t test was used.

RESULTS

Viability o j Hepatocytes

The cell suspension was predominantly free hepatocytes, which were 80 viable as judged by their ability to exclude trypan blue. Under the electron microscope, these cells appeared separate or in groups of two to four, with very good preservation of membranes and organelles.

ATP content of the hepatocytes was of 12.4 & 0.4 nmol/mg protein (39.3 5 0.8 nmol/106 cells). These results are similar to those reported by others [24].

ESfect of Preincubation with Glucagon on '251-glucugon Binding to Hepatocytes qf'Contvol and Glucagon- Treated Rats

Initial '251-glucagon binding to hepatocytes of rats treated with 40 pg of glucagon-Zn-protamine/lOO g body weight every 8 h for 4 days (2.3 & 0.4 fmol/106 cells) was markedly reduced as compared with the values of non-treated animals' (3.4 0.4 finol/lOh cells; P < 0.05). When the hepatocytes of control rats were preincubated for 4 h with glucagon (28 nM) the binding of 1251-glucagon to these cells decreased significantly in relation to the initial binding (Table I) , which was more marked after 6 h of preincubation ( P < 0.05). Acute exposure of hepatocytes to glucagon (2 h) had no effect ( P > 0.05).

Conversely, when the hepatocytes of glucagon-treated rats (40 pg of glucagon-Zn-protamine/lOO g body weight each 8 h during 4 days) were preincubated in the absence of glucagon (Table I) , the binding of '251-glucagon to these increased markedly with respect to the initial binding. Addition of glucagon (28 nM) to the incubation medium produced a time-dependent decrease over and above the initial reduced '251-glucagon binding ( P < 0.05).

Efrect of Concentration of Glucagon on Receptor Loss

When isolated hepatocytes of non-treated rats were preincubated in the absence or presence of glucagon (from 1-28 nM) for 6 h at 37"C, washed, and incubated with ''ST- glucagon and unlabelled hormone (0- 1 .O pg/ml) for 40 min at 2 0 T , the '251-glucagon was bound to the cells in an inverse relationship with the amount of hormone added during the preincubation period. Independently of the glucagon concentration that had been present (Fig. l), each concentration of unlabelled glucagon in the binding assay always produced the same percentage reduction in the specific binding of '251-glucagon to receptors with all sets of cells, suggesting that the decreased binding of '2'I-glucagon was due to a diminution in the number of receptors sites per cell rather than any change in affinity.

Eflect of Preincubation with Glucagon on '25Z-insulin Binding to Hepatocytes qf Non-treated Rats

In order to elucidate if the glucagon-dependent reduction of 12' I-glucagon binding to isolated hepatocytes was the consequence of a specific phenomenon, we studied the lZ5I-

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Table 1 Effect of preincubation with glucugon on 125 I-glutagon binding 10 isolated hepatocyter of control and glucagon-treated rats IEoldted cells ( 2 x 106/mI) of control and glucagon-treated rats, were preincubated with 199 medium, 2 bovine serum albumin, penicillin (2000 U) and streptomycin (2 mg/ml) in the absence or presence of glucagon (28 nM) At the indicated times, dliquots of the incubation medium were taken Unlahelled glucagon was dissociated by washing and mcubating cells a t 37 "C for 30 min Cell viability remained constant throughout the 6 h of incubation After washing, cells were incubated with '251-glucagon (0 1 nM) for 40 min at 20 C Each point represent the mean 2 S E M of the data obtained with hepatocytes of six different rats Stalistical comparisons were done with the data obtdined in the absence or presence of glucagon

Time of pre- incubdtion

Binding of '251-glucagon

cells from control rats _ _ _ ~ _ - - - - - - - - ~ - ~ _ ~ - - - ~ - _ ~ ~ _ _ - _ _ ~ - _ _ - - - _ ~

cells from glucagon-treated rats _ _ _ _ _ - - _ _ - - ~ _ - ~ ~ _ _ - - - _ ~ _ _ ~ - _ _ - _ - - ~ ~ _ - ~ ~ -

without glucagon with glucagon P without glucagon with glucagon P

11 fmol/lo6 cells fmol/lo6 cells

0 3 4 F 0 4 3 4 + 0 4 > 005 2 3 + 0 4 2 1 + 0 4 > 005 2 3 8 2 0 4 4 3 +_ 0 7 > 005 2 3 + 0 4 2 3 + _ 0 4 > 005 4 3 9 & 0 5 3 0 0 4 5 0 0 5 2 8 + 0 5 1 3 + 0 2 < 005

