University of Toronto T-Space · 2020. 4. 7. · Characterization of the trophic properties of GLP-2, a novel intestinal growth factor. Thesis MSc 1996. Anne Chun-Hui Tsai, Institute

C haracterization of the trop hic properties of GLP-2, a novel intestinal growth factor

Anne Chun-Hui Tsai

A thesis submitted in conformity of the requirements

for the Degree of Master of Science

Graduate Institute of Medical Science

University of Toronto

O Copyright by Chun-Hui Tsai 1996

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Characterization of the trophic properties of GLP-2, a novel

intestinal growth factor. Thesis MSc 1996. Anne Chun-Hui

Tsai, Institute of Medical Science, University of Toronto

Abstract

The molecular factors that reg1 liate intestinal growth. de ivelopmen t

and regeneration remain poorly understood. The experiments carried

out in this thesis test the hypothesis that GLP-2 is a novel intestinal

growth factor. GLP-2 induces bowel growth in male and fernale mice

of different ages and at least 2 strains of mice. Daily injections over 3

months produced intestinal growth without causing hfstological

abnormaiities or tumorigenesis. The GLP-2 effect on bowel growth

regresses following cessation of administration. GLP-2 did no t cause

teratogenic effec t w hen adminis tered to pregnan t mice. Twenty-

three analogues were screened, and some were more effective than

the natural peptide. GLP-2- treated animals had increased cryp t ce11

proliferation rates and decreased apoptotic cells compared to

controls.

Taken together, the results derived [rom these studies provide an

initial characterization of GLP-2 in p hysiological and pharmacological

aspects and facilitate the understanding of intestinal growth and

adaptation.

Acknowledgement

1 would like to thank Dr. Dan Dmcker for his time, energy,

encouragement, support during the past two years. In addition 1

would like to thank my cornmitee members: Dr. S. Asa who offered

me intellectual inspiration, psychological support, technical

assistance, and photos; Dr. P. Brubaker who spent tremendous time

and effort in helping with my statistical testing, and who used her

unique teaching style to force me to think; as well as Dr. Qing Shi. I

thank Mary Hill, Kelvin So and Susanna Tjan for technical assistance.

iii

Table of Contents

Chap ter One Introduction ~ m ~ ~ m ~ ~ m m m m m a ~ e o m o e ~ ~ m m ~ o ~ ~ e m ~ ~ o ~ m m ~ o o o e s s 1

C hap ter Two Experimental questions ........ ......................... 24

C hap ter T hree Mate rials and Me thods ................... ....m..o...om 2 5

......... Reagen ts ............................................................................................... 2 5

Peptides .....................................................~...........o................e...................o... 2 5

........... Animals .,., .....,.,................................................................................ 25

Animal and Tissue Processing .... ... ................................................ 26 In tes tinal Micro me try ......................................................................... 27

......................................................................... Imm u noc y toc he mis try 27

Proli ferating Cell Nuclear An tigen ...,........................................................... 2 8 In situ identification of apoptotic cells ................,........................................ 29

3 . 7 S tatis tics ........................................................................................................ 30

.................................................... C hap ter Four Resul t s ....,........o..*o..... 32

4.1 What is the effective range of concentrations for ..................................................................... GLP-2 bioac tivity? 32

4.2 When can bowel growth first be detected after initiation of GLP-2 administration? How frequently

............................................................ should GLP-2 be given? 39

4.3 What is the best route and vehicle for administration? ............................m.....................................m....... 42

4.4 Does GLP-2 work in different strains of mice or mice of different sex and age groups? ................................... 46

4.5 1s GLP-2 safe for long term treatment? 1s it .............................................................................. carcinogenic? 52

4.6 Will the GLP-2 induced bowel epithelial hyperplasia regress with cessation of GLP-2 administration? ..............a.....~...................~............~........m~.......... 55

4.7 1s GLP-2 teratogenic? .................~.......~...~.........m.......~....... 59

4.8 What is the optimal structure of GLP-2 related ............................... peptides that exhibit maximal efficacy? 62

4.9 What are the mechanisms for GLP-2 induction of ............................................................................ bowel growth? 86

5 .3 Apop tosis, Proliferation and Tumorigenesis ....~.................. 108

5.4.1 Short bowel syndrome in rat mode1 m m a a m ~ m m a m m m o ~ m a a o o m m ~ m m m m m o ~ m m m ~ m a m m m m m m m m o 1 1 1 ............................................................................................. 5 B. 2 Human S tudies 1 1 2

Chapter Six Conclusion and Future ....................................................................... Exp e r i m e nt s ..................,mm.moa...oo. 1 i 4

Table and Figure Titles

Table:

Table 1: Sequence cornparison of GLP-2 analogues.(P.65)

Figures:

Fig 1: Representative drawing of the human proglucagon

molecule and the proglucagon-derived peptides.(P.ll)

F i g 2: Summary of results of mouse experiments involving

expression of proglucagon-SV40 T-antigen tran~genes~(P.19)

Fig 3: Administration of synthetic rat proglucagon-derived

peptide~~(P.23)

Fig 4 Composite pictures of GLP-2 treated and control small

bowel (P.35)

Fig 5: Bowel weight and crypt plus villus height after GLP-2

administration.(P.37-38)

Fig 6. Temporal increase of small bowel weights and histology

after GLP-2 treatment of a variety of durations and

frequencies.(P.41)

vii

Fig 7. Compatison between different routes and vehicle for

GLP-2 administration. (P. 45 )

Fig 8. Age and sex response to GLP-2 administration.(P.49-51)

Fig 9. Long tem effect of GLP-2.(Pm54)

F i g 10 Regsession of epithelial hyperplasia following

cessation of GLP-2 administration.(P.57-58)

Fig 11: Photomicrograph (intestine) of a mouse fetus from a

pregnant mother that received GLP-2.(Pm61)

Fig 12: Effect of GLP-2 analogues 108, 109, 110 and 111 on

bowel weights and crypt plus villus heigh~(P.67)

Fig 13: Effect of GLP-2 analogues 112, 113, 114, 115, 116,

and 117 on bawel weights and crypt plus v i l l u s height.p.69)

Fig 14: E f f e c t of GLP-2 analogues 118 and PEN 7167 compared

to 109 on bmel weights and crypt plus villus height.(P.îl)


bowel weights and csypt plus villus height.(P.73)

Fig 16: E f f e c t s on b e l weight and histology of analogue

120.(P.75)

viii

Fig 17: Effects on b e l weight and histology of analogue

121. (P.77)

Fig 18: Effect of analogues 294 and 295 compared to 109 on

bowel weights and crypt plus villus h e i g h ~ ( p . 7 9 )


and 306 compared t o 109 on bowel weights and crypt plus

v i l l u s height . (P. 8 1)

Fig 20: Antagonist effect of analogue 114.(Pm83)

F i g 21: Antagonist effect of analogue 111 and 302.(P.85)

Big 22: C r y p t ce11 proliferation rates of GLP-2 treated mice

compared to controL(P.90)

Fig 23 Composite photomicrograph (200X) of small bowel

(inmiunostained for PCNA) from a GLP-2-treated and control

mice. (P.92)

Fig 24: Percent apoptotic cells in the small bowel of GLP-2-

treated and control mice.(P.94)

Fig 25: Composite photomicrograph (1000X) from small bowel

(immunostained for apoptotic cells) of GLP-2-treated and

control mi~e~(P.96)

Abbreviations

ag

Ach

ANL

BBS

C

CCK

CRE

DAB

DNA

E l

EDTA

CIP

GLUC

GLI

GLP- I

GLP-2

GRPP

IP- 1

IP-2

MPGF

antigen

acety 1

analogues

Born besin

base pair

carboxyl terminal

cholecystokinin

cyclic-AMP responsive elemen t

diamino-benzidine- tetrahydrochlonde

deoxyri bonucleic acid

exon one

ethylene diaminetetra acetic acid disodium salt

g='ams gastric inhibitov peptide

glucagon

glucagon-like immunoreac tivity

glucagon-like peptide one

glucagon-like peptide two

glicentin-related pancreatic peptide

growth hormone

hepatocyte nuclear factor 3 8

intervening peptide one

intervening peptide two

major proglucagon fmgment

MSBR

mRNA

N

ODC

PGDP

PBS

PCD

PCNA

PYY

RINlOSGA

RNA

R.T.

SEM

9/40 large T

TPN

TGF-u

TGF-fi

Tris

TdT

TED

TUNEL

1%

massive small bowel resection

messenger RNA

arnino terminal

ornithine decarboxylase

proglucagonderived peptides

phosphate buffered saline

programmed ce11 death

proliferating ceIl nuclear antigen

peptide tyrosine tyrosine

radiation-induced insulinoma cells

ribonucleic acid

room temperature

standard error of the mean

simian virus 40 large T antigen

total parental nutrition

transforming growth factor alpha

transforming growth factor beta

trizrna

terminal deoxynucleo tidyl tramferase

Trasylol EDTA Di pro tin-A

TdT-rnediated dUTP-bio tin nick end labeling

micrograns

Chapter One Introduction

1.1 Intestinal Growth Factors

Unders tanding the molecular factors that regula te in tes tinal growth,

development and regeneration will provide insight into the

pathophysiology of intestinal diseases and furthermore offer

opportunity for designing better treatments. However, to date, the

majority of these factors remain poorly understood In addition to

minor factors such as changes in mucosal blood flow, neural factors,

and bacterial flora etc., nutrition, pancreatic and biliary secretions,

local growth factors and hormones are major influences that regulate

mucosal growth 34.

Luminal nutrition

Luminal nutrition has long been recognized as one of the most

important factors in both maintaining intestinal integrity and

intestinal adaptation. In the fasting animal, villi were found to be

atrophic and intestinal enzyme function was reduced 10.34.749 12? In

ileal-jejunal transposition experiments, the ileal villus hyperplasia

was thought to be secondary to increased exposure to luminal

nutrients . Among the various nutritionai components, fatty acids,

specifically long chain fatty acids appeared to be most important lmw However, it is difficult to separate the direct topicai nutritional

effects Erom those of endogenous growth factors or intestinal

hormones. The rapid onset of compensatory intestinal hyperplasia

despite absence of oral intake clearly suggests the presence of

additional non-nutritional influences 19.

Pancreatico biliary secretions.

The presence of a proximo-distal gradient of villus size and gene

expression indicates that bile and pancreatic secretions may have a

trop hic effec t on the proximal small intestine 4 GoT " o m Evperimen ts

that transplant the duodenal papilla into the ileum result in

increased ileal ce11 proliferation, suggesting that these secretions

have a stimulatory effect on small intestinal growth 4. These effects

may be indirectly mediated by the effect of chyme which has a

provocative effect on the secretion of intestinal growth factors and homones 150.34.74.

Humoral and luminal growth factors

Afthough both luminal nutrients and pancreaticobilian, secretions

appear to influence mucosal growth, none of these factors, by

themselves controls intestinal growth, regulation and adaptation.

Intestine removed as an isolated vascularized loop (the thiry-vella

loop) undergoes mucosal hypoplasia; however, in the presence of

massive small bowel resection (MSBR) , the mucosa of the isolated

loop shows a significant degree of hyperplasia clt 141. This implies

that there was some factor secreted into the blood Stream after MSBR

which stimulated the loop to grow. These factors include polyamines,

peptide W (PYY), insulin-like growth factors, cholecystokinin (CCK),

neurotensin, epidermal growth factor, bombesin and enteroglucagon 150*

During the adaptive response of the residual intestine after 50%

resection, polyamine synthesis within the intestinal mucosa is

thought to be required for the initiation of the rapid cellular growth

and differentiation. This phenornenon however is strongly linked

with the activity of the enzyme ornithine decarboxylase (ODC) W.

