Effect of highly purified porcine gut glucagon-like immunoreactivity (glicentin) on glucose release from isolated rat hepatocytes

Biochimica et Biophysica Acta, 675 (1981) 163-170

163

Elsevier/North-HollandBiomedicalPress BBA 29620 EFFECT OF HIGHLY PURIFIED PORCINE GUT GLUCAGON-LIKE IMMUNOREACTIVITY (GLICENTIN) ON GLUCOSE RELEASE FROM ISOLATED RAT HEPATOCYTES HERLUFI.D. THIEDENa, JENS J. HOLSTb,., JOHN DICH a, ALISTERMOODY c and FINN SUNDBY c a Department of Biochemistry A and b Institute of Medical Physiology C, University of Copenhagen, The Panum Institute, Blegdamve] 30, 2200 Copenhagen N and c The NO VO Research Institute, Bagsvaerd (Denmark)

(Received July 24th, 1980) (Revised manuscriptreceived February 20th, 1981)

Key words: Glicentin; Glucagon.like activity; Glucose release; (Rat hepatocyte)

We studied the effect of the highly purified gut peptide glicentin on the glucose production and cyclic AMP accumulation of isolated rat hepatocytes. Glicentin at 2 • 10 -7 mol/l had the same effect on glucose production as maximally effective concentrations of glucagon, but did not stimulate cyclic AMP to the same extent; furthermore, glicentin apparently had only 1/100 of the potency of glucagon on glucose production. During incubation with hepatocytes glicentin was degraded to low molecular weight fragments some of which were chromatographically very similar to fragments of glucagon. It is suggested that glicentin exerts its glucagon-like effects on hepatocytes only after degradation to glucagon-like fragments. The results also demonstrate that the coupling between cyclic AMP accumulation and glucose production depends on the nature of the stimulatory peptide.

Introduction The intestinal mucosa produces a number of peptides with glucagon-like immunoreactivity [1]. One of these has been purified [2] and shown to consist of 100 amino acids. The peptide has been named glicentin [3]. Partial sequence analysis [3] showed that the sequence of its 10 C-terminal amino acids corresponded closely to C-terminal sequence of the 'proglucagon fragment' isolated from extracts of the pancreas by Tager and Steiner [4]. This sequence includes the residues 27-29 of the glucagon molecule. In addition, immunochemical studies with region-specific glucagon antisera indicated that glicentin contained the glucagon sequences 1-17 and 19-29, and it was suggested that glicentin might contain the entire giucagon sequence [5-6]. Glicentin reacts with antiglucagon sera specific for the mid to N-terminal sequence of the giucagon molecule, but not with anti glucagon sera specific for the C-terminal * To whom correspondenceshould be addressed.

sequence, in agreement with the suggestion that the C-terminal antigenic site is masked by the C-terminal extension of the glucagon sequence [5]. The N-terminal 63 amino acid sequence of glicentin does not react with antisera against glucagon. Studies of the biological effect of the glucagon-like gut peptides have been hampered by the lack of pure peptide preparations; because of the similarity to glucagon, the interest has been focused mainly on glucose metabolism (for review see Ref. 1). We report here the results of the effect of glicentin on cyclic AMP accumulation and glucose release of isolated rat hepatocytes. In addition, these studies provide further evidence that glicentin contains the giucagon sequence. Materials and Methods Porcine glicentin was isolated as described by Sundby et al. [2]. Liver cells from fed rats were prepared by the method of Berry and Friend [7], except for the omission of hyaluronidase in the perfusion medium.