- _ _ _ _ _ _ - - - -~ - _ _ - ~ - _ ~ _ _ _ ~ _

6 3 6 + 0 4 2 5 + 0 3 < 005 3 2 + 0 6 1 2 + _ 0 3 < 002

5

" 1 10 100 1000

Glucagon concent r a t i o n (ng i m l )

Fig. 1, Effect of concentrurion ofglucugon on rcwcytor loss. Isolated hepatocytes of control rats were preincuhated in the absence (t -e) or presence of glucagon (1 n M 0 -0; 5.7 nM t - m ; 28 nM CT- -0) for 6 h at 37"C, washed and incubated with '251-glucagon (0.2 ng/ml) and unlabelled hormone (0-1000 ng/ml) for 40 min at 20°C. Each value represent the mean of the data obtained with hepatocytes of thrce different rats. Errors of measurement range F 3-7%

insulin binding to these cells previously preincubated in the absence or presence of glucagon (28 nM). As shown in Fig. 2, preincubation of hepatocytes with glucagon for 6 h at 37 "C does not modify the i251-insulin binding to its receptors. In contrast, preincubation of hepatocytes with cycloheximide produces a significant decrease of i2sI-insulin binding.

Ejyect o j Preincubation with Glucagon on L-( 14C] Valine Incorporation into Hepatocyte Proteins

Since glucagon has been reported as a powerful inhibitor of hepatic protein synthesis, the glucagon-dependent regulation of glucagon receptors may be due more to a general effect of the hormone than to a specific mechanism. In an attempts to elucidate this question the hepatocytes of non- treated rats were incubated with ~- [ '~C]val ine , in the absence or presence of glucagon (50 nM).

At all the times studied (15 min, 30 min, 60 min and 120 min; n = 3), the incorporation of ~- [ '~C]vaI ine into hepatocyte proteins (in counts/min per lo6 cells) was slightly less in the cells incubated with glucagon (1 5 min : 5803 i 600,

7 t

Fig. 2. Eflect qf preincuharion ioitlz glucagon a n '251-insulin binding / a hepaiocytes of con/ro/ ruts. Isolated hepatocytes were preincubated in the absence (.--.) or presence (& -0) of glucagon (20 nM) or with cycloheximide (W- -m) during 6 h at 37 'C, washed and incubated with 1251-insulin (0.09nM) at the indicated times at 30°C. Each value re- presents the mean of the data obtained with hepatocytes of three different rats. Errors of measurement ranged + 5 - 10

30 min : 1 1 760 & 790, 60 min : 22948 f 370, and 120 min : 36 902 5 2700) as compared with the hepatocytes incubated in the absence of the hormone (15 min: 6470 f 570, 30 min: 13435 & 930, 60min: 24942 i 680, and 120min: 43099

1600). However, at the same concentration of glucagon added to the incubation medium the reduction of 12'1- glucagon binding to hepatocytes was greater than the decreased incorporation of ~- [ '~C]val ine into proteins. In contrast, the addition of cycloheximide to the incubation medium blocked the iiicorporation of ~-["C]valine into proteins (15 min: 538 41, 30 min: 859 & 57, 60 min: 1555 & 18, and 120 min: 2702 2 188).