Inhibition of ODC activity by di-a-di-fluoromethylornithine (DMFO)

decreases polyamine synthesis and mucosal hyperplasia 10. ODC

mRNA levels increase after MSBR 99914? The observed increase in

polyamine synthesis must itself be triggered by factors such as

luminal nutrition, pancreatic secretions or other humoral and growth

factors 150, and the mechanism of this dramatic increase in ODC

activity remains to be elucidated.

PYY is a 36 amino acid peptide. It is synthesized in and secreted

from the intestinal L-cell, primarily located in the ileum and colon. It

has long been linked to intestinal growth 58,143; however, infusion of

PYY to achieve both pharmacological and physiological plasma levels

after MSBR failed to demons trate growth- modulating effects by

peptide W itself 16. Recently, PW has been postulated to

function as an "ileal break" instead of a trophic factor because it

inhibits gastric emptying and intestinal mo bility, reduces mesenteric

blood flow and inhibits pancreatic exocrine secretions. 143TG53+9 16.

rnsulin-like yrowth factor

The insulin-Iike growth factors ( IGFs) I and II are homologous

peptides related to insulin by structure and function 71. The IGFs are

powerful mitogens for a variety of mammalian ce11 types. IGF-1 is

thought to mediate the growth-promoting effects of growth hormone

while the role of IGF-II appears to be restricted to fetal growth 71.

The actions of IGF in vivo are modulated by specific binding proteins

(IGFBPs) 71. Parenteral administration of IGF-1 resulted in increased

bowel growth and IGF-II enhanced mucosal adaptation after small

bowel resection In vitro studies on IEC-6 cells (rat jejunal crypt-

derived ce11 line) revealed that the cells proliferate in response to

exogenous IGF-I and IGF-II 118. Though IGF-1 mRNA levels do not

increase after MSBR, the precipitous and early fdl in ileal IGF-

binding protein-3 (IGFBP-3) mRNA levels suggests a fa11 in IGFBP-3

levels may increase local IGF-1 bioactivity 2. The role of IGFs in

adaptation is likely to be largely paracrine 150.71~2S.

This peptide is secreted by 1-cells of the duodenum and upper

jejunum in response to products of fat and protein digestion 70. It

stimulates the exocrine secretion of the pancreas and contracts the

gall bladder to exrete bile into the intestinal lumen 34. CCK was

postulated to be a trophic peptide; however, Dowling did not observe

hyperplasia when CCK was infused into the intestinal lumen M. The

trophic role of CCK may be through its stimulation of the exocrine

pancreas and biliary system leading to effects on intestinal growth 61,65,34. losm

Neurotensin is a tridecapeptide found mainly in the central nervous

system and in gut endocrine (N-cells) in jejunum and ileum 70.

Subcutaneous administration of neurotensin can reverse the mucosal

hypoplasia of small intestine in rats 50. The trophic mechanisms of

neurotensin were mediated bo th indirec tly by stimulation of

pancreatic juice and directly via a systemic effect on mucosa 50.

Eoidermal growth factor (EGF) and tfêpsformigeprowth factors a

d (TGF-rr TGF-6)

EGF is a 53 arnino acid peptide that stimulates ce11 mitotic activity,

differentiation and inhibits gastric acid secretion 130. EGF has k e n

demonstrated to induce ornithine decarboxylase (ODC) which in turn

will influence polyamine synthesis 51. The biological effects of EGF

are dependent on the route of administration 56. When given

inrravenously, ileal villus hyperplasia was O bserved in to ta1

parenteral nutrition (TPN)-treated rats but not at the degree

required to compensate for the lack of luminal contents 5.

Intraperitoneal administration of EGF resulted in trophic effects on

intestines 3. Schwartz et al found a slight but not statistically

significant increase in intestinal DNA concenmtion afier luminal

exposure to EGF 144 EGF acts via a membrane receptor, the level of

which plays a dynarnic role in the response to EGF in the intestine 128-

TGF-a is a 50 amino acid polypeptide which binds to the EGFR at a

different site than EGF. TGF-a is produced in cells of the intestinal

villi while TGF-ft is produced by cells that resides in the crypt base

10Gv91. TGF- has been reported to be trophic to the intestine of TPN-

fed rats 20. TGF-f! inhibits proliferation and stimulates differentiation

and is thought to play an autocrine or local paracrine inhibitory role 15O.9le

Bombesin (BBS/GRPL

This nidecapeptide was originally isolated from amphibian skin. The

mammalian counterpart, GRP, is located along the digestive tract.

BBS/GRP has a trophic effect on pancreas and enhances pancreatic

enzyme secretion 95, and stimulates gastrin release and gastric acid

secretion "Oe It has been reported to stimulate intestinal growth and

differentiation in the suckiing rat 95. However, the mechanism may

be secondary to the stimulation of enteroglucagon and CCK secretion.

Bombesin/GRP also produces significant rises in plasma insulin,

neurotensin, and enteroglucagon which in tum influence intestinal

adaptation l509142.

Proglucagon-derived peptides (PGDP1s) have been the hormones most

closely associated with intestinal growth. In the intestine,

posmanslational processing of proglucagon liberates a wide variety

of peptides, many of them with unknown functions. The finding of

increased levels of enteroglucagon immunoreactivity after small

bowel resection has been confirmed in many studies of intestinal

adaptation 149 349 81. There is both clhical and experimental evidence

that PDGP's may be trophic to the intestine. In 1971, a patient with a

renal glucagonoma presented with massive enlargement of small and

large intestine due to biopsy-proven epithelial hyperplasia. This

tumor secreted PGDP1s which were elevated in the patient's plasma

and with the removal of the renal tumor, the blood level of PGDP's

retumed to normal 55. Patients with active Celiac disease u and

inflammatory bowel diseases 14 have also been found to have

elevated PDGP1s. Experimental models such as massive small bowel

resection demonstrate immediate post-surgery elevations in PGDP's

14* 74. Inuaperitoneal injection of glucagonoma tumor extract

resulted in intestinal enlargement in mice 34* Therefore, it appears

that intestinal regeneration is associated with both the synthesis and

secretion of the PGDP's in both clinical and animal models. Because of

the complexity of proglucagon processing and secretion, the following

section will be focused on the proglucagon gene and its derived

products.

1.2 Glucagon Superfamily

The glucagon superfamily includes glucagon, secretin, vasoactive

intestinal peptide (VIP), gastric inhibitory peptide (GIP) and growth-

hormone releasing factor (GHRF) 11. This family of peptides is found

in the gut as well as the central and peripheral nervous systems,

which suggests that they act not only as hormones but also as

neurouansmitters or neuromodulators.

The Proglucagon Gene

Structure

The hamster proglucagon gene was first cloned in 1983 9, and found

to encode a prohormone containing the sequence of glucagon and two

glucagon-like peptides. This gene organization is seen aiso in humans

and rats 68. 10. The rat proglucagon gene consists of six exons and

five introns. The 5' and 3' untranslated regions of preproglucagon

mRNA are located in exons 1 and G respectively 69-68.

Preproglucagon is composed of 180 amino acids (Fig 1). The signal

peptide and part of the amino terminal portion of glucagon is

contained in exon 2. Exons 3 ,4 , and 5 contain the coding sequences

for the proglucagonderived peptides 68. The first 20 amino acids

form the leader sequence. The glucagon sequence is present at

position 33-6 1 of proglucagon. GLP-1 is located at positions 72- 108.

The biologically active foms of GLP-1 are the mncated forms, GLP-

1 (7-37) and GLP-1 (7-36) amide 110~66. GLP-1 (7-36) amide is the

predominant naturd form 117. GLP-2 is present at 126-15 8. In the

pancreas, 64-158 forms the major proglucagon fragment (MPGF) and

residues 1-3 0 forms glicentin- related pancreatic peptide (GRPP. ) 99

69968.39. In the intestine, glicentin and oxyntomodulin correspond to

positions 1-69 and 33-69 of proglucagon respectively (Fig 1) g96gP68.

39.

Bioloev & Phvsiolw

The proglucagon gene is expresseci in the A-cells of the pancreatic

islets, the L-cells of intestinal mucosa, and in the central nervous

system 93,1129 108.95, In rnammals, a single proglucagon mRNA is

transcribed from identical promoters in the pancreas, intestine and

the brain 112. The precursor, proglucagon, is processed in a tissue

specific pattern, which results in di fferen t peptides being liberated in

each of the difierent tissues lo8P249 119. In the pancreas, glucagon is

the major bioactive peptide synthesized and to a lesser amount,

O ther peptides produced in the pancreas include glicen tin-related

pancreatic peptide, the major proglucagon fragment and small

amounts of GLP-124? 48. In the intestine posttranslational processing

liberates a differen t set of peptides: glicentin, oxyntomodulin,

glucagon-like peptide 1, IP-2, and glucagon-like peptide 2 108911?

The peptides produced from posttranslational processing of the

proglucagon gene are known as the proglucagonderived peptides

(Fig 1.)

Fig 1: Representative drawing of the human proglucagon

malecule and the proglucagon-derived peptides. Alternative

posttranslational processing of proglucagon in pancreas and

intestine results in different bioactive foms of peptides.

The numbers refer to the relative amino acid positions within

proglucagon. GRPP=Glicentin-related pancreatic polypeptide,

IP-l=intervening peptide 1, IP-2-intervening peptide 2,

MPGF=major proglucagon fragment.( Modifieci frorn Taylor et al

1994 Iso and Holst et al 1994 7 5 )

MPGF Glicentin

30 33 61 64 69 72 78 108 1 1 1 123 126 158

- c -

Nm GRPP Glwagon IP-1 GLP-1 IP-2

PANCREAS

GLP-2

Glucagon is a 29 arnino acid pancreatic hormone that stimulates

gluconeogenesis and glycogenolysis for maintaining glucose

homeostasis 123.80. It is synthesized and secreted by the A cells of

the islets of Langerhans, and its major site of action is the liver.

Glucagon secretion from the A cells is stimulated by Iow blood

glucose, epinephrine (and O ther beta adrenergic stimuli), and arnino

acids; inhi bi ting factors include elevated levels of blood glucose,

insulin and somatostatin 125. Abnonnal action of glucagon has been

implicated in the pathogenesis of type II diabetes mellitus 6. GLP-1

(7-36 NH2 and 7-37) is a physiological incretin in man 92. It also

inhi bits gasMc motility and gastric and pancreatic secretions 759 1 G 4

1% GLP-1 (7-36) amide has recently been proposed as a new

treatment modality for type II diabetes because of its glucose-

dependent insulinotropic actions on the endocrine pancreas 369 661%

Oxyntomodulin is able to interact with the gasaic oxyntic gland and

inhibit gastric acid secretion 8.95 as well as to stimulate insulin

secretion in the pancreatic B cells at supraphysiological

concentrations 82, Although the physiological role of glicentin is not

clearly established, it also appears to be a gastric acid inhibitor 88.

Semi-purified glicentin was reported to stimulate intestinal growth

in vitro 14. Intestinal PGDP's are secreted in response to gastric

inhibi tory peptide (GIP) , bombesin, calcitonin gene related peptide,

glucose (carbohydrates) and fats (especiaüy long chah fatty acids) 23.

