Effect of glicentin on gut mucosal growth in rats with jejunal and ileal Thiry-vella fistulas

E f f e c t o f G l i c e n t i n on G u t M u c o s a l G r o w t h in Rats W i t h J e j u n a l and Ileal T h i r y - V e l l a F i s t u l a s By Naoki Hashimoto, Harumasa Ohyanagi, and Sasaki Kazuyuki

Osaka, Japan

Background/Purpose: Glicentin stimulates growth of normal and atrophic small bowel mucosa, the mechanisms for this trophic effect of glicentin are not completely known. The purpose of this study was to determine whether the trophic effect of glicentin is mediated by mechanism involving luminal or nonluminal factors and determine whether glicentin exerts a differential trophic effect on either jejunal or ileal mucosa. Methods: Thirty-two male wistar rats underwent construction of either a jejunal or ileal Thiry-vella fistula ('I-VF). After 10 days recovery period, rats were further subdivided into groups to receive either saline (control) or glicentin (100 /~g/kg/d). Rats were killed on day 8, and "I-VF as well as corresponding segments of intact jejunum or ileum were removed. Mucosa was scraped, weighed, and analyzed for

N 1971, Gleeson et al t reported an endocrine tumor that resulted in remarkable thickening of the small intestinal mucosa and constipation. Bloom 2 subsequently showed that it was an enteroglucagon-producing tumor. Since then, various experiments have shown that enteroglucagon has a t r o p h i c effect on the small intestinal mucosa. 3 Banba et al 4 has also reported that enteroglucagon is associated with the proliferation of rat small intestinal mucosa after experimental massive resection of the small intestine, or after ingestion of dietary fiber, pectin. Northern blot analysis has confirmed also the increased expression of enteroglucagon mRNA in resected models of the small intestine. Glicentin, a product of enteroglucagon, was isolated from the porcine small intestine and was purified by Sundby et al 5 in 1976. It was later found that glicentin consisted of 69 amino acids. Preproglucagon,.a precursor of enteroglucagon, is known to be processed into a glucagonslike peptide I(GLP-1), GLP-2, and glicentin in L cells in the intestinal tract. However, it has been difficult to examine the effects of glicentin alone because of the technological problems associated with purification. Human glicentin has been synthesized by gene recombination techniques, and subsequently rat glicentin was synthesized. These findings suggest an important role for glicentin as a potent enterotrophic factor, but the mechanisms responsible for glicentin-induced gut growth are not completely known.

I

Journal of Pediatric Surgery, Vol 38, No 4 (April), 2003: pp 579-584

protein content and Alp activity. In addition, representative sections of full-thickness bowel from each group were examined histologically.

Results: In the jejunal TVF group, glicentin increased mucosal growth measurements in both the TVF and the intact jejunum. However, in the ileal "I-VF group, glicentin stimulated proliferation of intact ileal mucosa only; it had no effect on ileal mucosa in the "rVF.

Conclusions: These results suggest that glicentin exerts a systemic effect independent of luminal factors on the proliferation of proximal gut mucosa in addition to an indirect effect produced by stimulation of endogenous luminal secretions. In contrast, an indirect mechanism appears to be the predominant action of glicentin on growth of distal gut mucosa. J Pediatr Surg 38:579-584. Copyright 2003, Elsevier Science (USA). All rights reserved.

The complex regulation of gut mucosal growth involves a multitude of factors, including the indirect actions of luminal nutrients and pancreaticobiliary secretions and the effects of unidentified humoral factors. 6-8 Most studies examining the trophic effects of various gut hormones have been performed in animals with gut in continuity with the luminal stream, thus, making it difficult to dissect out the specific factors responsible for stimulating gut growth. The purpose of this study was to determine whether glicentin exerts its effect on intestinal structure through stimulation of endogenous luminal secretions or by nonluminal systemic factors that do not require small bowel in continuity. In addition, we determined whether glicentin exerts a differential trophic effect on either proximal or distal segments of the intestine. To accomplish these goals we constructed loops of jejunum and ileal ThiryVella fistulas (TVFs) that were isolated from the luminal stream. Stimulation of mucosal growth in the TVF by From Kinki University, School of Medicine, Department of Second Surgery, and the Nisshin Kyourin Research Center, Osaka, Japan. Address reprint requests to Naoki Hashimoto, Second Department of Surgery, Kinki University, School of Medicine, 377-20hno-Higashi, Osaka Sayama, Osaka 589-8511, Japan. Copyright 2003, Elsevier Science (USA). All rights reserved. 0022-3468/03/3804-0012530.00/0 doi: l O.1053/jpsu.2003.50126 579

580

HASHIMOTO,OHYANAGI,ANDKAZUYUKI

g l i c e n t i n w o u l d s u g g e s t a m e c h a n i s m that d o e s not r e q u i r e the a c t i o n o f e n d o g e n o u s l u m i n a l secretions.

