Glucagonlike peptide-2 analogue enhances intestinal mucosal mass after ischemia and reperfusion

Glucagonlike

PeptideAnalogue After Ischemia

Enhances Intestinal and Reperfusion

Mucosal

Mass

By Rajeev Prasad, Karim Alavi, and Marshall Z. Schwartz Philadelphia,

Pennsylvania

Background/Purpose:Glucagonlike peptide(GLP-2). a product of the posttranslational processing of proglucagon, has been shown to enhance mucosal mass and function in both normal intestine and in the residual intestine after massive small bowel resection. This study was designed to determine if a synthetic, protease-resistant analogue of GLP-2 (GLP-2a) can enhance mucosal mass in small intestine after ischemia and reperfusion (I/R) injury. Methods: Ten young adult male Sprague-Dawley rats underwent laparotomy and superior mesenteric artery occlusion for a period of 40 minutes. During this period of ischemia, each rat underwent placement of a jugular venous catheter that was connected to a subcutaneously placed osmotic pump designed to deliver its contents over 3 days. The rats were divided into 2 groups based on the contents of the pumps: group 1, saline at 1 uL/h (n = 6) and group 2, GLP-2a at 100 uglkgld (n = 4). Three days after insertion of the pumps the small intestine was harvested from the surviving rats for determination of mucosal DNA and protein content.

I

NTESTINAL ISCHEMIA llnd reperfusion (I/R) injury, 3s occurs with necrotizing enterocolitis and midgut volvuh~s, often is encountered in neonatal and pediatric patients. In addition. harvesting and reimplantation of the donor graft during intestine transplantation results in I/R injury. The mechanism of injury after I/R has not been elucidated. However, it is known that oxygen-derived free radicals. leukocyte-derived mediators of inflammation. and cllterations in the bioavailability of nitric oxide contribute to 3r-1increased rate of primary cell necrosis and apoptosis.’ Furthermore, there is no accepted standard treatment for intestinal I/R injury. Current management includes volume resuscitation, antibiotics. and parenteral nutrition. Surgical resection of irreversibly injured intestine may be the only option in many patients, often resulting in inadequate intestinal absorption and the short bowel syndrome. Glucagonlike peptide- (GLP-2) is a 33-amino acid peptide known to enhance mucosal integrity in normal intestine and in intestine that has undergone adaptation after massive (80%) small bowel resection (MSBR). No studies have investigated whether GLP-2 is able to preserve or restore intestinal integrity after significant inflammation or injury. The purpose of this preliminary study was to explore the potential efficacy of a synthetic, Journal

of Pediatric

Surgery,

Vol35,

No 2 (February),

2000: pp 357-359

and Wilmington,

Statistical test.

Delaware

analysis

was

performed

using

unpaired

t

Student’s

Results: After I/R injury to the small intestine, a 3-day systemic infusion of GLP-2u significantly increased mucosal DNA content 41% (P < .05) and mucosal protein content 60% (PC .05) when compared with saline-treated controls. In addition, infusion of GLP-2a reduced mortality from 50% to 25%. Conclusions: These data show for the first time that GLP-2u enhances mucosal mass following I/R injury to the small intestine. GLP-2a may be of benefit to patients with intestinal ischemia syndromes such as necrotizing enterocolitis and midgut voIvuIus. J Pediatr Surg 35:357-359. Copyright o 2000 by W.B. Saunders Company. INDEX WORDS: lschemia and reperfusion injury, like peptide-2, growth factor, intestinal mucosa.

glucagon-

protease-resistant GLP-2 analogue (GLP-2ol) on the small intestine after I/R injury. MATERIALS AND METHODS Ten young Indianapolis, All animal

adult male Sprague-Dawley rats (Harlan Sprague-Dawley, IN) each weighing 175 to 200 g were used in this study. care and procedures

conformed

by the Animal Care and Use Committee Programs. Each mt was anesthetized with

to the regulations

set forth

of the Nemours Research ketamine (70 mg/kg) and

xylazine (13 mg/kg) and underwent a midline laparotomy and bowel evisceration. The duodenum and pancreas were reflected anteriorly and superiorly. exposing the superior mesenteric artery (SMA). was occluded for 40 minutes using a double-looped ligature. period jugular

of warm venous

placed (Model

osmotic 1003D.

divided (control

into Z groups based on the contents of the pumps. Group I rats group) had a systemic infusion of saline at a rate of I ~LI?I

(n = 6). Group

From

intestinal catheter,

The SMA During this

ischemia. each rat underwent placement of a which was connected to a subcutaneously

pump designed to deliver its contents Alza Scientific Products, Palo Alto. CA).