Effect of Glucagon and Cyclohexirnide on the 1251-glucagon Binding to Hepatocytes of Non-treated Rats

As compared with control cells, when hepatocytes were exposed to cycloheximide a loss of receptors ( P < 0.05), the same or greater than that observed in the presence of glucagon ( P < 0.05), was observed (Table 2). When both cycloheximide

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Table 2. cffiw of prcincziharion 4vith glucagon and/or cyclolwx-imidc on the '2SI-glucugon binding to isolated hepatoc~ytes of control ruts Isolated cells ( 1 . 5 ~ lo6 ml) of control rats were preincubated a t 37 C with 199 medium, 2 % bovine scrum albumin, penicillin (2000 U) and streptomycin (2 mg/ml) in the absence or presence of glucagon (28 nM), cycloheximide (1 pg/ml) and glucagon plus cycloheximide (28 n M and 1 pgiml, respectively). At thc indicated times, aliquols of the incubation mcdiuni were takcn. Unlabelled glucagon was dissociated by washing and incubating cells at 37°C for 30 min. Cell viabiljty remained constant throughout the 6 h of incubation. After washing, cells were incubated with '2'1-glucagon (0.1 nM) for 40 min at 20°C. Each point represent thc mean * S.E.M. of the data obtained with hepatocytes of four different rats. Statistical comparisons were done between the data obtaincd at 0 h versus those obtained at different times of preincubation with the same cell treatment

Time '*'I-Glucagon bound of pre- ~

InCUbdtlOn control With with with ~ - - - -

glucdgon cycloheximidc glucagon and cycloheximide

h fmol/1O6 cells

0 3 3 * 0 2 3 3 1 - 0 2 3 3 * 0 2 3 3 * 0 2 2 3 7 f 0 3 3 4 T 0 2 4 0 + 0 4 2 5 + 0 4 " 4 3 9 + 0 4 2 3 f 0 4 ' 2 9 * 0 4 2 3 * 0 4 ' 6 3 6 * 0 3 2 2 03' 2 5 + 0 4 h 1 7 + 0 4 "

- - _ _ - - -~ ~ ~

P = 0.05. P < 0.05. P < 0.02.

and glucagon were added together to the cells, an additive time-dependent loss of receptors ( P < 0.02) was observed, suggesting that cycloheximide inhibited the synthesis of new receptors while glucagon accelerated the degradation of receptors.

Comparison of Aijinity of '251-glucagon ,for Isolated Hepatocytes Preincubated with Glucagon and Cy c [ohexim ide

When the percentage of '251-glucagon that was bound by the cells was plotted as a function of glucagon concentrations (Fig. 3) a gradual decrease in the glucagon binding to hepatocytes preincubated with glucagon, cycloheximide or both together as compared to the control was observed over the range of 0.3- 1000 ng/ml. To determine if this reduced 1251- glucagon binding could be related to changes in the capacity of glucagon receptor populations, the bound/frec ratio of the labelled hormone was plotted as a function of the bound hormone of all the groups of hepatocytes according to Scatchard. Thc results obtained with this procedure could not be fitted to a single straight line but gave a curvilinear graph, compatible with negative cooperativity or at least two orders of binding sites : high-affinity/low-capacity and low-affinity/ high-capacity. To calculate it, we followed the procedure previously reported by others [25,26]. Similar affinity constants (P > 0.05) with all groups of cells were obtained. Thus, the high affinity constants were 0.78,0.71, 1.26 and 0.73 x lo9 M-' and the low affinity constants were 0.90, 0.92, 1.5 and 1.5 x 10' M-' , for control, glucagon-treated, cycloheximide- treated and (glucagon plus cyc1oheximide)-treated hepatocytes, respectively. However, there was a different binding capacity in each population of cells. Thus, the total number

Glucagon concen t ra t i on (ng irnl)

I B

n l I . -_ I " 0.0 2 0.2 0.4

Glucagon bound ( n g / 1 0 6 ce l l s )

Fig. 3. Competiiion curves ( A ) and Scatchard nnulysis (8) of glucupn binding to isolated hepatocytes of confrol rai.s previously preincuhaicd with glucagon andlor cycloheximide. Isolated hepatocyies (1.5 x lo6 C C I I S ~ mi) were preincubated in the absence (0-0) or presence of glucagon (28 n M ; B --m), cycloheximide (1 pg/ml; & -0) and glucagon plus cycloheximide (28 n M and 1 pg/ml, respectively; D---o) for 6 h at 37"C, washed and incubated with '251-gl~~cagon (0.1 nM) and unlabclled hormone (0- 1000 ng/ml) for 40 min at 20 ' C. Each value represent thc mean of the data obtained with hepatocytes of four different rats

of binding sites in ng/106 cells, were significantly greater in control (0.70 i 0.14) than in hepatocytes preincubated with glucagon (0.45 & 0.13, P < 0.05), cycloheximide (0.38 F 0.003, P < 0.02) and glucagon plus cycloheximide (0.20 & 0.03, P < 0.01), respectively. Furthermore, the binding sites with higher affinity accounted for 17:; of the values.