25,137. Somatostatin inhibits secretion of PGDP's from the intestinal L

cells 22.23. No physiological role for GRPP and MPGF have been

demonsmted. The biological functions of the other peptides

produced in the intestine or pancreas such as intervening peptides 1

and 2 remain unknown.

Pancreatic Proglucagon Gene Regulation

The factors that regulate pancreatic proglucagon gene expression have been extensively studied 119.39. 35.12 1.122.89. 84,90.120.4O,28.

161. DNA elements important for transcriptionai control of the

proglucagon gene in the endocrine pancreas have been identified 35.

121. There are four distinct elements within the first 300 b.p. 5'-to

the transcriptional start site important for vanscriptional regulation

359 '21*89*84m The elements are designated, Cl (-65 to -97). G2 (-1 74

to -192), G3 (-238 to -268) and GJ(4.00 to -140) 89984.28. In

addition, a cyclic AMP responsive element (CRE) (-29 1 to -298) was

also identified within this region in the rat 83, but not in the human

glucagon gene 10.

Further experiments dernonstrated that G1 is critical for A ce11

proglucagon gene expression. G2 and G3 are islet cell-specific control

elements 3g930. The G2 element contains a specific DNA sequence that

binds HNF-3p (hepatocyte nuclear factor 3p), and mediates inhibition

of proglucagon gene transcription 859124. The G3 element has been

further subdivided into two domains: A (roughly -258 to -252) and B

(-2 47 to -2 3 4) m. Mu tational analysis experiments demonstrated

that domain A is essential for islet ceil gene expression 123.90 and

insulin-responsiveness of the glucagon gene (designated IRE on the

glucagon promoter region) 123. G4, an islet-ce11 specific element

shares common binding sites with elements in the insuh gene

promoter and G1, is located upstream of G128. G 4 contains at least three protein-binding sites: two E-box motifs E2, E3 and IS (

intervening sequence). It is suggested that Cl and G 4 may have

functional relevance during islet ce11 differentiation 28.

Recent studies suggest a role for homeobox genes in the control of

islet cell-specific gene transcription. cdx-2/3 (whic h designates the

same homeobox pmtein called cdx-2 for mice and cdx-3 for hamster)

is a novel recently identified homeo box pro tein. Elec trop horetic

mo bility shift assay experiments using specific antisera identified

cdx2/3 as a major component of the G 14c2 complex in islet and

intestinal cells 6. cdx2/3 mediates the cell-specific expression of the

proglucagon gene in islets and intestine by specifically activating the

G1 element 8% This experiment also reveaied that the first half of G1

is an important element for glucagon gene regulation. isl-1 was

recently reported to function as a positive regulator of proglucagon

gene transcription in the endocrine pancreas 161. isl- 1 activates

transcription through the Gb/Gc element in the proglucagon gene

proximal promoter 161972.

Gene transfer experiments in glucagon-producing immortalized ce11

lines suggested that the rat proglucagon gene was regulated through

a protein kinase C-dependent pathway 1199120. Transfection of the 5'-

region of the rat proglucagon gene into InRI-G9 cells also supported

the hypothesis that the rat proglucagon gene was activated by a

CAMP-dependen t pro tein kinase A pathway m. Deletion analyses

mapped the site of activation of the glucagon-CAT fusion gene to the

CRE element in the rat proglucagon 5' flanking region 89~40.

Jntestiaal Proolucagon Gene Exbression

In contrast to the pancreatic studies, little is known about the factors

that regulate gene expression, biosynthesis, and secre tion of PGDP's

in the intestine. The initial lack of an intestinal-derived

proglucagon-producing ce11 line contributed to the difficulty in

studying this gene in the intestine.

Studies with primary fetd rat intestinal cultures demonstrated that

secretion of the proglucagonderived peptides was stimulated by GIP

in a dose dependent fashion a. The regulation of proglucagon gene

expression in primary fetal rat intestinal cultures was shown to be

modulated by a CAMP-dependent pathway 38. However, the

intestinal L cells in primary cultures constitute only a small

proportion of the total cells and therefore are not an ideal mode1 for

gene transfer studies 223.21.

STC-1 is a mouse intestinal ce11 line, derived frorn the progeny of the

Rat Insulin Prornoter-Simian Virus 40 (SV40) large T antigen/Rat

Insulin Promoter-Polyoma large T antigen transgenic mice 53. 135.62.

These mice develop intestinal neuroendocrine tumors. STC-1 is a

plurihomonal tumor cell Iine that produces signifiant quantities of

secretin, PGDP's, somatostath, P U , amylin and cholecystokinin 53.

STC-1 cells have b e n used to further investigate the molecular

contml of the intestinal proglucagon gene. It has been shown in STC-

1 cells that proglucagon gene expression is also regulated through a

cyclic AMP-dependent pathway, as in the prirnary intestinal culture

mode1 53.

GLUTag is a novel ce11 line derived from intestinal tumors of a

proglucagon gene-simian virus 40 large T-antigen transgenic mouse

wT84742g43. GLUTag cells express the proglucagon and CCK genes,

consistent with the pattern of lineage-specific enteroendocrine

differentiation described for mouse intestine. Experiments with

GLUTag cells demonstrated direct induction of proglucagon gene

transcription by a cAMPdependent pathway 42. The GLUTag ce11 line

is a useful mode1 for the analysis of the molecular determinants of

enteroendocrine gene expression 43.

Recent studies in transgenic mice using transgenes containing 5'

flanking sequences of the rat proglucagon gene have contributed

important information about the differences between intestinal and

pancreatic proglucagon gene regulation and possible biological

func tions of the proglucagonderived peptides 94.24.6. The first

" proglucagon" transgenic mouse carried a 1,300 bop. fragment of the

5'-flanking region of the rat pmglucagon gene fused to the SVJO

large T antigen , GLUTag (1.3) 46-42. This transgene was expressed in

the brain and pancreas but not the intestine. These transgenic mice

eventuaiiy developed endocrine tumors of the pancreas. A second

transgenic rnouse strain canying 2,300 b.p. of the 5' flanking region

of the rat proglucagon gene fused to the SV40 large T antigen,

GLUTag (2.3 ) , resulted in transgene expression in the brain, pancreas

and the intestine 94.84942. These experiments suggest that the cis-

acting sequences that speci@ intestine-specifc glucagon gene

expression are different from those that direct expression in brain

and the pancreas. The intestinal specific element may restde in the

DNA sequences between -2292 and -1253 upstrearn from the start of

transcription 84 (Fig 2). These transgenic mice developed an invasive

proglucagon-producing neuroendocrine carcinoma of the large bowel

as well as a prominent small bowel M. The plasma levels of

proglucagonderived peptides in these transgenic mice were

considerably elevated in association with tumor growth 24. The

posttranslational processing of the proglucagonderived peptides

from the large bowel tumors represented a pattern that was

intermediate between the pattern of peptides seen in the pancreas

and intestine 24.

Based on the findings from studies of the transgenic mice, it was

reasonable to suggest that proglucagon-producing tumors secreted

some factor that modulates small bowel growth. As the epithelium of

the GLUTag transgenic mice contains cells expressing the SV40 T

an tigen w hich may accoun t for the intestinal proliferation, therefore,

a second experiment was conducted in which neoplastic cells from

this large bowel proglucagon-producing neuroendocrine carcinoma

were injected subcutaneously into nude mice 41. These cells

reproducibly fonned su bcutaneous tumors. These tumors

F i g 2 : Summary of results from mouse experiments involving

expression of proglucagon-SV40 T-antigen transgenes. GLmag,

glucagon-SV 40 T antigen; El, exon 1; IWF-36, hepatocyte

nuclear factor-3g. (~odified from Dnicker et al 1994 42)

Transgene expression in pancreas and brain

Insulin-response element

GLUTag(2.3)

Transgene expression in pancreas, brain and intestine

( C W ECO RI Kpn I TGACGTCA

Intestine specific (GU€)

HM-3 ~ - b ~ ~ cdx-2/3

I m (-300) (-258) (-1 90)

63 G 2 G 4 G 1 E l

synthesized and secreted proglucagon-derived peptides into the

plasma. Three distinct observations were noted in these nude mice :

marked suppression of endogenous pancreatic glucagon gene

expression in the tumor-bearing mice: islet area was markedly

reduced 41, and the small intestine in the nude mice with the

proglucagon-producing tumors was enlarged, compared to

controls 4.

To further determine the association between proglucagon-derived

peptide and small bowel growth, Drucker et al .i4 studied nude mice

carrying 3 hormonally distinct subcutaneous endocrine tumors that

express the proglucagon gene in vivo. This experiment would

precisely elucidate PGDP's role in stirnulating bowel growth in the

absence of SV40 antigen. Three proglucagon-producing neoplastic

endocrine ce11 lines were cultured in vitro and transplanted

subcutaneously into nude mice. The three proglucagon producing

endocrine cell lines were tested: InRi-G9 37,147, a BK vims-

transformed hamster islet ce11 line; RIN1056A 119983, a rat islet ce11

line derived from radiation-induced insulinomas; and STC-1 5 3 9 1359

a, a mouse intestinal ce11 line that also synthesizes and secretes

PGDP's. Each rnouse was injected with only one ce11 line.

Subcutaneous tumor nodules formed and after thirty days the nude

rnice were sacrificed. At the end of the experiment aiI tumors

continued to synthesize and secrete large amounts of PGDP's u.47.

Elevated plasma levels of PGDPfs were associated with marked

increases in small bowel weight, villus height, and cqpt ceil

proliferation 14y47.

Individual ~eot ide testinn in v i v ~

To discover the identity of this novel intestinal growth factor, PGDP's

were synthesized and injec ted subcutaneously in 6-week-old CD 1

female mice. over ten days. The result of this experiment revealed

that the mice receiving glucagon-like peptide 2 (GLP-2) had

increased bowel weight, and c y p t plus villus height *. This

experiment strongly suggested that GLP-2 is an intestinal growth

factor (Fig 3).

1.3 Study Hypothesis

Therefore, the following hypothesis was generated:

" CLP-2 is a novel intestinal growth factor that is

efficacious in mice."

Fig 3: Administration of synthetic rat proglucagon-derived

peptides: Glicentin, GLP-1 [7-361 amide, IP-2, and GLP-2

(43.75 pg peptide per injection in 16% gelatin, twice a day

for 10 days to 6 week old CD1 female mice (n=4-6)). The mean

f S.E.M. of the (A) small bowel weights and ( B ) C r y p t plus

v i l l u s height from each peptide-treated group compared to

control are shown. EW, b m e l weight of PJ, proximal jejunum;

DJ, dis ta l jejunum; D I , distal ileum. The asterisks denote a

s t a t i s t i c a l l y significant difference compared to control,

*=p<0.05, **- 0.01, ***=p<0.001.( Adapted from Drucker et

al 1996 )

Chapter Two Experimental questions

To examine the hypothesis that CLP-2 is a novel intestinal growth

factor and to characterize this peptide the following questions were

genera ted:

1. What is the optimal dose of GLP-2 treatment?

2. When can the induced bowel growth first be detected after GLP-2

administration? How frequen tly should it be g iven?

3. What is the best route and vehicle for GLP-2 administration?

4. Does GLP-2 work in animals of difierent sex and age ?

5. 1s GLP-2 safe for long term treatment? Is it carcinogenic?