2 rats (experimental

the A.I.

duPonr

Hospirnl

group)

for

had a systemic

Children,

Thomas Jefferson University, Philadelphia, Presented at rhe 30th Annual Meeting

over 3 days The rats were

infusion

Wilmington.

PA. of the American

DE,

of

and

Pediatric

Surgical Associatiotb Ranch0 Mirage. California. May 16-19. 1999. Address reprint requests to Marshall Z Schwartz. MD, A.I. duPont Hospiral for Children. Wibningron. DE 19803. Copyright

Departmenr

Q 2000 by WB.

Saunders

of Surgeq

1600

Rockland

Rd.

Con1pan.v

0022-3468/00/3502-0040$03.00/O 357

PRASAD,

358

GLP-2x

at a rate of 100 pgikg/d

residue suhstituted Genemed Synthesis.

(n = 4). GLP-3~.

for alaninc at the 2 position. Inc. San Francisco. CA. After

which

has a glycinc

80 -

ALAVI,

AND

SCHWARTZ

l

was obtained from a J-day infusion. the

entire small intestine of each surviving rat was harvested. and mucosul scrapings were ohtaincd for determination of DNA and protein content as a measure of mucosal mass. DNA content was determined

using

a standard

diphenylaminc

procedure as described by Burton2 and moditied by Giles Protein content was derermined using the standard melhod by Lowry milligram unpaired

et al.’ mucosa Studenr‘s

and Myers.’ as described

Results were expressed as mean micrograms t SEM. Statistical analysis was performed

per using

t test.

RESULTS

Forty minutes of SMA occlusion resulted in the death of 3 animals in the control group (50% mortality rate) and I animal in the experimental group (25% mortality rate). All of these deaths occurred less than 48 hours after ischemic injury. Among survivors. the mean mucosal DNA content was 0.992 + 0.098 pg/mg in control animals and 1.399 ? 0.074 pg/mg in GLP-7ol-treated rats (Fig 1). Thus. after GLP-2a treatment there was a 41% increase above the control group in mean mucosal DNA content (P < 0.05). The mean mucosal protein content was 41.84 -C 4.08 pg/mg in animals in the control group and 66.85 + 7.2 1 pg/mg in rats in the experimental group (Fig 7), representing a 60% increase (P < .OS). DISCUSSION Intestinal I/R injury remains a formidable challenge and often results in significant patient morbidity. There are limited data in the literature supporting potential medical treatments that may diminish l/R injury OI enhance the restoration of intestinal integrity or function. Diclofenac sodium has been shown to have a significant cytoprotective effect on intestine subjected to I/R injury.’ O’Donnell et alh showed that oxygenated intraluminal perfluorocarbon. an intestinal imaging agent, protects intestinal mucosa from I/R injury when it is delivered during the ischemic period. Furthermore. it has been 2

1

1

2

Fig 1. Mean mucosal DNA content for each group. The GLP-ZUtreated group (group 2) had a statistically significant increase when compared with the saline-treated group.

1

2

Fig 2. Mean mucosal protein content for each group. The GLP-2~ treated group (group 2) showed a statistically significant increase when compared with the control group.

shown that heparin-binding EGF-like growth factor exerts a cytoprotective effect on intestinal epithelial cells during hypoxia in vitro.’ GLP-7 is a 3%amino acid peptide that is liberated from the carboxy-terminus of proglucagon in small and large intestine L cells by the action of tissue-specifc proteases. GLP-2 belongs to a specific class of compounds referred to as proglucagon-derived peptides (PGDPs). products of the posttranslational processing of proglucagon. A role for PGDPs as intestinal growth factors was postulated when small intestinal hyperplasia and hypertrophy were noted in 2 patients with PGDP-secreting tumors.“,” It was shown subsequently that nude mice carrying subcutaneous PGDP-producing tumors had a significant increase in small intestinal weight.“’ The PGDP responsible for the intestinal epithelial proliferation in these mice was identified as GLP-2. Subsequent studies confirmed that the naturally occurring form of GLP-2 induces growth in normal intestine.“.I3 Drucker et ali4 further showed that a synthetic. protease-resistant form of GLP-2 was a potent agent for increasing small and large intestine mass in normal rats. Findings in our laboratory have shown that both GLP-215 and GLP-2a (unpublished data) increase mucosal mass and absorptive function in intestine after MSBR. We hypothesized that a growth factor might preserve or restore intestinal integrity after significant inflammation or injury in vivo. Because GLP-2a appears to be a significant intestinal growth factor. we designed this preliminary study to evaluate its potential benefit after small intestine I/R injury. Rats subjected to 40 minutes of mesenteric artery occlusion were treated with either systemic saline or GLP-2a. There was a significant increase in mean mucosal DNA and protein content in rats treated with GLP-2a when compared with the control group. To our knowledge, this is the first demonstration that GLP-3~ can increase mucosal mass in

