J Gastroenterol 1997; 32:300-305 Journal of Gastroenterology �9 Springer-Verlag 1997

Trophic effects of glicentin on rat small-intestinal mucosa in vivo and in vitro

SATOSHI MYOJO, TOMOYUKI TSUJIKAWA, MASAYA SASAKI, YOSHIHIDE FUJIYAMA, and TADAO BAMBA

Second Department of Internal Medicine, Shiga University of Medical Science, Tsukinowa, Seta, Otsu, Shiga 520-21, Japan

Abstract: To define the role of glicentin the active site of enteroglucagon, we evaluated the trophic effects of re- combinant rat glicentin on rat small intestine and IEC- 6 cells. I n vivo, a significant increase was observed in jejunal wet weight, protein content, DNA content, and alkaline phosphatase activity after the subcutaneous administration of 100~tg/kg per day of glicentin for 2 weeks. In the ileum, however, there were no significant differences between the control versus glicentin groups in any of these parameters. Ornithine decarboxylase (ODC) activity 3.5h after an intraperitonea! injection of glicentin was increased in the jejunal mucosa, but not in the ileal mucosa. In vitro, glicentin, at a dose of more than 100ng/ml, significantly increased both tritium-thymidine incorporation and the number of IEC-6 cells. These findings indicate that glicentin exerts direct trophic effects on the rat small-intestinal mucosa and on the rat small-intestinal cell line, IEC-6, and that this peptide appears to be an active site of enteroglucagon.

Key words: enteroglucagon, glicentin, ornithine decar- boxylase (ODC), rat small intestine, IEC-6

enteroglucagon is associated with the proliferation of rat small-intestinal mucosa after experimental massive resection of the small intestine, or after ingestion of the dietary fiber, pectin. Northern blot analysis has also confirmed the increased expression of enteroglucagon mRNA in resected models of the small intestine. 7 How- ever, some studies have failed to show these trophic effects of enteroglucagon on small-intestinal mucosa. 8-1~

Glicentin, a product of enteroglucagon, was isolated from the porcine small intestine and was purified by Sundby et al. ~I in 1976. It was later found that glicentin consisted of 69 amino acids. 12 Preproglucagon, a pre- cursor of enteroglucagon, is known to be processed into a glucagon-like peptide 1 (GLP-1), GLP-2, and glicentin in L cells in the intestinal tract. 13a4 However, it has been difficult to examine the effects of glicentin alone because of the technological problems asso- ciated with purification. Human glicentin has been synthesized by gene recombination techniques, is and subsequently rat glicentin was synthesized. To define the role of glicentin as the actiVe site of enteroglucagon, we examined the trophic effects of glicentin on rat small-intestinal mucosa and the rat small-intestinal epithelial cell line, IEC-6.

Introduction

In 1971, Gleeson et al. 1 reported an endocrine tu- mor that resulted in remarkable thickening of the small-intestinal mucosa and constipation. Bloom 2 sub- sequently demonstrated that it was an enteroglucagon- producing tumor. Since then, various experiments have shown that enteroglucagon has atrophic effect on the small-intestinal mucosa. 3,4 We 5,6 have also reported that

Offprint requests to: S. Myojo (Received Aug. 9, 1996; accepted Nov. 22, 1996)

Materials and methods

Glicentin administration for 2 weeks

Four-week-old male Wistar rats (Clea Japan, Tokyo, Japan) were used. An elemental diet; (ED; Elental; Ajinomoto, Tokyo, Japan) was dissolved in water to yield 1 kcal/ml, and rats received this ad libitum through a water-feeding bottle. The animals were housed in wire-bottomed cages in an animal room maintained at 22~ with 12 h of light daily (8 a.m.-8 p.m.). After being fed on ED only for 4 weeks, the rats were randomly divided into control and glicentin groups.

S. Myojo et al.: Trophic effect of glicentin 301

Rat glicentin was obtained from Nisshin Flour Mill- ing (Saitama, Japan). Glicentin was dissolved in saline and injected subcutaneously, at a dose of 50 ~tg/kg every 12 h, for 2 weeks. ED was given isocalorically to both groups.