6. Will the GLP-2 induced intestinal epithelial hyperplasia regress

with cessation of administration?

7. Is GLP-2 teratogenic?

8. What is the optimal structure of GLP-2 related peptides that

exhi bit maximal efficacy? Are there antagonists?

9. What are the mechanisms for GLP-binduction of bowel gmwth?

Chapter Three Materials and Methods

3.1 Reagents

Al1 chernicals were from Sigma Chernicals ( S t Louis, MO) or Baxter

Travenol Canada (Toronto, Ontario, Canada). immunohis tochemical

reagents were obtained from Novocastra La b, Ltd. (U.K. ), Vec tor La b,

Inc. (Buriingame, CA, U . S . k ) and Oncor Inc. (Gaithers burg, MD, U.S.A.)

3.2 Peptides

GLP-2 was synthesized by the American Peptide Company, Inc.

(Sunnyvale, CA, U.S.A) and the GLP-2 derivatives were from

California Peptide Research, Inc. ( Napa. CA. U.S.Ao).

3.3 Animals

The CD1 mice were obtained from Charles River iaboratory (Ontario,

Canada). The CD1 mice were age-matched females (n=3-4 per

group), 6 weeks of age, unless othenvise specified. The animals were

allowed a minimum of 24 hours to acclimatize to the laboratov

facility before the initiation of each experiment. Animals were

identified by ear punch. The mice were not restricted by diet or

activity during the experiment. The lightldark cycle was 12 hours,

between 6 pm to 6 am. The majority of the injections used 12%

gelatin or PBS as vehicle. Controls were age- and sex-matched (n=3-

4) animals that were injected with PBS or Gelatin. Each peptide was

prepared at a specific concentration, dissolveci in 0.5 cc of vehicle.

The peptides were injected su bcutaneously, unless otherwise

indicated, and mice were monitored daily in the laboratory facility.

Animais were sacrificed 14 days after injection, unless otherwise

indicated and were fasted at least 20 hours before sacrifice.

3.4 Animal and Tissue Processing

The mice were anesthetized with CO2 and exsanguinated by cardiac

puncture. Blood was collected in 75 pl of TED (Trasylol; EDTA (5000

KIU/ml: 1.2 mg/ml); Diprotin-A [0.1 nM] ), the blood was centrifuged

at 14 k x g for 5 minutes and the plasma was stored at -70 prior to

analysis. The mal1 intestine was removed from the peritoneal cavity,

frorn pylorus to cecum, cleaned, weighed and measured. For

comparative purpose, sections from each animal were obtained from

the identical anatomical position. Fragments each measuring 1.5-2.0

cm in length were obtained 812 cm, 18t2cm, and 3252cm from the

pylorus for histomorphometry representing proximal jejunum, distal

jejunum and distal ileum 479 Each small bowel fragment was opened

longitudinally on its anti-mesenteric border in a tissue block and

then placed in 10% formalin (vol./vol.) overnight, then transferred to

70% ETOH. The large bowel was also processed in a similar way in

specific experiments for histology.

3.5 Intestinal Micrometry

5pm thick sections were cut and stained with hematoxylin and eosin

for tissue identification; these sections were used for micrometry and

morphometric analysis. The sections were processed by Ms. Aida

Stefan in the Department of Pathology, Mount Sinai Hospital.

Intestinal micrometry was performed using a microscope with a

video camera( Leitz, Wetzar, Germany) connected to a cornputer

monitor. The microscope was calibrated at 4x, 10x, 25x

magnification and the same microscope was used for al1 evaluations.

Crypt plus villus height was measured by examining at least 20

longitudinally-oriented villi from each slide for proximal and distal

jejunum and distal ileum and is expressed in prn+S.EM.

3.6 lmmunocytochemistry

Separate 3-5 pm sections of srnaIl intestine were taken for

immunostaining 165 for the nuclear antigens-Proliferating Ce11

Nuclear Antigen (PCNA) 78 and modified TUNEL method (TdT-

mediated dUTP-biotin nick end labeling) (Apoptagrhfv Oncor) 5. A

minimum of 15 to a maximum of 30 longitudinally oriented

crypt/villus axes were counted per section per animal for PCNA

while 20-22 high power fields per animal were examineci for the

apoptosis analysis. For PCNA staining, the results are expressed as a

percentage of positive staining crypt cells over total ce11 numbers in

a crypt for each animai. For the apoptotic analysis, the ratios were

obtained for the number of positive nuclei to total intestinal nuclei in

each lOOOX field. Final results are means t S.E.M. for peptide-treated

and control mice. Technical assistance with staining was obtained

from the Pathology Department of Mt. Sinai Hospital, Toronto. The

PCNA staining was done by Mr. Kelvin So, and the apoptosis analysis

was done by Ms. Susanna Tjan.

Protiferatin? Ceil Nuclear Anrj~en

Proliferating Ce11 Nuclear Antigen may be used as an indicator of ceil

proliferation. PCNA is a 36 kD nuclear protein associated with the ce11

cycle. It functions as a cofactor for DNA polymerase 138. 139T33967. It

is synthesized in greater amounts during the S-phase and has a half-

life of - 20 hours. PCNA expression can be used to identiQ the

proliferative cornpartment in normal intestine. In the experimen ts

described here, PCNA is detected using mouse monocional antibody,

clone PC- 10 67,131 (Novocastra Lab, Ltd.). Results are expressed as a

percentage of positive staining crypt cells in longitudinaily orien ted

sections (n= 15-30 villi per section for each animai).

5 pm sections were cut ont0 sialine-coated slides and dried at room

temperature overnight After dewaxing the slides, and microwave

heattng for antigen retrieval for 20 min., 3% (vol./vol.) aqueous

hydrogen peroxide was incubated with each section in a moist

chamber to block endogenous peroxidase activity ( 15 minutes).

After washing the section with tap water, the slides were incubated

with 10% (vol./vol.) normal horse serum. The excess horse serum

was then decanted off the slide and 1:1500 dilution of anti-PCNA

antibody was incubated with the section (in an humidified chamber)

at 370 C for one hour. The slides were washed in PBS and then a

1 :MO diiution of biotinylated horse anti-mouse secondary antibody

(Vector Laboratories, Inc., Burlingame, CA, U.S.A.) was incubated with

the section for thirty minutes at room temperature. Following

another wash in tap water, the section was incubated with a 150

dilution of Vector Staining Elite Avidin Biotin-Peroxidase Cornplex Kit

(Vector Lab, Inc. Burlingame) for 30 minutes. Freshly prepared

dianiinobenzidine-hydrochloride (DAB) (for 50 ml, 1 g of DAB is

diluted in 200 ml of Tris, a single 5 ml aliquot of this is mixed with

45 ml of Tris/HCl pH 8 and 10 pl of 30% hydrogen peroxide) was

employed as a chromogen to develop the slides followed by counter-

staining with light hernatoxylin.

The existence of apoptosis may be inferred from gel electrophoresis

of pooled DNA extracts, as programmed ce11 death (PCD) is associated

with DNA fragmentation. Based on this observation, in situ detection

of PCD may be carried out by the TUNEL rnethod (TdT-mediated

dUTP-biotin nick end labeling) 54 . The study on apoptosis in this

thesis used the Apop tag kit (Oncor) which had some modification of

the staining system.

3-5 pm paraffin sections were cut and adhered onto a sialinized slide.

Mter deparaffinizing procedures s4 nuclei of tissue sections were

stripped of proteins by immersing with 20 p g h l proteinase K (PK)

(Sigma Chernical Co.) for four times. Endogenous peroxidase was

inactivated by covering the sections with 2% H2CQ for 5 min., at

room temperature (R.T). followed by washing with PBS for 5 min.,

two times. The sections were then covered with Terminal

Deoxynucleotidyl Tramferase (TdT) working solution ( Bufier 80 mh4

Trizma base, pH 7.2,140 mM sodium cacodylate, 1 mM Cobalt

Chloride plus TdT (0.3 e. u./ml) enzyme and digoxigenin-dUTP) , and

were incubated in a humidified chamber at 3 7°C for 60 min. The

reaction was terminated by transferring the slides into Stop Wash

buffer (Oncor Inc., Gaithersburg, MD) for 20 min., at 37°C. The

sections were then rinsed with distilled water (DW) and were

incubated with Anti-digoxigenin Peroxidase (Oncor, Inc.) in a

humidified chamber at R.T. followed by washing slldes in 4 changes

of PBS. The sections were covered with D M substrate for 3 min., and

washed in 3 changes of water, then were counterstained with

hematoxylin. Al1 the tec hnical work was done by Ms. Susanna Tjan.

Positive and negative controls were used with each incubation.

Positive controls were from a mouse mammary tumor (Oncor) and

negative controls were same tissue sections without using the

enzyme TdT in procedures.

3.7 Statistics

Al1 data are given as mean t S.E.M. Multiple cornparisons between

samples were made using the Tukey's Studentized Range Test.

Multiple cornparisons with controls were made using the ANOVA

(Analysis of variance). Software used for analysis is the Statistical

Analysis System Package for personal computers (SAS Institute, Inc.,

cary, NC)

Chapter Four Results

4.1 What is the effective ange of

bioactivity?

Drucker et al 44 have demonstrated

concentrations for GLP-2

that GLP-2, in concentrations

ranging from 6.25 to 42.5 pg bid. was effective in promoting small

bowel growth in mice. In order to detemine the full range of

effective GLP-2 concentrations sufficient for the induction of bowel

growth in vivo, mice were treated with varying amounts of GLP-2.

Two sets of evperiments were done. In the first set, G week old CD 1

female mice were divided into 6 groups ( n=3); each group received O

(gelatin only), 0.1 pg, 0.25 pg, 0.5 pg, 1 pg. and 5 pg GCLP-2

respectively via su bcutaneous route every 12 hours for 14 days

using 12% gelatin as vehicle. This experiment reproducibly

demonstrated that GLP-2 induced bowel growth both grossly and

histologicaily (shown in Fig 4) at varying concentrations. However,

as shown in figure 5 (a), no statistically significant effect was observed till the dose of GLP-2 reached 1-5 pg. As well, the

histomorphometry findings varied somewhat in the 0.5 and 1 pg

groups. A second set of experiments contained 4 animals ( n=4 ) per

group and each group received one of the following GLP-2

concentrations : O pg, 0.25 pg, 0.5 pg, 1 pg, 2.5 pg, or 5 pg. As shown in

figure 5 (b), GLP-2 induced bowel growth (both grossly and

histologicaiiy ) at different concentrations of peptide tested. Since

crypt plus villus height was increased between 1 and 2.5 pg, 2.5 pg

was selected as the concentration for subsequent experinients. The

discrepant results at 0.25,O.S and 1 pg of GLP-2 may be attributed to

technical inexperience and smaU sample numbers appiied In the first

set of experiment.

Fig 4 Composite photomicrograph of proximal jejunum from

control and GLP-2 treated animals following administration of

vehicle (a) or 2 . 5 pg GLP-2 in PBS, twise a day, for 10

days (b) .

Fig 5: Bowel weight ( A ) and c ryp t plus v i l l u s height

(B)(C)(D) after GLP-2 administration. GLP-2 was tested at O ,

0 . 1 , 0 .25 , 0 . 5 , l and 5 pg (a) or at 0, 0.25, 0.5, 1, 2.5 and

5 ug (b) The mean f S.E.M for groups of peptide tseated and

control CD1 mice are shown. Results are expressed as percent

change of control. C, control: receiving 12% gelatin only;

BW, bowel weight; PJ, proximal jejunum; RI, d i s t a l jejunum;

01, d i s t a l ileum. The asterisks denote a statistically

significant difference compared to control, *=p<0.05, **=p<

0.01, ***=p<0.001.