Degradation of i251-glucagon by the Hepatocytes Preincubated with or witliour Glucagon

Degradation of 1251-glucagon by the hepatocytes preincubated in the presence (28 nM) or absence of glucagon was similar during the 60 min of incubation. These results support the conclusion that the hormone-dependent reduction in '251-glucagon binding was not due to incrcased glucagon degradation during the incubation period.

Stirnulalion of Cyclic A M P Production by Glucagon and Epinephrine in Hepalocyte.y Pveincubaied vvith or without Glucugon

In order to evaluate if the reduced binding of glucagon by the hepatocytes preincubated with this hormone could generate a decreased biological response, the production of cyclic AMP by these cells was studied under basal conditions, and when stimulated by glucagon and epinephrine. In order to avoid the phosphodiesterase activities, 2 mM theophylline was always added to the incubation medium.

6 76

1 0

I I I

1d10 s,,d10 l$ a-8 167

Glucagon concentration (M)

Fig. 4. Glucagon-stirnulaird cyclic, A M P produc,rroti b.v isolated heparo- q t e s preiricuhated with or without glucagon. Isolated cells (1.5 x 106 cells/ ml) of control rats, were preincubated with (28 n M ; &-o) or without (0- -0) glucagon for 6 h at 37 'C, washed and incubated for 2.5 min at 37 f with Krcbs-Ringer bicarbonate pH 7.4, containing 4';; defatted bovine serum albumin, 10 mM alanine, 2 mM theophylline in the absence or presence of glucagon. Each value represent the mean i- S.E.M. of the data obtained with hepatocytes of five different rats. Statistical comparisons between both groups a t 0.5 nM, 30 nM and 0.1 pM of glucagon, P i 0.05 and at 1 nM, P < 0.01.

As shown in Fig.4, the stimulation of cyclic AMP production by glucagon in both populations of hepatocytes was a function of the hormone concentrations added to the incubation medium. However, a smaller dose-response curve was obtained with the hepatocytes previously preincubated with glucagon (statistical comparisons between both groups at 0.5 nM, 10 nM andO.l pM, P < 0.05 and at 1 nM, P < 0.01). The concentrations of glucagon that elicited half-maximal (2.5 nM) and maximal effects (0.1 pM) on the content of cyclic AMP were similar for hepatocytes preincubated in absence or presence of glucagon, indicating a specific role of glucagon in decreasing target-cell sensitivity.

Also, epinephrine (50 pM) stimulation of cyclic AMP production by hepatocytes previously preincubated with or without glucagon was studied. Cyclic AMP production was similar ( P > 0.05) with both populations of cells at all times (2.5 min and 10 min) studied, suggesting a specific role for glucagon in modifying target cell sensitivity.

DISCUSSION

At present, there is contradictory evidence about the role of glucagon in regulating its own receptor concentrations. Most studies have been done in vivo, under circumstances in which either endogenous or exogenously induced hyperglucagonemia is associated with marked changes of other hormones and metabolites, making it difficult to obtain definitive proof of a direct hormonal effect. To avoid this difficulty, we developed a model in vitro with isolated rat hepatocytes. These cells, the main target cells for glucagon, retain full activity of the glucagon-sensitive adenylate cyclase, and when incubated in vilro for an appropriate time (0-6 h) maintain the relationship found in vivo between receptors and circulating levels of glucagon, making them suitable for these studies. When isolated cells from rats treated with a long-acting glucagon preparation were incubated in the presence of glucagon they retained the decreased glucagon receptor concentrations already observed in vivo but this