After 2 weeks, the rats were anesthetized with ethyl ether, and decapitated. The small intestine between the ligament of Treitz and the ileocecaI valve was rap- idly excised and washed in chilled saline. To measure the intestinal length precisely, the bowel was suspend- ed under a uniform 10-g tension, as previously de- scribed? 6 Two 10-cm segments of the bowel were identified, one starting 2cm distal to the ligament of Treitz (jejunum) and the other 2cm proximal to the ileocecal valve (ileum). The mucosa was scraped from these specimens with a glass slide on a chilled plate. The mucosal wet weight was measured and the mucosa was homogenized.

The protein content was measured by Lowry's 17 method, and DNA content was determined by the ethidium bromide method? 8 Alkaline phosphatase (ALP) activity was measured by Kind and King's ~9 method.

For histological analysis, the jejunum and ileum were fixed in 4% formalin, embedded in paraffin, and then cut into 4-~tm-thick segments. After hematoxylin-eosin staining, the villus height and the crypt depth were measured.

Single glicentin administration

Eight-week-old male Wistar rats were used in this study. The rats were fasted for 24h before the experi- ment, and were injected intraperitoneally with saline or 100~tg/kg glicentin. They were killed 3.5h after the in- jection. Intestinal specimens were then prepared in the way described above.

�9 Ornithine decarboxylase (ODC) activity was assayed by a slight modification of the micro-method of Colarian et al., z~ as previously described. 21 L-[1-14C]- ornithine (specific activity, 58.0mCi/mmol) was pur- chased from New England Nuclear; (Boston, MA, USA). One enzyme unit was defined as 1 pmol of ~4CO2 released per mg protein per h. The protein concentra- tion was determined by the method of Bradford. 22

Addition of glicentin to IEC-6 cells

IEC-6 cells were obtained from the American Type Culture Collection (Bethesda, MD, USA). The cells were maintained as monolayer cultures in 25-cm 2 flasks in Dulbecco's modified eagle medium (DMEM) supple- mented with 5 % fetal calf serum (FCS; GIBCO Labora- tories, Grand Island, NY, USA), and were incubated at 37~ in a humidified atmosphere containing 5% CO2.

The cells were seeded at 30000 cells/well in 24-well plates (DNA synthesis assay) or at 80000 cells/well in 12-well plates (cell counts). The cells were made quies- cent in the G0/G1 phase by serum deprivation for 24 hfl 3 The medium was then changed to DMEM with glicentin (0-1000 ng/ml), and the cells were incubated for another 48h.

For the determination of DNA synthesis, we mea- sured the incorporation of tritium-thymidine (specific activity, 84.6 Ci/mmol; New England Nuclear) into the trichloroacetic acid (TCA)-precipitable fraction, as pre- viously described24 The cells were then counted by hematocytometry.

Statistical analysis

Values were expressed as means __ SEM. Significant differences between the means were analyzed by Student's two-tailed t-test. Differences with P values of less than 0.05 were considered to be significant.

Results

After the administration of glicentin for 2 weeks, there was no significant difference in body weight between the glicentin group (G) and the control group (C). We examined the wet weight, protein, and DNA content, as well as the ALP activity of the jejunum and ileum as parameters of mucosal growth. The wet weight (G;39.7 --_ 1.64, C;35.0 ___ 0.83 mg/cm), protein content (G;5.46 ___ 0.19, C;4.95 _+ 0.09mg/cm), and DNA content (G;240 ___ 12.4, C;209 ___ 3.18~tg/cm) were significantly higher in the jejunal mucosa of the glicentin group than in the control group. ALP activity (G;6.46 -+ 0.22, C;5.46 ___ 0.32 x 103 IU/g protein) was also significantly higher in the jejunum of the glicentin group. However, there were no significant differences between the two groups in any of the ileal parameters measured (Fig. 1). Histological examination showed that the villus height of the je- junum was higher in the glicentin group than in the control group, although the difference was not signifi- cant (P = 0.08). In the ileum, there were no differences between the two groups (Table 1).

The ODC activity of the jejunal mucosa 3.5 h after the glicentin injection was 477.69 ___ 53.06 (CO2pmol/h per mg protein), significantly greater than that in the con- trol group (233.59 __+ 46.88). However, there were no significant differences between the glicentin and the control groups in ileal ODC activity (Fig. 2).