0 304-4165/81/0000-0000/$02.50 © Elsevier/North-HollandBiomedicalPress

164

Experimental design. The incubation medium consisted of Krebs-Henseleit buffer containing 0.5% (w/v) gelatine to minimize the degration of glucagon [9]. The cell concentration was 30~40/.d of tightly packed cells per ml suspension. Incubation was performed under continuous shaking (90 rev./min) in Warburg flasks at 37°C under 95% 02/5% CO2. All the suspensions were preincubated for 10 min, total incubation time usually being 70 min. Cyclic AMP was determined as described earlier [10,11] and glucose was determined by the hexokinase method [12]. Radioimmunological determination of glucagonlike immunoreactivity was performed as previously described using antisera 4304 and 4317 [ 13-15 ]. Antiserum 4304 binds to the 6 - 1 5 sequence of glucagon. It does not bind to any of the tryptic fragments of glucagon, but readily binds the 1-17 fragment, as well as duck glucagon, which is modified at position 16; assuming that the binding site can accomodate a chain of maximally eight amino acids, antiserum 4304 should bind to the 6 - 1 5 sequence of glucagon. Antiserum 4317 binds to the 19-29 sequence of glucagon but it does not bind to other fragments, and does not bind turkey glucagon, which is modified at position 28. Antiserum 4304 binds labelled and unlabelled glucagon and glicentin with identical energy [14], whereas 4317 does not bind intact glicentin. Assay buffer was the buffer described below for column experiments; separation was performed using plasma coated charcoal [16]. Detection limits of the assays were 2 - 5 pmol/1 and within assay coefficient of variation better than 10%. 12SI-labelled glucagon and glicentin were prepared and purified according to Jorgensen and Larsen [17] so as to obtain mono-iodinated moieties. Gel filtration. Degradation of glicentin to components of lower molecular size was examined by gel t'titration of hormone-containing incubation medium on 0.9 ×60 cm columns (K 9/60, Pharmacia Fine Chemicals, Uppsala, Sweden) packed with BioGel P 30 (Biorad Laboratories, Richmond, CA), equilibrated with 0.125 mol/l NH4HCO3 containing in addition 0.1 mol/1 NaC1 (flow-rate, 12 rnl/h; I - 2 ml fractions). In some experiments K 16/100 columns (100 ×

1.6 cm; Pharmacia) packed with Sephadex G-50 superfine and equilibrated with the above buffer adjusted to pH 9.1 and supplemented with 0.2% human serum albumin (Behringwerke, Marburg, F.R.G.) were employed to increase the power of resolution, The columns were operated at ambient temperature and calibrated with labelled and unlabelled glicentin and glucagon, as well as 12s I-labelled human serum albumin (Amersham) and 22NaC1. The last two markers were also added to all samples for internal standardization (flow-rate, 15 ml/h). Results

Kinetics of glucose relase and cyclic AMP accumulation Glicentin as well as glucagon stimulated the release of glucose into the medium at a constant or slightly gLucose release pmol/ mt packed celts

100

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15

30

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70

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Fig. 1. Effect o f glicentin and glueagon on the time-course of glucose release from (upper panel) and cyclic AMP levels (lower panel) in h e p a t o c y t e s isolated from fed rats. Each point represents m e a n * S.E. o f four experiments. Pcptides were added at zero time to give the following concentrations: Glicentin: a ~, 10 -7 mol/1; gincagon: o o, 10 -9 mol/1; • -- represents controls w i t h o u t peptide addition. The constant cyclic AMP concentrations in t h e controls were 0.75 ± 0.08 nmol]108 cells.

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Fig. 2. Effect o f varying concentrations of glucagon and gli-

centin on the glucose release and cyclic AMP level. Glucose release was calculated as mentioned in Materials and Methods and given as percentage of the controls. Cyclic AMP was measured 3 min after addition of glucagon or glicentin and also expressed as percentage of the control. The figures are means-'! S.E. of three experiments, o o, glucagon; e-e, glicentin.

increasing rate for the first 30 min whereafter the rate o f release approached that o f the control hepatocytes. Both peptides also stimulated cyclic AMP accumulation. (The time course is shown in Fig. 1).