effect disappeared after the removal of the hormone from the incubation medium. Convcrsely, cells from non-treated animals showed a reduced '251-glucagon binding after the incubation with exogenous glucagon. This reproducibility of findings in vivo in our system in vitro permitted investigation of the different steps involved in the process, and enabled us to demonstrate that the concentration of glucagon receptors was directly related to the glucagon concentration to which the cells were exposed and to duration of exposure. The observed decrease in binding is fully accounted for by reduced receptor concentrations, as the receptors were functionally normal by various different criteria. Specificity of this effect was assessed by demonstrating that insulin receptors were unaffected by the presence of glucagon in the incubation medium. Also, since glucagon inhibits hepatic protein synthesis, and this could affect glucagon receptor concentrations non-specifically, we evaluated the relationship between this effect of hepatocyte protein synthesis and receptor concentration. Since valine is poorly metabolized by hepatic cells [27], it was the amino acid selected for measuring rates of protein synthesis. The incorporation of ~ - [ ~ ~ C ] v a l i n e into proteins was slightly reduced when glucagon was added to the incubation medium, compared to incubation without the hormone. However, at the same glucagon concentrations there was greatly reduced '251-glucagon binding to hepatocytes, suggesting a specific role for glucagon in regulating its own receptor concentrations.

The number of receptors would be reduced if glucagon caused a decrease in the rate of synthesis or degradation or if it modified subunit and storage pools of receptors. To estimate the rate of degradation, cycloheximide was used to inhibit the synthesis of new receptors, with the assumption that cycloheximide does not modify the degradation rate and that receptor synthesis does not proceed when overall protein synthesis is inhibited. Our studies with cycloheximide show a progressive loss of glucagon receptors; however, the effect of cycloheximide was additive to the receptor loss induced by glucagon, suggesting that cycloheximide inhibited synthesis of receptors while glucagon increased the degradation rates. Furthermore, the glucagon receptor appears to be a very stable protein, since even with 90% blocking there was only a 35% decrease in the number of receptors. As with glucagon, both insulin and growth hormone seem to increase receptor loss by increasing the degradative rate [3,13]. In addition, thyroliberin mediated loss of thyroliberin receptors by a process requiring active protein synthesis [28]. Also, occupancy of only a small percentage of receptors produced significant receptor loss (e.g. 1 nM). However, greater glucagon concentrations (50 nM) with almost complete occupancy of receptors produce only a 35 % receptor loss.

Since hepatocytes contain a heterogenous population of saturable glucagon-binding sites and occupancy of only a limited number of these can produce full stimulation of cyclic AMP formation, the reduced glucagon receptor concentrations induced by the homologous hormone could not be expressed in the target cells by a reduced biological response to glucagon. This is supported by the work of Rosselin et al. [29], who reported a nonlinear relationship between binding and cAMP production, with half-maximal effect when only 8 % of receptors were occupied. Birnbaumer and Pohl [30] have found that 80-900/, of the binding sites did not take part in the activation process and therefore were not true receptors. Also, Sonne et al. [25 ] showed that accumulation of cAMP is proportional neither to receptor occupancy nor to the rate of association. However, Rodbell et al. [31] found

677

that complete activation of the enzyme required full occupancy, indicating that all binding sitcs are true receptors. Nevertheless our results show that the reduced concentrations of glucagon receptors induced by the homologous hormone closely correlate with the decreased CAMP production induced by this hormone. In addition epinephrine stimulation of CAMP accumulation was similar in the hepatocytes preincubated in the presence or absence of glugacon, suggesting a specific role for glucagon in regulating its own receptor concentrations and as a consequence decreasing the target- cell sensitivity to the hormone.

The authors wish to thank D r M. Serrano Rios for his critical reading of the manuscript and Ms S. Pastor and Mrs A. Ortiz for their excellcnt technical assistance. This work has been supported by grant from Fundacion Rodriguez Pascual and Comisihn Asesorn para rl DtJsar- rolh de lu InvestiguciOn.

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Direct Evidence of a Glucagon-Dependent Regulation of the Concentration of Glucagon Receptors in the...

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