4.2 When can bowel growth first be detected after initiation of

GLP-2 administration? How frequently should GLP-2 be given?

To determine when the growth-promoting effect of GLP-2 becomes

clearly evident, G week old female CD1 mice, (n=3-4), were tested.

Each animal received 2.5 pg GLP-2 SC for 1, 2,4,6,10, 11, 13, or 14

days in PBS. For each time course we set up individual control

groups. Figure G(A) shows the percent change in bowel weight for

each group in comparison to its interval-specific control. After 6 days

administration of GLP-2, the GLP-2-treated group demonstrated a

statistically significant increase in bowel weight in comparison to

control. The increment in bowei weight reached a plateau after 1 1

days of peptide administration.

To study the importance of time intervals between individual

peptide injections, 16 6-week-old female mice were divided into 4

groups; each received a total amount of 70 pg GLP-2 over 14 days (

evcept for the control group). The exact amount and frequency of

peptide administered was 2.5 pg every 12 hours, 5 pg once a day,

and 10 !tg every other day. The injections were given subcutaneously

(using PBS as vehicle). As shown in figure G(B), al l3 different

treatment regimens produced a significant increase in bowel weight. However, 2.5 pg twice a day produced the greatest increase in bowel

growth, particularly when assessed at the crypt plus villus height in

proximal jejunum (PJ), distal jejunum (DJ) and ileum.

Fig 6. (A) Temporal increase of small bowel weights in paired

groups of 6 week old female CD1 mice treated with PBS alone

(Control) or synthetic GLP-2 (2.5 pg in PBS, twice a day,

subcutaneously for 1, 2, 4, 6, 10, 11, 13, and 14 days (B)

Changes in small bowel weight and histology after various

G U - 2 treatment frequencies. PBS, phosphate-buffered saline;

q12h, every 12 hou; qd, once a day; q@ ; every other day;

WI, bowel weight; PJ, proximal jejunum; IN, distal jejunum;

DI, distal ileum. The asterisks denote a statistically

significant difference compared to control, *=p<0.05,

**=p<O.Ol, ***=p<0.001.

(A) onset of effect

(8) Frequency of administration

Days of Injection

Crypt plus villus height I i

4.3 What is the best route and vehicle for administration?

To test if different routes of GLP-2 administration are effective for

induction of bowel growth, 24 animals were divided into 3 groups

(n=8), and then subdivided into 2 smaller groups (n=4, as control and

experimental ). Three experimental groups received GLP-2, using PBS

as vehicle, via subcutaneous (SC), intramuscular (IM), and

intraperitoneal (IP) routes, respec tively. Bowel weigh ts from the

peptide-treated groups were compared to the mean of bowel weights

of control animals. Statistical testing was done to compare differences

between control and treatment groups by ANOVA and between one

another by TUKEY. As shown in Fig 7(A) subcutaneous appeared to

be the best route of administration; however, inuamuscular and

intraperitoneal routes were al1 effective for GLP-2 administration.

Initial experiments were done using 12% gelatin as vehicle for its

slow-release effect. Because of technical inconvenience and the

tendency for infection, phosphate buffered saline (PBS) was

subsequen tly tested. Figure 7(B) demonstrates the difference

between the peptide-treated (n=4) and control (n=4) animais. GLP-2

was also able to induce bowel growth when dissolved in PBS. To

further compare Gelatin and PBS as vehicle, three groups of animals

(n=4) were tested at the same time, using gelatin as control. As

show in Fig 7(C), GLP-2 in PBS was about 10% less effective

compared to GLP-2 in Gelatin. The results of these experiments

demonstrated that the efflcacy of GLP-2 in promoting small bowel

growth is not restricted to a gelatin formulation.

Fig 7. Comparison between different routes and vehicle for

GLP-2 administration (A) Percent change of bowel weight after

SC (subcutaneous), IM (intramuscular) and IP

(intraperitoneal) administration, al1 values were compared to

SC control. C, control; T, GLP-2 treated. (B) Percent change

of bowel weight and crypt plus villus height between animals

receiving GLP-2 or control (using PBS as vehicle). (C)

Comparison between Gelatin and PBS as vehicle for GLP-2

injection. G, gelatin; PBS, phosphate-buffered saline; BW,

b e l weight; PJ, proximal jejunum; DJ, distal jejunum; DI,

distal ileum. The asterisks denote a statistically

significant difference compared to control, *=p<0.05, **=p<

0.01, '**=p~0.001.

(A) Routes of administration

(C) Vehicles for GLP-2 administration ( PBS vs gelatin )

4.4 Does GLP-2 work in different strains of mice or mice of

different sex and age groups?

Initial experiments were done on 6 week old CD1 rnice. To ascertain

whether GLP-2 was effective in mice of different seu and age groups,

the following two sets of experiments were done. 48 CD 1 mice were

subdivided into 8 groups (n=6): 4 week old male, 4 week old female,

8 week old male, 8 week old female, 12 week old male, 12 week old

femaie, 16 week old male and 16 week old female mice. Each group

contained 3 control and 3 peptide-treated animals. GLP-2 in PBS was

given at the dose of 2.5 pg subcutaneously, twice a day every 12

hours over 14 days. The results are shown in fig 8 (A)-(H). GLP-2

administration produced a statistically-signifiant change in bowel

growth in al1 groups of mice.

A second experiment using older mice was carried out with 64

C57BLK femaie mice from 6 months to 24 months. Fig 8 (1)-(L)

demonstrates that GLP-2 was effective in inducing bowel growth in

mice of various ages, and both sexes. ALI the animals were subjected

to a complete autopsy at the time of sacrifice. Of interest, two 24

month old rnice had visible tumors over face and skin before the

initiation of the experiment. Another two 24-month-old mice (one

control and one experimental) died 6 days after treatment and were

found to have multiple tumors and nodules in the intemal organs on

autopsy. One 18- month-old treated mouse was found to have partial

intestinal obstruction and dilatation of the proximal jejunum at the

time of sacrifice. This animai was excluded from this study and the

biopsy of the nodule sampled from the lesion proved to be a

lymphoma. The histologicai study on colons of al1 animais sacrificed

at the designated time were normal. There were no other histological

abnormaiities found.

F i g 8. Age and sex response to GLP-2 administration. (A)- (H)

Sex-matched GLP-2 treated animals from 4 to 16 weeks of age

were compared to t h e i r own controls for both bowel weight and

histology. (1)-(L) female anbals aged 6 months to 2 years

old. C, control: T, G U - 2 treated. BW, bowel weight; PJ,

proximal jejunum; DJ, distal jejunum; DI, distal ileum. The

asterisks denote a statistically significant difference

compared to control, *=p<0.05, **TC 0.01, * * * ~ 0 . 0 0 1 .

(A) 4 week old male

Csrpt plus villus heig ht r

PJ DJ 1

01

8, i

IO0 Y C:

8 b a

o . . . . . . . . . . . .

(C) 8 week old male

Crypt plus villus heig ht

49

(6) 4 week old female

Crypt plus villus height

( i l ) 8 week old female

Crypt plus villus heig ht

w

(E) 12 Week old male


(G) 16 Week old male

2ool 1 Crypt plus villus height

BW . PJ DJ DI '

(F) 12 week old female


(H) 16 week old female


(1) 6 month old female

Crypt plus viilus height w

y? DJ Dl

(K)18 month old female


51

(5) 12 month old female


(L) 24 month old female

Ciypt plus viilus height r 1

B iao

Y C

8 L

Ptl

O

4.5 Is GLP-2 safe for long term treatment? Is it carcinogenic?

As GLP-2 appears to function as a growth factor, we studied the

consequence of long term GLP-2 administration. 24 six -week- old

fernale CD1 mice were treated with 5 pg GLP-2 in 12% gelatin

subcutaneously, continuously once a day for 4 , 8 , and 12 weeks . The

control was treated identically with 12% gelatin only. As shown in

figure 9,GLP-2 was effective in producing and maintaining increased

smail bowel weight and villus hyperplasia for geriods of time

ranging from 4 to 12 weeks. AU the animals received a complete

autopsy at the time of sacrifice and there were no histological

abnomalities found in any of these animals.

Fig 9. Long tem effect of GLP-2. GLP-2 was administered at a

dose of 5 pg every day continuously (A) for 4 weeks, (B) for

8 weeks and (C) for 12 weeks, comparing treated animals(T)

with control(C) for the bawel weights and histology. BW,

bmel weight; PJ, proximal jejunum; DJ, distal jejunum; DI,

distal ileum. Percent change of control are shawn. The


compared to control, *=p<0.05, **=<O.Ol, ***=p<0.001.

(A) 4 week term

Crypt plus villus height I i

BW Pl DJ 01 t

(8) 8 week term Crypt plus villus height

(C)12 week term


4.6 Will the GLP-2 induced bowel epithelial hyperplasia regress

with cessation of GLP-2 administration?

64 C57BLK femaie mice (6 months to 24 months of age) were divided

into 2 groups (n=32). Each group was subdivided into 4 age-specific

groups (n=8; age =G, 12,18 and 24 months). In each age group there

were 4 controls and 4 peptide treated animals. Each peptide treated

animal received 2.5 pg G U - 2 per 25 gram body weight in PBS bid

for 14 days while the contml mice received vehicie only. The first

group of treated rnice were sacrificed on day 15 after initiation of

GLP-2 administration. The results are shown in figure 8 (1)-(L) and

Fig 10. The second major group was treated with GLP-2 for 15 days,

then left untreated in the animal facility and sacrificed 10 days later.

As shown in Figure 10, except for the oldest age group, the effect of

GLP-2 on bowel weight regressed &ter ten days. However, the GLP-2

effect on villus hyperplasia did not completely regress in the older

(18 and 24 rnonths) animals. This could be explained by the higher

dose administered to the older group and/or the fact that the older

animals may have slower rates of bowel turnover than the younger

ones.

Fig 10 Regression of epithelial hyperplasia following

cessation of GLP-2 administration. X - a i s represents percent

change in bowel weight and crypt plus villus heigkt vs.

control. Animals of 4 different age groups: 6 months, 12

rnonth, 18 months, and 24 months were tested. The first two

bars for each age group represents the effect after GLP-2

administration (control vs. experimental group), C, control;

E, experimental. The last two bars of each age group

represents the difference between the control and GLP-2

treated group 9.5 days following cessation of GLP-2

administration BW, bowel weight; PJ, proximal jejunum; DJ,

distal jejunum: DI, distal ileum. Percent change of control

is shown. The asterisks denote a statistically significant

difference compared to the appropriate control, *=p<0.05,

**=<O .01, ***=p<O.OOl.

4.7 Is GLP-2 teratogenic?

Four female CD1 mice were found (unexpectedly) to be pregnant

after initiation of peptide administration. They each received 2.5 pg

GLP-2 every 12 hours starting on approximately the 3,4, 7, and 9th

day of gestation respec tively. The latter two mice delivered heal thy

looking litters and the babies appeared normal up to 3 weeks of age.

The former two were sacrificed before delivery and the fetuses were

analyzed at autopsy (n=20) The fetai intestinal developmenc was

normal ( Fig 11). It is not known whether GLP-2 crosses the Placenta.