MATERIALS AND METHODS Experimental Design Thirty-two male wistar rats (300 to 350 g) were divided randomly into 2 groups of 8 rats each. They were maintained in accordance with the recommendations of the Guide for the Care and Use of Laboratory Ammals, and the experiments were approved by the Kinki University Medical Faculty. Animal Care Committe. All groups were placed in individual wire-bottomed cages, acclimated for one week at a constant temperature of 22°C with 12-hour light/dark cycles, and fed standard chow ad libitum. After an overnight fast, the rats underwent construction of either a jejunal or ileal TVF as previously described 9 and as illustrated in Fig 1. Briefly, the small bowel was divided at 10 cm and 30 cm distal to the ligament of Treitz (jejunal TVF) or 10 and 30 cm proximal to the ileocecal junction (ileal TVF), taking care not to A ) Jejunal TVF

damage the neurovascular structures within tile mesentery. The two ends of 20-cm segments of either jejunal or ileal TVF were exteriorized and sutured to the abdominal wall. Intestinal continuity was restored by end-to-end enteroenterostomy. On postoperative day 2, rats were allowed regular chow ad libitum. On postoperative day 10. rats were further subdivided to receive subcutaneous injection of either saline (control) or ghcentin (50 p.g/kg), administered every 12 hours for I week. Rat glicentin was obtained from Nisshin kyorin (Saitama, Japan). After 1 week of treatment, rats were weighed and killed. The abdomen was opened by a midline incision, and 18-cm midsegments from TVFs as well as 10-cm segments of intact jejunum from the jejunal TVF group and 10-cm segments of intact ileum from the ileal TVF group were removed. The segments of small intestine then were opened longltuidinally, blotted dry, and the mucosa was scraped carefully from the underlying seromuscular layer using a glass slide as a scraper. The mucosa was weighed and immediately frozen at -20°C until assayed for alkahne phospbatase (Alp) and protein. For biochemical analysis, the mucosa was homogenized in 10 volumes of extraction media using a microhomogenizer at 0 to 4°C. Protein in the homogenates was assayed by the method of Lowry et al. m Alp activity was assayed by Kind and King's metbodJ I Fullthickness segments from the jejunal and ileal TVFs were removed. fixed in 10% buffered formalin, and embedded in paraffin. Sections were stained with H & E and examined.

Statistical Analysis Values are expressed as the mean - SEM and analyzed using analysis of variance at the .05 level of significance.

RESULTS

B ) ileal TVF

Body Weight

Tr+ltz

T h e r e w e r e n o d i f f e r e n c e s in initial a n d final b o d y w e i g h t s o f control a n d g l i c e n t i n - t r e a t e d rats.

Effect of Glicenth~ on Jejunal TVF and hTmct Jejunum

)P ~ TVF

~

Control

(n = 8) ~

(n =16)

(n = S) I

I

-IOD

t

Operation

11

S e v e n days o f g l i c e n t i n a d m i n i s t r a t i o n to rats with j e j u n a l T V F s significantly i n c r e a s e d m u c o s a l w e i g h t a n d p r o t e i n b y 4 9 % a n d 7 6 % , r e s p e c t i v e l y c o m p a r e d with the c o n t r o l group, w h i c h r e c e i v e d s a l i n e i n j e c t i o n s (Fig 2). Also, A l p was i n c r e a s e d b y 7 3 % w i t h a d m i n i s t r a t i o n o f glicentin. In addition, g l i c e n t i n s t i m u l a t e d m u c o s a l g r o w t h in j e j u n u m that w a s in c o n t i n u i t y with the rem a i n i n g gut. M u c o s a l w e i g h t w a s i n c r e a s e d by 2 1 % , p r o t e i n c o n t e n t b y 17%, a n d A l p by 7 1 % , c o m p a r e d w i t h rats g i v e n saline.

I

0D

t

Treatment started

Glicentin 100 p.g/kg 11

Effect of Glicentin on Ileal TVF and h+tact Ileum I

7' D

I'

Sacrificed

Fig 1. Schematic diagram of jejunal and ileal TVFs as described in detail in Materials and Methods. Segments (hatched lines) from proximal jejunum, distal ileum, and TVFs were excised, and mucosa was weighed and analyzed for protein content and Alp activity.