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;o 7'0m ° 0 10 30 70 min ' 10 Fig. 3. Degradation of glucagon and glicentin by liver cells. The figure shows the changes in 6-15 region immunoreactivity ( ) and in 19-29 region immunoreactivity (. . . . . -) after addition of glucagon (left) or glicentin (right). Hormones were added to the assay at the start of the experiment. The values are means of three experiments ±S.E. Glucagon: • • 10 -10 M; • -% 10 -s M; • •, 10 -6 M. Glicentin: • • and • A, 10 -8 M; o 0 and z~ ~, 2 • 10 -~ M. centration was approx. 5 - 10 -11 mol/1, whereas glicentin stimulated glucose release at concentrations above l 0 -9 mol/l only and with a half-maximal effective concentration o f approx. 2 • 10 -s mol/1. Both peptides stimulated the cyclic AMP accumulation. Significant stimulation was obtained with glucagon in concentrations above l 0 -l° mol/1, and half-maximal effect was reached at 3" l 0 "9 reel/1. Significant stimulation was obtained with glicentin at or above l 0 -7 reel/1 only.

Fate of glucagon and glicentin during incubation Dose-response relationships Dose-response curves are shown in Fig. 2. Although a full dose-response curve for glicentin was not obtained because o f limited supplies, the results suggest that glucagon and glicentin have similar maximal effects on glucose release, but that they differ in potency b y at least 102, glucagon being the more potent. Glucagon significantly stimulated glucose release at 10 -12 mol/l and half-maximal effective con-

(a) Fig. 3 shows the concentration o f immunoreactivity in the incubation media as a function o f incubation time during incubation o f giucagon and glicentin with isolated hepatocytes as measured with regionspecific antiglucagon sera directed against the 6 - 1 5 or 1 9 - 2 9 sequence o f the glucagon molecule. During glucagon incubations the 1 9 - 2 9 region immunoreactivity decreased rapidly, regardless o f the initial concentration, and with identical rates of decrease

166 (Fig. 3, left). The decrease undoubtedly represents degradation of the glucagon molecule *). The rate of degradation of glicentin, as measured with 6 - 1 5 region specific antisera was much slower and apparently no degradation took place at initial concentrations of 10 -8 mol/1. By contrast, a significant and identical fractional increase in the concentration of 19-29 region immunoreactivity was found during incubations of glicentin at both initial concentrations (Fig. 3, right). (b) The immunoreactive products of degradation of glicentin were studied after separation by gel illtration as shown in Fig. 4. Panel A shows the elution patterns of non-incubated glicentin and glucagon (separate experiments) as measured with 6 - 1 5 region and 19-29 region immunoreactive antisera, respectively. The degradation seems to consist of a change from an apparently homogeneous exclusively 6 - 1 5 immunoreactive moiety, with elution position at Kay 0.25 (in this experiment some heterogeneity is indicated by the presence of a 'shoulder' at Kav 0.37) to (1) an exclusively 6 - 1 5 immunoreactive moiety eluting at 0.37 and (2)an exclusively 6 - 1 5 immunoreacrive moiety at 0.7 and (3) a mainly 19-29 immunoreactive moiety eluting at Kay 0.7-0.8 (panels B and C). Panel D shows that very limited changes take place after incubation of glicentin without hepatocytes. The results of eight similar experiments showed that 14.4 -+ 1.7% (mean +- S.E.) and 2.7 ± 0.8% of the applied amount of incubated glicentin appeared at Kay 0.6-0.8 when measured with region-specific antisera against the 6 - 1 5 and 19-29 regions, respectively. A similar degradation of glucagon takes place during incubation with hepatocytes; 7.4-+ 1.6% of the applied amount of glucagon was recovered at Kay 0.7 as 6 - 1 5 region immunoreactivity, and 1.8 -+ 0.2% at Kay around 0.8 as 19-29 region immunoreactivity. (n ---4). We then studied the degradation products of glicentin on columns of somewhat higher powers of * It might be inferred that the observed changes could also reflect other changes in the incubation medium (accumulation of proteolytic enzymes e.g.) with nonspecffic effects on the radioimmunoassay. However, the incubation media were assayed diluted up to 10 4 times which would tend to eliminate nonspecific interference; furthermore such interference usually is reflected as increasing rather than decreasing values.