Fig 11: Photomicrograph ( i n t e s t i n e ) of a mouse ( feta l day 2 0 )

from a pregnant mother who received GLP-2 for ten days.

4.8 What is the optimal structure of GLP-2 related peptides that

exhibit maximal efficacy?

To understand the structure of GLP-2 necessary for maximal efficacy

and stability, the following strategies were applied to synthesize

derivatives. These strategies include: 1. terminal deletions, to

ascertain the minimal size of peptide required for bioactivity; 2.

terminal modifications, to avoid degradation and increase s tability of

peptides; 3. select mutations, of several presumed unstable residues

that might be prone to cleavage or oxidation; 4. alanine scanning, to

determine which specific residues or domains are crucial for GLP-2

bioactivity by arbitrarily replacing every single residue by alanine;

5. glucagon replacement, to again determine which specific residues

or domains are crucial for GLP-2 bioactivity by replacing GLP-2

sequences with homologous peptide sequences from glucagon. Table

1 shows the structure of al1 analogues tested to date. A sumrnary of

the route, vehicle, concentration and duration of experiments is

shown. Al1 the analogues were compared by assessing murine small

bowel growth in vivo, using bowel weight as an index of relative

efficac y. Analogues were analyzed by HPLC by the manufacturer

before testing in animals.

The effects of each GLP-2 analogues on bowel weight and crypt plus

villus height compared with control (vehicle only) and the biological

form of GLP-2 are shown in figures 12-19 (n-3-4). Each figure shows

data for analogues tested simultaneously in the same experiment.

The effect of each analogue on bowel weight compared to control

were summarhed in table one. As shown, some of the derivative

peptide demonstrated apparently better efficacy than the biological

GLP-2 such as analogues 112, Penn 7167, 120.121,294,295,305,

306 while the rests appeared to be no better than the biological

fonn. By analyzing the modified change on the residues, the key

amino acids and structural motif responsible for the GLP-2 molecule

in inducing bowel growth are revealed. Since analogue 120 and 12 1

demonstrated outstanding potency, better characterization in the full

range of effectiveness of these two potent analogues, with focusing

on the dose response effect at lower concentrations were. At one fifth

the concentration of the original form, both analogues achieved same

potency as the original one. The detailed results are shown in Figures

16 and 17. As well, analogues 295 and 306 achieved higher efficacy

than 109. (Figures 18-19) On the contrary, some analogues such as

1 1 1, 1 14 and 3 02 did not demonstrated the biological ac tivity in

inducing bowel growth.

Based on the receptor competing and signal blocking rationales,

analogues 1 1 1, 1 14 and 302 were selected as po tential antagonists.

Each candidate antagonist was administered alone or with GLP-2

( 109) at a ratio of 10:l. As show in Figure 20, analogue 114

inhi bited GLP-2-induced bowel growth. However, su bsequent testing

of analogue 114 at a ratio of 114:109 of 8:l, 6:1,1:1,2:1 and 1:l did

not achieve successful inhibition of GLP-2 activity. Analogues 1 1 1

and 3 02 also demonsmted antagonist activity, although somewhat

less than analogue 114 (Figure 21).

Table 1: Sequence cornparison of GLP-2 analogues. 0, gelatin;

Pr PBS. ~njections were given twice a day at according doses,

routes, concentrations and duration as sumxnarized i n t h e

table.

- 5RZLLOGO - 108

109

110

111

112

113

114

115

116

117

118

PENN 71 67 119

120

121

294

295

299

300

302

304

305

306

MODIFICATION

N- 4 deletion

N- 3 ddetion

N- modification

N- 2 ddetjon

N- modification

C- 3 ddetion

N- 8 ddetion

C- modification

Buman GLP-2 G modification N- modification

Sdect mutaüon

Sei- mutation

3dacî mutation

~ c t c c ( mutation

~clect mutation

select mutation

N- 1 ddetion

Seiect mutetion

Sdsct mutation

Select mutatlon

PEPTIDE

IP-2

Preproglucagon 126-158



ah-preproglucagon 126-158

Angelerfish GLP-1


~g+reproglucagon 126-158

Preproglucaqon 1 2 6 4 5 5

Preproglucagon 12 6-1 5 0

Preproglucagon 126-158-NH2

[Alal 0 1 Preproglucagon 126-15 &Ug

kg%- Preproqlucagon 126-158

[DAlaZ ] Preproglucagon 126-158

[ G I Y ~ 1 Preproglucagon 126-158

f 1 Preproglucagon 126-158

[Va12 1 Preproglucaqon 126-158

[Ma4 1 Preproglucagon 126-1 5 8

[Glu3 1 Preproglucagon 126-158


:Leu10 ] Preproglucagon 126-158

[NlelO Preproglucagon 126-158

[Lys201 Preproglucagon 126-158


bowel weights and crypt plus villus height. C , control; BW,

bowel weight; PJ, proximal jejunum; Ri, d i s t a l jejunm; D I ,

distal ileum. Percent change o f conttol are shown. The


compared to control, *=gK0.05, **=<0.01, ***=~S0.001.


m m o r - m b ) O r - Q b U ¶ O r - a 0 0 - g o O c r E O O 2 0 O e~ O F F C i r r r r W r r r r ~ , r r r r w r r r p

g d d A A a d a a d C J A J z t z " f a * Z Z Z 2 2 2 u a a a 8 ; 2 2 3


and 117 on bowel weights and crypt plus villus height. C,

control; BW, bowel weight; PJ, proximal jejunum; Rf, d i s ta l

jejunum; D I , distal ileum. Percent change of control is

shown. The asterisks denote a s t a t i s t i c a l l y s igni f icant

difference compared to control, *=p<0.05, **=<0.01,

***=p<0.001.

Percent Change

Percent Change n O

b Percent Change

B Percent Change z n 4

O g E ro O J 1 7 2

Fig 14: Effect of GLP-2 analogues 109,118, PEN 7167 on bowel

weights and crypt plus villus height. C, control; BU, bawel

weight; PJ, proximal jejunum; RI, distal jejunum; DI, distal

ileum. Percent change of control is shawn. The asterisks

denote a statistically significant difference compared to

control, *=p<0.05, **=<0.01, ***=p<0.001.

Analogue -3 ANL PEN 71 67,118


Fig 15: Effect of GLP-2 analogues 119,120,121 and 109 on

bawel weights and crypt plus villus height. Each peptide was

tested at 2 doses (0.5 and 2.5 pg) EW, bowel weight; PJ,

proximal jejunum; RI, distal jejunum; DI, distal ileum.

Percent change of control is sham. The asterisks denote a

statistically significant difference cmpared to control,

* = ~ < 0 , 0 5 ~ **=<O.Olr ***np<0.001

Dose PBS 0.5 2.5 0.5 2.5 0.5 2.5 0.5 2.5 k e PIS 0.5 2.5 0.5 2.5 0.5 2.5 0.5 2.5 1IiIi- 1 . 1 i Ii d

ANL 109 119 120 121 ANL 109 119 120 121

200

8 C z 100 Y C QI

P

O Dose

ANL IIIIid

IO9 119 120 121 AhiL

Fig 16: Effects on bowel weight and histology of analogue

12O.(A) ANL 120 was tested at 0, 0.025, 0.050, 0.1,0.25 and

0.5 pg. Results are expressed as mean I SoEmM for groups of

peptide treated and control CD1 mice. P88, control; BW, bawel

weight; PJ, proximal jejunum; DJ, distal jejunum; DI, distal

ileum. The asterisks denote a statistically significant

difference compared to control, *=p<O.OS, **=p< 0.01,

***=p<0.001.

F i g 17:

121. (A)

0 . 5 pg.

peptide

weight ;

Effects on bowel weight and histology of analogue

ANL 121 was t e s t e d a t 0 , 0 .025 , 0 .050 , 0 . 1 , 0 . 2 5 and

Results are expressed as mean 2 S.E.M for groups of

treated and control CD1 mice. PBS, control; BW, bawel

PJ, proximal jejunum; DJ, d i s t a l jejunum; D I , d i s t a l

ileum. The a s t e r i s k s denote a statistically significant

difference compared to control, *=p<0.05, **=p< 0.01,

***=p<O.OOl.

f ercent change h

-. A -. 4 n O -i. lu W - O O O

# s O O ;R d ! L

Percent change

Percent ohmge

Percent ehaage

F i g 18 : Effect of analogue 294 ( A ) or 295 (B) compared to

control and 109 and on bowel weights and c q p t plus villus

height. ANL 295 was tested at 2 doses (0.5 and 2.5 pg). C,

control, receiving PBS only. Bn, bowel weight; PJ, proximal

jejunum; DJ, d i s t a l jejunum; Dr, dista l ileum. Percent change

of control are shown. The asterisks denote a statistically

significant difference compared to control, *=p<0.05,

**=<0.01, ***=p<O.OOl.

Crypt plus villus height I 1

(B) Analogue6 ~ ~ ~ 2 9 s

Crypt plus villus heig h t

F i g 19: Effect of GLP-2 analogues 299, 300, 302, 304, 305,

306 and 109 on bowel weights and cryp t plus villus height BW,

bowel weight; PJ, proximal jejunum; Percent change of control

is shown. The asterisks denote a statistically significant

difference compared to control, *=p<0.05, **=<0.01,

***=p<0.001.

Analogue7

(A) Bowel Weight

81

ANL 299, 30Q 302,304, 305, and 306

PBS 109 299 300 302 304 305 306 ANL

(B) Crypt plus villus height PJ

PBS 109 299 300 302 304 305 306 ANL

-Fig 19-

Fig 20: Antagonist effect of analogue 114. A range of

concentrat ions of ANL 114 were administered with GLP-2. The

ratio of 114/109 (pg/pg) is shown. Controls are PBS only and

GLP-2 alone. BW, bawel weight. Percent change of control are

shown. The asterisks denote a statistically significant

difference compared to c o n t r o l , **0.05, **=<0.01,

***=p<0.001.

Antagonist-1 ANL114 vs ANL 109

Fig 21: ~ntagonist effect of analogues 111 and 302. Tan-fold

excess concentrations of ANL 111(A) and ANL 302 ( B ) were

administered wi th GLP-2. The effects on bowel weights and

crypt and qrillus height were compared to control and GLP-2-

treated mice (ANL 109 ) . Bn, bowel weight; PJ, proximal

jejunum; ht, distal jejunum; DI, distal ileum. Percent change

of control is shown. The asterisks denote a s t a t i s t i c a l l y

significant difference compared to control, *=p<O.OS,

**=<O.Ol, ***=p<0.001.

Control ANL 11 1 ANLl09 /ANL109

Control ANL 1 1 1 ANL109 /ANL109

Control ANL 302 ANLl09 /ANL109

4.9 What are the mechanisms for GLP-2 induction of bowel

growth?

The possible mechanisms for elongated villi incl ude ei ther cell

proliferation or/and inhibition of programmed cell death. Thus the

balance between these two activities reflects the size of the viilus. To

examine these two possibilities, paraffin sections of small intestine

from GLP-2-treated and controls ( PBS) (n=4) were sacrificed to

immunostaining with antisera against nuclear antigens Proliferating

Ce11 Nuclear Antigen (PCNA) for proliferation analysis and antisera

against digoxigenin-labeled DNA fragment for apoptosis analysis

( Apo p tag Tbl, Oncor).