In c o n t r a s t to the j e j u n a l T V F , in w h i c h g l i c e n t i n s t i m u l a t e d m u c o s a l g r o w t h , g l i c e n t i n h a d no effect on m u c o s a o f the ileal T V F (Fig 3). H o w e v e r , ileal m u c o s a in c o n t i n u i t y w i t h the l u m i n a l s t r e a m was s t i m u l a t e d b y a d m i n i s t r a t i o n o f glicentin. M u c o s a l w e i g h t w a s inc r e a s e d by 15%, p r o t e i n c o n t e n t b y 10%, a n d A l p by 8 4 % c o m p a r e d with the c o r r e s p o n d i n g ileal s e g m e n t s o f rats r e c e i v i n g saline.

EFFECT OF GLICENTIN ON MUCOSA

581

Mucosal

weight

maitre

mylcm

Mucosal

40

40

35

35

30

30

25

25

20"

20

15"

weight

15 |

10 5

I

0

I

control

mglcm

Pro(eln

t0

gllcentln

control

gllcentln

Protein

content

conlent

20

15

1,0

control

control

gllcenlin

mUting

ALP

gllcenlln

ALP

mUtmg

+

6000 t

6000 t 5000

+

4000

5000 40Of' 3001

300 1

200

200 ' | i

tO01 '

i

I

i

Ji i control

J

100

; ! I

i gllcentln

Jejunal TVFs

conllol

gllcentln

intact jejunum

Fig 2. Mucosal weight and Alp activity and protein content of jejunal TVFs and intact jejunum from rats treated with injection of saline and glicentin, 100/~g/kg. *P < .05 versus control.

Villus Height Histologic examination showed that the villus height of the jejunal TVFs was significantly higher in glicentin group than in the control group (Fig 4). In the ileal TVFs, there were no differences between the 2 groups. DISCUSSION The gut mucosa has a high rate of cell turnover, with proliferation tightly regulated by many factors that in-

clude luminal nutrients, pancreaticobiliary secretions, and humoral factors such as certain gut hormones, lz Construction of an isolated, exteriorized loop of small bowel facilitates analysis of gut mucosa that is not exposed to luminal contents but has an intact neurovascular supply. A trophic response of the mucosa in the TVF would suggest a mechanism that does not require exposure of the gut mucosa to the luminal environment. We have shown that glicentin stimulates mucosal growth

582

HASHIMOTO, OHYANAGI, AND KAZUYUKI

Mucosal

mgtcm

weight

mg/~m

40

40"

35

35

30

30

25

25"

20

20"

15

15"

10 5" 0

weight

10"

l

I

control

I I

Protein

mglcm

Mucosal

!

gllcentln

I

5" O'

gllcenlln

control

Protein

content

content

2.0

t.5

///i

1.0.

0.5.

0.0

control

control

gllcentln

mUIm9

ALP

mulmg

6000

5000"

5000"

4000"

4000

3000"

3000"

2000"

2000"

1O00"

I000"

F i g 3.

Mucosal

weight

100/~g/kg.

, .---~,...,

I! • Im

O

¢onlrol

gllcentln

¢onllrol

ileal TVFs

glicentin,

ALP

6000!

O

gllcentln

and Alp activity and protein content

gllcenlln

intact ileum o f ileal T V F s a n d i n t a c t i l e u m f r o m

rats treated

with

injection

of saline and

* P < .05 v e r s u s c o n t r o l .

of both intact jejunum and ileum that are in contact with the luminal stream. We have also shown that glicentin stimulates mucosal growth in isolated Thiry-vella loops of jejunum but not of ileum, suggesting that the effects of glicentin on the proximal gut mucosa are caused by a combination of nonluminal and luminal factors. However, the effect of glicentin on ileal mucosa appears to be secondary to indirect stimulation of endogenous luminal secretions.

To better determine whether the trophic effect of glicentin on small intestinal mucosa is mediated by a nonluminal, systemic effect, we constructed either jejunal or ileal TVFs. The mucosa of the TVF is not subject to the same luminal environment as the intestine in continuity, although it has a common neurovascular supply. ~3 A response in the fistula would suggest a systemic effect, whereas a response confined to the intestine in continuity would implicate local mechanism,

583

EFFECT OF GLICENTIN ON MUCOSA

~rn

Villus

height

Villus

/~rn

height

500'

500 t 400

400'

300

300.

200

200.