Tritium-thymidine incorporation in IEC-6 cells was significantly augmented with the addition of glicentin at more than 50ng/ml (Fig. 3). At l~tg/ml of glicentin, thymidine incorporation was about 300% higher than in the control group (0ng/ml). The cell counts were also

302 S. Myojo et al.: Trophic effect of glicentin

Table 1. Effect of subcutaneous glicentin administration (100~tg/kg per day) for 2 weeks on villus height and crypt depth in rat small intestine

Jejunum Ileum

Control Glicentin Control Glicentin

Villus height (pm) 474 ___ 12 515 _+ 14 279 • 15 287 _+ 11 Crypt depth (gm) 126 _+ 14 134 • 5 84 • 8 88 • 9

Values are expressed as means +_ SEM (n = 5)

[A] Wet Weight 4 5 *

[ - - 1

4 0

35

30

525 s 2 0

15

I0

5

0

[B] 6

5

4

L

2

I

0

,_.I,.

JEJUNUM ILEUM JEJUNUM I LELIM

Pro te in 700

It] DNA [D] ALP 300 8 ,

250 [ - - I I

,-= 6 l

.x. o" 200 o

S

150 %

2 100

50 JEJUNUM ILEUN 0 JEJUNUM ILEUM

Fig . 1A-D. Effect of subcutaneous glicentin administration (100 Ixg/kg per day) for 2 weeks on rat small-intestinal mucosal parameters. A wet weight; B protein content; C DNA content; D alkaline phosphatase activity. White columns, Values are expressed as means _+ SEM (n = 5). Control (saline); gray columns, glicentin. *P < 0.05

significantly increased by glicentin, at more than 100 ng/ ml (Fig. 4).

Discussion

Only a few studies have reported the physiological actions of purified glicentin, including its effects on glucose release from isolated rat hepatocytes, 25 its in- hibitory effects on gastric acid secretion in rats after the administration of pentagastrin, 26 and its inhibitory efo

O t . O.

E

O E r

600

500

400

300

200

1 O0

i

i

I

]

JEJUNUM ILEUM

Fig. 2. Effect of intraperitoneal glicentin administration (100 pg/kg) on ornithine decarboxylase activity of rat small- intestinal mucosa 3.5 h after injection. Values are expressed as means _+ SEM (n = 7). White columns, Control (saline); gray columns, glicentin. *P < 0.05

fects on glucagon secretion in dogs. 27 Since the purifica- tion of glicentin has been difficult, its other physiologi- cal actions, particularly its trophic effects, have remained unclear. Recently, gene recombinant tech- niques have made it possible to synthesize human

S. Myojo et al.: Trophic effect of glicentin 303

0 0

350

300

250

200

150

1 O0 mE 5O

0 0 20 50 100 500

g l i c e n t i n c o n c e n t r a t i o n (ng/ml)

1000

Fig. 3. Effect of glicentin on tritium- thymidine incorporation in IEC-6 cells. Values are expressed as means -+ SEM (n = 5). **P < 0.01 versus 0ng/ml

0 .~a C 0 0

q - 0

300

250

200

150

I O0

5O

0 20 50 1 O0 500

g l i c e n t i n c o n c e n t r a t i o n (ng/ml)

1000

Fig. 4. Effect of glicentin on cell counts of IEC-6. Values are expressed as means -+ SEM (n = 5). **P < 0.01 versus 0 ng/ml

glicentin. 15 Ohneda et al. 28 have found an increase in insulin release from the dog pancreas with recombinant human glicentin.

In a preliminary study of recombinant human glicentin, we found that the administration of 100 ~g/kg per day for 2 weeks had a positive trophic effect, but that 20 ~tg/kg per day had no effect (data not shown). Therefore, in this study, we chose a dose of 100~tg/kg per day for rat glicentin. In the atrophic small intestinal mucosa of rats fed an ED, 16 glicentin increased the wet weight, protein and D N A content, and ALP activity only in the jejunal mucosa. All parameters were in- creased from 110% to 115%. We have also demon- strated that 30 ~tg/kg per day of epidermal growth factor (EGF) administered subcutaneously for-2 weeks in- creased these same parameters by about 110%. 29 How- ever, Watanabe et al. 3~ showed that the continuous

administration of glucagon (1-21) caused only villous elongation of the ileum, with no change in the wet weight, protein, or D N A concentration. On the other hand, Goodlad et al. 9 showed that glucagon (1-21) re- duced intestinal epithelial cell proliferation in parenter- ally fed rats. Our study showed that various mucosal parameters, such as protein and D N A content, in- creased with the administration of glicentin. Therefore, it would appear that glicentin has a stronger trophic effect than glucagon (1-21).