Proliferation results from repeated cell division. Normal villus

growth begins in cells at the base of the crypt 33. Pluripotential stem

cells migrate up the crypt axis proliferating, and as these cells reach

the villus, proliferation rates decline and differentiation begins.

Normal proliferation is usually conflned to the cryp ts, occasionaily

extending to the crypt-villus junc tion 78. In the mice treated with

GLP-2, the srnall intestinal weight, length and villus height were

increased. Increased crypt ce11 proliferation rates may con tri bu te to

these observed increases. Figure 2 2 shows the crypt ce11 proliferation

rates in the proximal jejunum in the control and the GLP-2 treated

mice. Proliferation rates were assessed with PCNA. Results are

evpressed as the percent of positive staining/total crypt cells in each

crypt on longitudinally oriented section (n=15-30 viU per section for

each animai). Proliferation rates in the peptide-treated mice were

statisticaiiy increased over conwl mice. In the control mice, the

mean crypt ce11 proliferation rate in the proximal jejunum was

46.02 1.2%. In the GLP-2-treated mice, the proliferation rate for the

proximal jejunum was 5 7 t 5.5 % which was 124% of conuols. In

control mice, proliferation was confineci to the crypt cornpartment of

the bowel; the villi did not generally contain PCNA-positive cells

although a few lymphocytes were PCNA- positive in the villous

stroma. In the peptide- treated group, proliferating cells are detec ted

in the villi, and at the crypt-villus junction. Figure 23 is a composite

photomicrograph of PCNA-stained proximal jejunum of bowel from

control(a) and peptide-treated mice (b). Proliferating ceiis are those

whose nuclei are stained brown. Proliferation rates in the outer

serosal muscle layer were also examined and found to be equal in

the control and GLP-2 treated ones ( data not shown). Proliferation in

the fibrous tissue was not measured.

Apoptosis is a morphological involution caused by a "cellular suicide"

program, which is associateci with physiologicai or programmed ce11

death. Some examples of systems in which apoptosis occurs are:

maturation of the immune systern, embryonic development, hormone

deprivation of endocrine or other hormone-dependent or sensitive

cells, tissue turn over, etc. 54Recent research has revealed profound

regulatory interrelationships between the cell cycle, transformation,

and ce11 death 146.

In this anaiysis, the results are expressed as a percentage of positive

staining (brown) crypt cells in each IOOOX field (n=20) per section

for each animal . Final results are means of the peptide-treated and

control mice. Apoptotic rates in the proximal jejunum of peptide

treated mice were decreased cornpared control mice, as shown in Fig

24. In the control mice, the apoptotic cells were found mainly at the

tip of the villi or along the edges of the villi; rarely found in the crypt

cornpartment of the bowel. The mean t S.EwM of the positive stained

cells was 9.05t 2.0 % in the contml animals. In the GLP-2 treated

animais, the distribution of apoptotic cells was similar to controls but

positive cells were fewer, representing only 2.181 0.9%. Fig 25 shows

the composite photomicrograph of Apoptag-stained control (a) vs.

GLP-2 treated (b) under oil lem view (magnification 1000X).

However, the difference between the treated animals and controls

did not reach statistical signiflcance because there was a big

variation in the control group. To achieve statistical significance, a larger number of sarnples should be assessed.

Fig 22: C r y p t ce11 proliferation rates of GLP-2 treated mice

compared to control. Sections were immunostained by PCNA.

Results are expressed as mean 2 S.E.M. for groups of GLP-2

treated and control animals. 0.01

F i g 23 composite photomicrograph (200X) of jejunum

immunostained for PCNA from GLP-2 treated (14 days) (b) or

control (a) mice.

Fig 24: Apoptotic ce11 rate of GLP-2 treated mice compared to

control. Sections were irmnunostained by anti-digoxigenin

peroxidase. Apoptotic rates were counted as brown stained

cells divideci by total intestinal cells for each high pawer

f ie ld. Results are expressed as mean t S.E.M. for groups of

GLP-2-treated and control animals

Control GLP-2 treated

Fig 25: Composite photomicrograph (1000X) of jejunum

hunostained for apoptotic cells (arrws) in GLP-2 treated

(b) and control (a) mice (for 14 days).

Chap ter Five Discussion

5.1.1 Biological Role of GLP-2

No physiological role for GLP-2 has been described to date 74. GLP-2

is secreted into plasma, and is found at concentrations comparable to

that of GLP-1 116 , thus potentially it could act as a hormone. Both

GLP-1 and GLP-2 appear to be released in equivaient amounts from

the intestinal L ce11 74, and after meals the plasma Ievels of both

hormones increase 116. These results suggest that GLP-2 may play a

role in nu tritionai physiology. h i tial studies of proglucagon gene

expression did not suggest that GLP-2 be biologically important

because GLP-2 sequence was not detected in anglerfish islet

proglucagon cDNAs loi9 102- However, recent studies have shown that

the proglucagon of fish and birds do encode a GLP-2 sequence that is

con tained within a differentially spliced in testinal proglucagon mRNA

manscript 3. These results reemphasize the potential biological

importance of GLP-2. Recently, Drucker et al 44identified GLP-2 as an

intestinal growth factor. The high degree of conservation of GLP-2

sequences across various mammaiian species 79 is consistent with the

important biological role of GLP-2 as a growth factor. Review of the

literature suggests that CLP-2 activates adenylate cyclase in

hypothalamus and pituitary 76, activates gluconeogenesis in fish

hepatocytes 111, and may inhibit intestinal epitheLial growth l(w

Other studies found that GLP-2 had no influence on insulin secretion

by idet ce11 lines 36, on fatty acid metabolism 140, on secretion of

macromolecules from pulmonary arteries 107, or on changes of blood

pressure or heart rate when infused intmvenously 7

The studies in this thesis delineate GLP-2 as a novel intestinal

arowth factor and characterize the physiological and phannacological b

role of this peptide. The contradictory fmding from the litenture lw that GLP-2 might inhibit intestinal ce11 growth will be evplained in

the foliowing discussion.

5.1.2 The historical association between PGDP's and

intestinal adaptation.

The historical association between PGDP's and bowel adaptation is

further clarified from studies carried out in this thesis. Since 197 1

Gleeson et ai described the first patient with a glucagonoma and

intestinal villus hypertrophy 5 5 13 and subsequently a simihr case

was described 147. A series of studies were initiated to look for a

possible role for enteroglucagon in intestinal growth and adaptation

139 149 159 16934. In addition, MSBR, celiac disease, cold-induced

hyperp hagia, lactation, changes induced by fas ting-refeeding , fiber

ingestion, and colonic glucose infusions have been found to be

associated with elevated levels of PGDP's and accelerated intestinal

ce11 renewal and growth 74. However, several studies have

questioned the role of enteroglucagon in intestinal growth. In a single

unpublished study-cited by Bloom et al 34 purified glicentin had no

effect in intestinal growth. Goodlad et al 59 reported glucagon 1-2 1

reduces intestinal epithellal ceii proliferation in parenteraiiy fed rats.

Immunoneu traüzation with monoclonal anti bodies agains t

glucagoden teroglucagon and GLP- 1 had no effec t on in tes tinal

adaptation following bowel resection a. Johnson et al 116, reported

that rats fed gum arabic had elevated enteroglucagon levels but had

no increased mucosal ceii proliferation. Bristol et al 18 reported a 2-

to 3- fold increment in plasma levels of enteroglucagon after jejuno-

ileal bypass but no change in distal carcinogenesis in rat, concluding

that enteroglucagon is unlikely to play a role in intestinal growth . Finally, Gregor et al 64, conducted an in vitro evperiment by

showing that highly purifieci rat enteroglucagon inhi bited the

growth-promoting effec t of epidermal growth factor and concluded

that enteroglucagons have little to do with intestinal growth and

adaptation. If we take a close look at the nomenclature and pay

attention to the antisera used in the above experiments, the missing

role (as a trophic factor) for GLP-2 is clear. GLI, was first extracted

from the intestinal mucosa and was found to be different from

glucagon both physiochemically and biologically. GU ezrists in two

molecular forms: one has a C-terminal extension (oxyntomodulin)

and the other has an N-terminal and C-terminal extension (GIicentin).

The term enteroglucagon encompasses these two peptides. However,

the term PGDP's includes aii possible peptides generated from

posttranslational processing of proglucagon. In addition to

glucagonf enteroglucagon, PGDP's include GLP-1, GLP-2, IP-2, and

IP-2. Previous plasma assays using antisera RCSS and R59 to detect

GLI can not detect GLP-2 at all. It is therefore not surprishg that the

experiments using immunoneutralization with antisera that only

recognize or block the first half of proglucagon failed to demonstrate

a role for PGDP's in intestinal adaptation 15~59~63. According to our

studies here (Fig 3), as glicentin demonstrated some effect on

promoting bowel growth, more sMking effect in stfmulating bowel

growth was observed in GLP-2. Thus while the entity known as

"enteroglucagon" or "PDGP's" may have a trophic effect on intestinal

growth, the principal PGDP with growth factor-like properties

appears to be GLP-2.

5.1.3 Intestinal bowel growth factors VS CLP-2

Bowel adaptation is mediated by a cornplex interplay of factors

including luminal nutrition, pancreaticobiliary secretions, luminal

growth factors and humoral or endocrine factors W 141.125.

Severai molecules important for the modulation of intestinal

epithelial growth are peptide growth factors, such as EGF, TGFa and

IGF- 173- These factors are produceci locally and may regulate

proliferation and differentiation. Intestinal epithelium also contains

receptors for insulin-Iike growth factors, and IGF-1 has k e n shown

to stimulate srnaIl bowel growth and enhance mucosal adaptation

after small bowel resection 96973. Their role however, is likely to be

largely paracrine. The insuun-like growth factors (IGFs) have a

potent mitogenic action on the bowel; however, IGFs affect many ce11

types, from fibroblast to gonadal celIsg8. Transgenic mice

overevpressing bovine growth hormone, (in which many effects were

mediated by IGF-l), have enlarged organs tncluding enlarged

intestines 1539103m In contrast, GLP-2 injections result in a targeted

increase in small bowel weight and villus hyperplasia, but not in

non-specitic organornegaly.

The proliferative effects of EGF (on the intestine) were not associated

with increased plasma enteroglucagon and thus the two agents

probably exert their proliferative effect via separate mechanisms 57.

Gregor et al, conducted an in vitro experiment by incubating highly

purified rat GLI-I with primary rat intestinal celis and observed an

inhibition of the growth-promoting effect of epidermal growth factor

64. While the author suggested that PGDP's are not relevant to

intestinal adaptation, this experiment is, however, worthwhile

repeating with GLP-2 alone to determine its relationship and

interaction with EGF in regulating cell growth.

Polyamine synthesis is a critical component of the adaptive response.

The rate-limiting enzyme of polyamine synthesis is ODC. Inhibition of

ODC by DMFO decreases mucosal hyperplasia despite high levels of

PDGP8s 9% 1 0 0 9 149. This indicated that the function of GLP-2 is

upstream of polyamine synthesis. GLP-2 will positively regulate ODC

and stimulate polyamine synthesis. To test this hypothesis one might

analyze the gut of GLP-2-treated mice for the expression pattern of

the ODC gene. If the hypothesis is a ie , ODC expression will be

increased in GLP-2 wated small bowel.