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resolution. The results of six such experiments are summarised in Table I, which shows that some of the 6 - 1 5 region immunoreactivity remained at the elution position of intact glicentin (range 0.22-0.26) but new peaks appeared at positions 0.33-0.40; 0.58-0.67 and 0.72-0.76. C-terminal immunoreactivity ( 1 9 - 2 9 region immunorecafivity) appeared at 0.23-0.33 and at 0.80-0.82; some 19-29 region immunoreactivity coincided with 6 - 1 5 region immunoreactivity at Kd 0.62--0.71; these peaks also coincided with the glucagon marker (0.68-0.73). The majority of the 19-29 region immunoreactivity eluted at 0.80-0.82. Table I also shows that the actual presence of hepatocytes is not necessary for degradation; the substance(s) responsible for the degradation may be found in the incubation medium itself after separation of hepatocytes by centrifugafion. To characterize further the degradation product of giicentin, we studied the degradation of monoiodinated 12SI-labelled glicentin and giucagon during incubation with hepatocytes. The degradation products were separated by gel £titration and their immunore-

167 TABLE I DEGRADATION OF GLICENTIN BY HEPATOCYTES Glicentin (at 10 -7 mol/l) was incubated for 10-70 min with either a hepatocyte suspension or with the supernatant from hepatocytes incubated for 15 min with medium at 370(? and centrifuged. Incubations were terminated by centrifugation and[or freezing. A sample was applied to Sephadex colums (Sephadex G-50 SF, 100 X 1.6 cm, equilibrated and eluted with 0.125 mol/l NH4HCO3, containing in addition, 0.1% human serum albumin, pH 9.2 at 4°C). The eluted fractions were assayed in addition, 6-15 region immunoreactivity and 19-29 region immunoreactivity by region-specific radioimmunoassay. Samples of non-incubated glucagon or glicentin were also studied. Elution positions (kay value) of immunoreactive moieties

Incubation conditions

19-29 region immunoreactivity

6-15 region immunoreactivity Glucagon control (not incubated n = 7, range Glicentin control (not incubated) n = 8, range

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activity e x a m i n e d b y i n c u b a t i o n o f the eluted fragments w i t h surplus a m o u n t s o f region-specific antisera and d e t e r m i n a t i o n o f a n t i b o d y - b o u n d radioactivity (Fig. 5). The radioactivity elution profiles o f the' degradation p r o d u c t s o f glucagon and glicentin in the Kay interval 0 . 7 - 1 . 3 were remarkably similar. The m a j o r i t y o f the radioactivity eluted w i t h the salt peak o f the chromatograms, and probably represents

0.620.71 -

0.800.82 0.790.86

free 12sI-. A small a m o u n t eluted after the salt peak, and m a y represent 12SI-labelled tyrosine. The remaining fraction eluted at Kav 0.8 and displayed strong 6 - 1 5 region i m m u n o r e a c t i v i t y . Radioactive fragm e n t s w i t h 1 9 - 2 9 region i m m u n o r e a c t i v i t y were n o t identified e x c e p t for a small peak corresponding to the elution position o f intact labelled glucagon (Panel B), indicating that the relatively small a m o u n t s o f

TABLE II REINCUBATION EXPERIMENTS Glicentin (2 • 10 -7 M) and glucagon (2 • 10 -8 M) were incubated with 2 ml cell suspensions. After 15 min the contents of the flasks were centrifuged and 1 ml of the supernatants were incubated with a fresh preparation of cells (same number of cells and same volume). Fresh glucagon and glicentin were added to separate cell suspensions in amounts corresponding to the calculated amounts in the reincubated samples. Cyclic AMP and glucose contents of the incubation mixtures were determined at the times indicated and expressed in absolute values or as a percentage of the values obtained in control incubations without peptide addition. Values in parenthesis represent the number of experiments. Glucose concentration (30 min) (%) Control 100 Glucagon 10 -a M 440 Glucagon 10 -8 M, reincubated468 Glicentin 10 .7 M 421 Glicentin 10 -7 M, reincubated511

+ 166 • 89 -+ 153 -+ 88

(3) (3) (3) (3)

Cyclic AMP (/~mol/ml cells) Time (min): 3 1.40 15.65 2.79 1.82 7.34

10

25

1.22 8.26 2.11 1.61 4.68

1.20 4.72 1.57 1.44 2.68

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the peptides with hepatocytes, as compared to control experiments without preincubation of the peptides. The most conspicuous differences are the large cyclic AMP response to preincubated glicentin and the small cyclic AMP response to preincubated glucagon, responses which are not reflected in differences in glucose production.