100

100,

0 control jejunal

control

glicentin

glicentin ileal

TVFs

TVFs

Fig 4. Effect of glicentin administration (100 p.g/kg) on villus height of jejunal and ileal TVFs. *P < .05 versus control.

including influences of luminal contents and endogenous secretions. Furthermore, by creating both jejunal and ileal TVFs, the current study allowed us to examine whether glicentin exerts a differential trophic effect on either jejunum or ileum. Administration of glicentin stimulated the growth of mucosa by 76% in isolted loops of jejunum. This mucosal proliferation was completely independent of luminal nutrition and pancreaticobiliary secretions, suggesting that one of the mechanism leading to the trophic effect of glicentin in the proximal small bowel is by a systemic, nonluminal effect. This could be secondary to a number of mechanisms, including a direct effect of glicentin on jejunal mucosa or a number of growth promoting factors that could possibly by released by glicentin. In addition, indirect luminal mechanisms appear to contribute to the proliferation of jejunal mucosa in continuity. Measurements of mucosal weight and protein content of jejunum in continuity with the luminal stream were increased 28% to 70% in rats treated with glicentin compared with corresponding values from control rats treated with saline. In contrast, glicentin doses sufficient to produce jejunal mucosal growth did not stimulate mucosal growth in isolated loop of distal ileum; however, mucosal proliferation occurred in the

ileum in continuity with the luminal stream. These results suggest that the glicentin-induced growth of ileal mucosa is caused primarily by the indirect stimulation of endogenous luminal secretions and further show that glicentin exerts a differential trophic effect on proximal and distal small bowel rrmcosa. The finding that glicentin, present in greatest concentrations in the distal ileum, stimulates jejunal but not ileal mucosal growth in isolated TVFs coincides with other studies of endocrine physiology showing that trophic hormones do not affect their tissue of origin. For example, the trophic hormone gastrin, found in greatest concentration in the gastric antrum, stimulates mucosal growth of the fundus and duodenum but has no effect on the proliferation of antral mucosa. We have shown that glicentin exerts a differential trophic effect on mucosa of the proximal jejunum and distal ileum. Glicentin stimulates growth of jejunal mucosa by mechanisms involving both nonluminal and luminal factors. In contrast, glicentin stimulates growth of ileal mucosa primarily by stimulating endogenous luminal secretions. Glicentin may prove to be an important enterotrophic factor, especially for the proximal gut mucosa.

REFERENCES

1. Gleeson MH, Bloom SR, Polak JM, et al: Endocrine tumor in kidney affecting small bowel structure, motility and absorptive function. Gut 12:773-782, 1971 2. Bloom SR: An enteroglucagontumor. Gut 13:520-523, 1972 3. Sagor GR, Ghafe MA, AI-Mukhtaret al: Evidence for a humoral mechanismaftersmallintestinalresection.Gastroenterology84:902-906,1983 4. Banba T, Sasaki M, Hosoda S: Enteroglucagon-putativehumoral factor inducing pancreatic hyperplasia after proximal small bowel resection. Dig Dis Sci 39:1532-1536, 1994

5. Sundly F, Jacobsen H, Moody AJ: Purification and characterization of a protein from porcine gut with glucagons-likeimmunoreactivity. Horm Meta Res 8:366-371, 1976 6. Johnson LR, Copeland EM, Dudrick SJ, et al: Structural and hormonal alterations in the gastrointestinal tract of parenterally fed rats. Gastroenterology 68:1177-1183, 1975 7. AI-MukhtarMYT, Sagor GR, Ghatei MA, et al: The role of pancreatico-biliary secretions in intestinal adaptation after resection and its relationship to plasma enteroglucagon.Br J Surg 70:398-400, 1983

584

8. Johnson LR: Regulation of gastrointestinal growth, in Johnson LR, Christensen J, Jackson MJ, et al (eds): Physiology of Gastrointestinal Tract. New York, NY, Raven 1987. pp 301-303 9. Chung DH, Evers BM, Shimoda I, et al: Effect of neurotensin on gut mucosal growth in rats with jejunal and ileal thiry-vella fistulas. Gastroenterology 103:1254-1259, 1992 10. Lowry OH, Rosebrough NJ, Farr AL. et al: Protein measurements with the folic phenol reagent. J Biol Chem 193:265-275, 1951

HASHIMOTO, OHYANAGI, AND KAZUYUKI

I1. Kind PRN. King EJ: Estimation of plasma phosphatase by determination of hydrolysed phenol with amino-amipyrine. J Clin Pathol 7:322-326, 1954 12. Johnson LR: Regulation of gastrointestinal mucosal growth. Physiol Rev. 68:456-502. 1988 13. Hanson WR, Rljke RPC, Plaisier HM, et al: The effect of intestinal resection on Thiry-vella fistula of jejunal and ileal origin m the rat; Evidence for a systemic control mechanism of cell renewal. Cell Tissue Kinet 10:543-555, 1977