ODC is an initial rate-limiting enzyme in polyamine synthesis, and converts the amino acid ornithine into the diamine putrescine. An increase in O D C activity is one of the earliest biochemical events associated with cellular proliferation. Since ODC activity increases within 2-4 h after the administration of growth factors such as EGF, 31 we chose to measure ODC activity 3.5 h

304 S. Myojo et al.: Trophic effect of glicentin

after the administration of glicentin. Fitzpatrick et al. 31 showed a significant increase in the ODC activity of the small-intestinal mucosa after the intraperitoneal administration of EGF (150~tg/kg). We observed a significant increase in ODC activity of the jejunum in the glicentin group compared to the control group. It is, therefore, suggested that the significant induction in ODC activity after the intraperitoneal administration of glicentin may be important in initiating the cellular proliferation observed after the administration of this peptide.

The trophic effects of glicentin were observed in the jejunum, but not in the ileum. These findings were con- sistent with a report of marked mucosal proliferation of the jejunum in a patient with an enteroglucagonoma. 1,2 The reason that glicentin exerted trophic effects only on the jejunum remains unknown. Ohneda et al. 2s found that glicentin promoted the release of insulin from the pancreas, and Maudsley et al. 32 reported that insulin increased ODC activity in the small intestine. Accord- ingly, it appears that secondary action via an insulo- acinar axis can be excluded as a mechanism for the trophic effects of glicentin. However, it appears that the effect of glicentin on ODC activity may reflect levels of glicentin receptor expression in the jejunum, as, based on the present in vitro study, glicentin seemed to have a direct action on small-intestinal epithelial cells. Since we cannot obtain a glicentin antibody at present, to perform a receptor assay, this idea remains to be eluci- dated in future studies.

An increase in tritium-thymidine incorporation in IEC-6 and in the cell counts was observed at a glicentin concentration of 100ng/ml or more. This concentration is approximately ten fold greater than the physiological concentration. Ohneda 33 reported that circulating levels of plasma glicentin reached approximately 1 nmol/1 af- ter nutrient ingestion. This level is high compared to the optimal concentration for other growth factors. 24 It is possible that the number of glicentin receptors in ICE- 6 cells, may be reduced, or that the affinity of receptors may be decreased, or, alternatively, that glicentin may act in a paracrine manner.

In conclusion, these findings indicate that glicentin exerts its trophic effect not only on rat small-intestinal mucosa, but also on the small-intestinal epithelial cell line, ICE-6, and that this peptide appears to be an active site of enteroglucagon.

References

1. Gleeson MH, Bloom SR, Polak JM, et al. Endocrine tumour in kidney affecting small bowel struture, motility, and absorptive function. Gut 1971;12:773-782.

2. Bloom SR. An enteroglucagon tumour. Gut 1972;13:520-523.

3. Sagor GR, Ghatei MA, AI-Mukhtar MYT, et al. Evidence for a humoral mechanism after small intestinal resection. Gastroenter- ology 1983;84:902-906.

4. Miazza BM, A1-Mukhtar MYT, Salmeron M, et al. Hyper- enteroglucagonaemia and small intestinal mucosal growth after colonic perfusion of glucose in rats. Gut 1985;26:518-524.

5. Bamba T, Sasaki M, Hosoda S. Enteroglucagon--putative hu- moral factor inducing pancreatic hyperplasia after proximal small bowel resection. Dig Dis Sci 1994;39:1532-1536.

6. Chun W, Bamba T, Hosoda S. Effect of pectin, a soluble dietary fiber, on functional and morphological parameters of the small intestine in rats. Digestion 1989;42:22-29.

7. Rountree DB, Ulshen MH, Selub S, et al. Nutrient-independent increases in proglucagon and ornithine decarboxylase messenger RNAs after jejuno-ileal resection. Gastroenterology 1992;103: 462-468.

8. Gregor M, Stallmach A, Menge H, et al. The role of gut-glucagon- like immunoreactants in the control of gastrointestinal epithelial cell renewal. Digestion 1990;46(Suppl 2):59-65.

9. Goodlad RA, Ghatei MA, Bloom SR, et al. Glucagon 1-21 re- duces intestinal epithelial cell .proliferation in parenterally fed rats. Exp Physiol 1991;76:943-949.

10. Gregor M, Menge H, St6ssel R, et al. Effect of monoclonal anti- bodies to enteroglucagon on ileal adaptation after proximal small bowel resection. Gut 1987;28:9-14.