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Fig. 5. Degradation of 12 s I-labelled glucagon and 12 s I-labelled glicentin during incubation with hepatocytes. After incubation for 70 min the material in the incubation medium was subjected to gel filtration on Sephadex G-50. Panel A: Elution pattern of radioactivity from incubated 12s I-labelled glucagon. Panel B: Elution pattern of radioactive immunoreactivity from incubated 12 s Iqabelled glucagon. Panel C: Elution pattern of radioactivity from incubated 12 s I-labelled glicentin. Panle D: Elution pattern of radioactive immunoreactivity from incubated 12 Si.labelled glicentin. 6-15 immunoreactivity; • o, 19-29 region immunoreactivity.

o

o,

glucagon added to the hepatocytes in these experiments were almost completely degraded during incubation.

Effect of degradation on the biological effects of preincubation Table II shows the results of experiments in which the glucose and cyclic AMP responses to glucagon and glicentin were studied after a 15 min preincubation of

Discussion

The effect of glicentin on hepatic glycogenolysis Throughout this investigation we have used cells isolated from the livers of fed rats, which are known to be highly responsive to the glycogenolytic effect of ghicagon [18,19]. In this respect, glicentin was as effective as glucagon, but its potency was two orders of magnituded lower. This raises the question whether our glicentin preparation could be contaminated with glucagon to the extent of 1%. A number of factors refute this possibility. First, the isolation procedure used [2] effectively removes all glucagonsized and glucagon-charged contaminants. Secondly, by numerous gel filtration studies of freshly prepared glicentin, including those described in Table I and Fig. 4, glucagon has never been detectable; with a load of peptide of 40 pmol as little as 0.1% contamination should have been detectable. (The 19-29 region immunoreactivity measured initially in the degradation experiments (Fig. 3) does not represent contamination of intact glucagon, but 19-29 region immunoreactive components eluted after glucagon by gel f'titration (Fig. 4, Panel D). Thirdly, the marked differences in cyclic AMP responses to glicentin and glucagon also dictate against contamination. We therefore conclude that glicentin is indeed capable of stimulating hepatic glycogenolysis. Since this is the first report on the effects of a highly purified peptide of the gut glucagon-like family of peptides, our results are not comparable to those obtained previously with impure preparations (see Ref. 1 for recent review).

Mechanism of the effect of glicentin on hepatic glycogenolysis The results of incubation experiments with combinations of glucagon and glicentin were compatible with the interactions of two full agonists on the same receptor as far as the glucose response is concerned.

169 However, it has been shown convincingly that glicentin does not compete with glucagon for binding to the hepatic receptor [14,20]; neither has glicentin a liver cell membrane receptor of its own [14]. A prerequisite for the actions of glicentin therefore seems to be a degradation of the inactive intact molecule to biologically active fragments. Our results show that glicentin is indeed being degraded during incubation with hepatocytes. A slight degradation takes place even by incubation in buffer and probably reflects either instability of the molecule and/or proteolytic activity of the reagents (gelatin) used. The degrading activity is probably being released from the hepatocytes, since it can be recovered in the isolated incubation medium. Also glucagon was degraded by the hepatocytes and it is of considerable interest that some of its degradation products seemed to correspond to those of glicentin (Fig. 5). Both peptides give rise to two immunoreactive moities, which by gel f'tltration appear to be of lower molecular size than glucagon. The larger of the two shows 6 - 1 5 region immunoreactivity and the smaller, 19-29 region immunoreactivity. Since the degradation products of both peptides behave similarly chromatographically and immunologlcally, it is tempting to suggest that the corresponding pairs are identical. Both, then, must represent parts of the sequence of the glucagon molecule, which is in agreement with the recent demonstration that glicentin contains the entire glucagon sequence [5]. Limited tryptic degradation of glucagon and glicentin gives rise to similar fragments, among these the 1-17 and the 18 (19)-29 fragment [5]. Because of their immunological and chromatographical similarities, it is therefore suggested that the low molecular weight 6 - 1 5 and 19-29 immunoreactive fragments of glicentin are indeed similar to the 1-17 and 18 (19)-29 fragments of glucagon, and it therefore appears that the degradation of glicentin involves the 63-92 region which contains the entire glucagon sequence. If the entire glucagon sequence is liberated from glicentin during degradation by hepatocytes, then the biological activity of glicentin is readily explained. Indeed, very small amounts of glucagon-sized fragments with 6-15 region as well as 19-29 region immunoreactivity were identified in most experiments (Table I); further rapid degradation could explain why smaller fragments were dominant. How-