GLP-2a

ENHANCES

MUCOSAL

MASS

AFTER

ISCHEMIA

359

intestine that has been subjected to I/R injury. Thus, GLP-2ol may have a role in the treatment of intestinal injury that results from I/R, such as in necrotizing

enterocolitis and midgut volvulus. Furthermore, GLP-2cx may prove to be a useful adjunct for improving graft survival in intestine transplantation.

REFERENCES I. Shah KA. Shurey S, Green CJ: Apoptosis after intestinal ischemiareperfusion injury. Transplantation 64: I393- 1397, I997 2. Burton K: A study of the conditions and mechanisms of the diphenylamine reaction for the colorimeuic estimation of deoxyribonucleic acid. Biochem J 62:315-323. 1956 3. Giles KW, Myers A: An improved diphenylamine method for the estimation of deoxyribonucleic acid. Nature 206:93. 1965 4. Lowry OH, Rosebrough NJ, Farr AL, et al: Protein measurement with the folin phenol reagent. J Biol Chem 193:265-275, 1951 5. Schmeling DJ, Caty MC. Oldham KT. et al: Cytoprotection by diclofenac sodium after intestinal ischemia/reperfusion injury. J Pediatr Surg 29:1044-1048, 1994 6. O’Donnell KA, Caty MC. Zheng S. et al: Oxygenated intraluminal perfluorocarbon protects intestinal mucosa from ischemial reperfusion injury. J Pediatr Surg 32:361-365, 1997 7. Pillai SB, Tut-man MA, Besner GE: Heparin-binding EGF-like growth factor is cytoprotective for intestinal epithelial cells exposed to hypoxia. J Pediatr Surg 33:973-978, 1998 8. Gleeson MH. Bloom SR. Polak JM, et al: Endocrine tumour in kidney affecting small bowel structure, motility, and absorptive function. Gut 12:773-782, I97 I

9. Stevens FM, Flanagan RW, O’Gorman D, et al: Glucagonoma syndrome demonstrating giant duodenal villi. Gut 25784-791, 1984 IO. Drucker DJ, Ehrlich P, Asa SL. et al: Induction of intestinal epithelial proliferation by glucagon-like peptide 2. Proc Nat1 Acad Sci USA93:7911-7916, 1996 Il. Brubaker PL, Izzo A, Hill M, et al: Intestinal function in mice with small bowel growth induced by glucagon-like peptide-2. Am J Physiol272:El050-El058, 1997 12. Tsai C, Hill M. Asa SL, et al: Intestinal growth promoting properties of glucagon-like peptidein mice. Am J Physiol 273:E77E84,1997 13. Fischer KD, Dhanvantari S, Drucker DJ, et al: Intestinal growth is associated with elevated levels of glucagon-like peptide 2 in diabetic rats. Am J Physiol 273:88 15E820, I997 14. Drucker DJ. DeForest L, Brubaker PL: Intestinal response to growth factors administered alone or in combination with human [Gly?]glucagon-like peptide 2. Am J Physiol273:G1252-G1262, 1997 15. Kato Y, Alavi K, Yu D, et al: Glucagon-like peptideenhances intestinal function following massive small bowel resection. Gastroenterology I l4:A I 152, 1998

Discussion K.D. Anderson (Los Angeles, CA): How soon do you think this could be clinically applied, and how would you do this? R. Prusad (response): This is a preliminary study that represents a new direction for our laboratory and, as I pointed

out, GLP-2 has been used for short bowel. In this situation, ischemia and reperfusion, I would say that its clinical application is sometime off. We need to study this further, including whether GLP-2a affects function, and whether injury can be prevented or gut function can be restored.