11. Sundby F, Jacobsen H, Moody AJ. Purification and characteriza- tion of a protein from porcine gut with glucagon-like immunore- activity. Horm Metab Res 1976;8:366-371.

12. Thim L, Moody AJ. The primary structure of porcine glicentin (proglucagon). Regul Pept 1981;2:139-150.

13. Mojsov S, Heinrich G, Wilson IB, et al. Preproglucagon gene expression in pancreas and intestine diversifies at the level of post-translational processing. J Biol Chem 1986;261:11880-11889.

14. Orskov C, Hoist JJ, Knuhtsen S, et al. Glucagon-like peptides GLP-1 and GLP-2, predicted products of the glucagon gene, are secreted separetely from pig small intestine but not pancreas. Endocrinology 1986;119:1467-1475.

15. Imai S, Sasaki K, Satoh T, et al. Production of human glicentin in Escherichia coli. Biomed Res 1996;17:279-285.

16. Tsujikawa T, Bamba T, Hosoda S. The trophic effect of epidermal growth factor on morphological changes and polyamine metabo- lism in the small intestine of rats. Gastroenterol Jpn 1990;25: 328-334.

17. Lowry OH, Rosebrough NJ, Farr AL, et al. Protein measurement with the Folin-phenol reagent. J Biol Chem 1951;83:703-708.

18. Andersen KJ, Skagen DW. Fluorometric determination of DNA in fixed tissue using ethidium bromide. Anal Biochem 1977;83: 703-708.

19. Kind PRN, King EJ. Estimation of plasma phosphatase by deter- mination of hydrolysed phenol with amino-antipyrine. J Clin Pathol 1954;7:322-326.

20. Colarian J, Arlow FL, Calzada R, et al. Differential activation of ornithine decarboxylase and tyrosine kinase in the rectal mucosa of patients with hyperplastic and adenomatous polyps. Gastroen- terology 1991;100:1528-1532.

21. Sakamoto K, Fujiyama Y, Bamba T. Altered polyamine biosyn- thesis with aging after massive proximal small resection in rat. J Gastroenterol 1996;31:338-346.

22. Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of pro- tein-dye binding. Anal Biochem 1976;72:248-254.

23. Ginty DD, Seidel ER. Polyamine-dependent growth and calmodulin-regulated induction of ornithine decarboxylase. Am J Physiol 1989;256:G342-G348.

24. Baliga BS, Borowitz SM, Barnard JA. Effects of EGF and PMA on the growth and proliferation of IEC-6 cells. Biochem Int 1989;19:1045-1056.

25. Thieden HID, Hoist J J, Dich J, et al. Effect of highly purified porcine gut glucagon-like immunoreactivity (glicentin) on glucose

S. Myojo et al.: Trophic effect of gl icent in 305

release from isolated rat hepatocytes. Biochim Biophys Acta 1981;675:163-170.

26. Kirkegaard P, Moody A J, Hoist J J, et al. Glicentin inhibits gastric acid secretion in the rat. Nature 1982:297:156-157.

27. Ohneda A, Kobayashi T, Nihei J. Effect of glicentin-related pep- tides on glucagon secretion in anesthetized dogs. Diabetologia 1986;29:397-401.

28. Ohneda A, Ohneda K, Nagasaki T, et al. Insulinotropic action of human glicentin in dogs. Metabolism 1995;44:47-51.

29. Bamba T, Tsujikawa T, Hosoda S. Effect of epidermal growth factor by different routes of administration on the small intestinal mucosa of rats fed elemental diet. Gastroenterol Jpn 1993;28:511- 517.

30. Watanabe N, Namba M, Itoh H, et al. Trophic effect of glucagon- (1-21)-peptide on the rat small intestine and colon. Biomed Res 1990;11:307-312.

31. Fitzpatrick LR, Wang P, Johnson LR. Effect of epidermal growth factor on polyamine-synthesizing enzymes in rat enterocytes. Am J Physiol 1987;252:G209-214.

32. Maudsley DV, Leif J, Kobayashi Y. Ornithine decarboxylase in rat small intesine: Stimulation with food or insulin. Am J Physiol 1976;231:1557-1561.

33. Ohneda A. Response of plasma glicentin to intraduodenal admin- istration of glucose in piglets. Diabetes Res Clin Pract 1987;3:97- 102.