ever, it is also possible that the small 6 - 1 5 immunereactive fragment could be biologically active. The biologically active sequence of the glucagon molecule has repeatedly been shown to reside in the N-terminal sequence [21-23]. The 1-21 sequence of glucagon is a weak stimulator of adenylate cyclase activity [23]. It was demonstrated recently [24] that a synthetic peptide corresponding to the 64-100 sequence of the glicentin molecule was capable of binding to the glucagon receptors and activating adenylate cyclase of liver cell membranes; thus, masking of the C-terminus of the glucagon sequence does not interfere with biological activity, (but abolishes 19-29 region immunoreactivity), whereas extensions at the N-terminus destroys the biological activity without interference with the 6-15 region immunoreactivity. Glicentin's biological activity therefore seems to include a cleavage at the 63, 64 bond, whereby the N-terminus of the glucagon sequence and thus glucagon-like bioactivity is exposed. The molecule responsible for the biological activity may in turn be the 64-100 fragment (which might correspond to part of 6-15 region immunoreactivity detected at Kay 0.58-0.67 (Table I) or one or more of the smaller fragments thereof, including glucagon itself. The cyclic AMP response The qualitative differences between glucagon and glicentin as stimulators of glucose production and cyclic AMP accumulation remains to be explained. Fig. 2 clearly shows that with glucagon, maximal glucose production takes place at concentrations which give rise to only fractions of the maximal cyclic AMP response and a considerable glucose release takes place at glucagon concentrations which do not significantly stimulate cyclic AMP accumulation. Furthermore, glicentin at concentrations which give rise to maximal glucose production, causes considerably less cyclic AMP accumulation than does glucagon in concentrations which leads to the same glucose production (Figs. 1 and 2). As discussed above, fragments containing the free N-terminal of the glucagon sequence stimulate cyclic AMP accumulation, but they are not as potent as glucagon; if the biological activity of glicentin is associated with the forma. tion of such fragments (as opposed to intact glucagon) this might explain the discrepancy between the effect of glucagon and glicentin; support for this

170 assumption is found in the reincubation experiments (Table II) which show that degraded glucagon is indeed a weaker stimulator of cyclic AMP accumulation than of glucose release. However, this in turn means that a fraction of the cyclic AMP response to glucagon is dissociated from the mechanism which leads to glycogenolysis. Similar findings and discussions presented recently by other authors [ 1 9 , 2 4 27] might suggest that the mechanism of glucagon action on the hepatocyte is more complicated than generally believed.

7 8 9 10 11 12

Role of glicentin in physiology The physiological role o f glicentin and the other members of the glucagon-like family of gut peptides is unknown [1]. Because it may contain the entire glucagon, sequence it is tempting to look upon glicentin as a glucagon prohormone, and evidence has been presented for the presence of glicentin in the glucagon cells of the pancreatic islets [28]. Its ability to degrade to glucagon-like peptides makes it a candidate for a glucagon-like role in glucose metabolism.

Acknowledgements The expert technical assistance of Rikke Gronholt, Merete Hagerup, Liselotte Stummann, Else Thieden and Ida Cohrt Tonnesen is gratefully acknowledged.. This work was supported by grants from The Danish Medical Research Council (grants Nos. 512-6602 and 512-10227) and the P. Carl Petersan Foundation.

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13 14 15 16 17 18 19 20 21 22 23 24

24 25 26 27 28

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