5.1.4 Developmental role of GLP-2

Another interesting question to ask is " What kind of role does GLP-2

play in formation of embryonic intestine?" If an animai is deprived

of GLP-2 during development, wiil the intestine be dysplastic?

Because of the importance of glucagon and GLP-1, the targeted

disruption method that is widely applied in studying the biologicai

functions of genes may not be feasible for proglucagon. A aincated

or disrupted proglucagon mRNA will probably result in complete loss

of proglucagon func tion (no t on1 y for GLP-2 specifically.)

Alternatively, a site-specific mutation (which will no t interfere with

the expression of proglucagon and the processing of other peptides)

could be generated. An equally attractive approach would be to

isolate the GLP-2 receptor and target the GLP-2 receptor using

homoIogous recombina tion.

5.1.5 Does the GLP-2-treated small intestine function

normally?

The GLP-2 induced small bowel is heavier, and appears bigger in

caliber and thicker in texture. An interesting point to ponder is " Can

this GLP-2-induced bowel function normally? "

Based on observations of adaptation following small bowel resection,

the adapted bowel maintains its ability to absorb nutrients 34. There

is increased segmental absorption of water, electrolytes, mono-, di-

and oligo-saccharides, amino acids, di- and oligo- peptides and

probably water soluble vitamins M. Assuming that adequate luminal

bile acid concentrations are maintained, it is iikely that mono- and

di- glycerides, fatty acids, the fat soluble vitamins and steroids are

also absorbed. In other words, there is a non-specific increase in

segmen ta1 absorption 34.

Measurement of mucosal digestive enzyme activity gives an

indication of not only morphological but also functional

characterization of intestinal maturation or adaptation 159. Therefore,

to further ascertain that GLP-binduced bowel can function normally,

enzyme assays on bmsh border enzymes such as sucrase, lactase,

maltase, and assays of lipid binding proteins would be worthwhile.

If Our hypothesis is m e , the total enzyme amount that was extracted

from the GLP-2-treated animals should be increased while each unit

amount ( nomalized per cc or per gm) will be the same as in the

controL

A hyperaophied bowel may be beneficial for absorption and

digestion: however, if bowel motility is a bnonnal, pseudo obstruction

may occur and the bowel may not be functional. The patient with a

glucagonorna reported in 197 1 did develop some intestinal

obstruction 5s. However, this could probably be prevented by

carefully monitoring the CLP-2 levels or using medication to assist

normal motility. One might argue that the patient with the

glucagonoma had excess levels of other PGDP's in the blood and also

had other factors secreted h m the tumor that may have caused the

obstruction. For example, GLP-1 has been reported to delay gasvic

emptying as well as display an iieal brake effect 75.164. Therefore,

the intestinal stasis of the patient may have been due to GLP-1. None

of the 500 mice injected with GLP-2 or its analogues have developed

intestinal obsflic tion excep t one with lymphoma However, to

further address the effect of GLP-2 on motility, studies of bowel

motility &ter high dose GLP-2 administration should be performed.

Lipid malabsorption was also noticed in a patient with Glucagonoma

syndrome 55. Although we did not observe stool quality changes

between the control and peptide-treated animals, further researc h

on GLP-2 should incl ude careful nutrient absorption studies.

Given the potential importance of GLP-2 as discussed in this thesis as

a novel growth factor, it is crucial that the factors regulating the

synthesis and secretion of this peptide are understood. There is little

li terature on the physiological analysis of CLP-2. However, several

studies were done on the biosynthesis and secretion of PGDP's 21-25.

Since GLP-2 is one of the PGDP's, it is reasonable to presume that the

biosynthetic pathway for GLP-1 and GLP-2 will be the similar.

The intestinal L ce11 is an open type epithelial ceil a, which receives

a signal from the lumen at its apical membrane and secrets

endocrine hormones into the blood Stream. Although L cells are

diffusely disnibuted through out the gastrointesünal tract, they are

localized in greatest numbers in the distal ileum. Different

methodological approaches (cell Unes, in vivo , perfused intestinal

segment) on different species ( human, rat, dog, mouse) have k e n

utilized to study the synthesis and secretion of GLP-2. Intracellular

signais such as CAMP, nutrients, peptide hormones, and neural

mediators are major mediators of GLP-2 secretion 25.

5.2 What is the optimal structure of GLP-2 to produce maximal

bowel growth?

5.2.1 The desfgning of analogues

To understand the structure of GLP-2 necessary for maximal efficacy

and stability, the following strategies were applied to synthesize

derivatives: 1. Terminal deletions, to ascertain the minimal size of

peptide required for bioactivity. These include analogues 1 10, 1 1 1,

114,l 17, and 302.2. Terminal modifications, to assess degradation

and stabiiity of GLP-2. These include analogues 1 12, 1 15, 1 18, 7 167,

and 1 19 3. Select mutations of several presumed unstable residues

that might be prone to cleavage or oxidation 27. These include 304,

305, and 306.4. Alanine scanning, to determine which specific

residues or domains are crucial for GLP-2 bioactivity by arbitrarily

replacing every single amino acid by alanine. 5. Glucagon

replacement chimeras 72, to again determine which specific residues

or domains are crucial for GLP-2 bioacdvity by replacing

hornologuous GLP-2 peptide sequences wîth those from glucagon. The

experiments in the kt two categories are still in progress. To date,

we are able to conclude that the first few residues fmm the N-

terminus are very important for maintaining GLP-2 biological

activity. When they are deleted. the derivative peptide cm no longer

stimulate bowel growth.

5.2.2 Dipeptidyl peptidase IV (DPP IV)

Based on observations with GLP-1 and GIP, peptide bioactivity mav

be diminished following cleavage of peptide residues by dipeptidyl

peptidase N (DPP N) 1079 1,819 32. As GLP-2 is derived from the

sarne peptide superfamily and shares homologous sequences with

GIP and GLP-1, it is easy to suggest that this phenornena might also

apply to GLP-2.

DPP IV is a highly specialized aminopeptidase removing dipeptides

only from peptides with N-terminal penultimate proline or

alanine Io? Human DPP N is a 766 amino acid polypeptide with a

high degree of seauence similarity with the rat liver protein m. DPP IV occurs in human serum, as an ectoenzyme on the surface of

capillary endothelial cells and a variety of epithelial tissues such as

kidney , hepatocytes. thymocytes and most abundantly in the small

intestine 30.31. In the intestine, DPP N is associated with the brush-

border membrane enterocytes and exhibits a differentiation-

dependent expression along the rat ieiunum crypt-villus axis and in

human enterocyte-like colon cancer celis in culture 107. I t is

predominantiy located to the apical surface in epitheual ceils W. The

DPP IV mRNA level remains very low in undmerentiated ce11

populations and specificdiy increases in cells that undergo an

enterocytic differentiation 3431. DPP IV gene expression in human

intestine is highest in the distal small intestine and is regulated at

the posttranslational level 31929.

A N L 114, a synthetic GLP-2 analogue, has the same predicted

peptide sequence of a post-DPP IV-cleaved GLP-2, and ANL 114

demonstrated no intestinal growth promoting effect in vivo. This is

consistent with an important role for DPP N in GLP-2 cleavage.

Therefore, in analogue 120, the second alanine was changed from L-

to D- form to prevent the cleavage by DPP N. ANL 120 achieves an

equivalent or better effect on bowel growth compareci to natural

GLP-2. Analogues 121 and 295 were designed for the same purpose

and achieved a better effect than GLP-2 itself. This result suggests

that DPP N plays an important role in determining the biological

half life of GLP-2.

Kieffer et al used a strain of Japanese Fisher rat 1639151 (a DPP N

negative strain) to study the degradation of GLP-1. Their results

demonstrated a prolonged half life of GLP-1 in this strain of rat 19819

21). Therefore to ascertain whether DPP N is also a major enzyme

that inactivates GLP-2, one potential experiment to do is testing GLP-

2 in vivo using the DPP iV-negative rats. Alternatively, the cleavage

of GLP-2 and various analogues by DPP N such as ANL 120 and 12 1

can be assessed using assays in v l ~ o .

5.2.3 Antagonists

A useful way to detect candidate analogues involves experiments in

vivo. For example, ANL 1 14, is predicted ( from DPP N experiments)

to be the biological inactive product of GLP-2 and may compete for

GLP-2 receptors. As shown in the in vivo studies, the antagonizing

effect that analogue 1 14 demonstrated was consistent with this

expectation. With the availability of a known specific GLP-2 receptor,

it will be much easier to study this issue in vitro. However, this

receptor has not yet been isolated. The other strategy to find an

an tagonis t involves making predictions from known peptides of

similar sequence. Based on the similarity between GLP-2 and

glucagon (whose structural importance has been appreciated for a

long time), several predictions could be made from glucagon 1 3 6 154.

1% 16~.17.17,77,155,115.158,157. The carboxylic group of Dg 1%

interacts with Hl to promote signal transduction. E9 of GLP-2 may

therefore be important and substitutions might Iead to lack of

activity and an antagonist 156. D2 1 and Dl 5 are identicai in glucagon

and GLP-2 and can be weakly substituted 158. The corresponding

candidate antagonists in GLP-2 may be D8, E9, D2 1, D 15 and D3 3 77.

155.115.158,157m

5.3 Apoptosis, Proliferation and Tumorigenesis

The small intestine is perhaps one of the most extensively studied

organs from a ceiî kinetic point of view and yet, surprisingly,

relatively little is known about the factors that regulate cell

proliferation. differentiation and ceIl deaui. This rapidly proliferating

tissue, which constitutes about 75090% of the intestinal tract, rarely

develops cancer as ( about 3 50 new cases per year) 129 which

suggests efficient regulation of proliferation, differen tiation and ceil

death, or the existence of other efficient protec tive mechanisms 134.

During rapid proMeration, the chances of errors from DNA replication

will be higher. Fortunately, most of these errors are checked by p53 ,

and subsequently repaired or ce11 apoptosis (through the action of

bcl-2 and/or other genes ) is initiated 1469129m However, not al1 the

apoptosis is p53 dependent 12%nd not al1 the cells programmed to

die have a genetic mutation. Some are programmed to die as a result

of contact inhibition n? It has been suggested that the levels of

spontaneous apoptosis in crypt cells represent the removal of

occasional stem cells that are produced to excessive requirements,

perhaps as a consequence of occasional symmetric division in

population which is normally characterized by asyrnmetric division

129* Genes such as bcl-2 and myc in the presence of high levels of

growth factors have k e n implicated in suppressing apoptosis and

favoring proliferation 49. According to our data, apoptotic ce11

numbers were decreased in GLP-2-treated animals and clinically,

these mice did not develop tumors, suggesting that GLP-2 reduces

but does not completely inhibit enterocyte apoptosis .

The decrease in apoptosis raises the question of whether GLP-2

might predispose to tumorigenesis with t h e . Wfth peptide

administration continuously for 3 months, we did not observe any

neoplastic growth in GLP-2-treated animals. One rnight argue that 3

months is not long enough to develop cancer, therefore higher doses

or administration for a longer duration ( i.e. 2 years ) may be

worthwhile as a future experiment to address this question. The

apoptotic and proliferative rate assessments describeci in this thesis

were done on the same group of 6 week old animals treated with

GLP-2 for 14 days. One may easily ask " Does the inhibition of

apoptosis decrease with time? " or " Does the proliferation effect of

GLP-2 decrease with time while inhibition of apoptosis persists?" As

we have 500 paraffin blocks from mice of different ages and ses

treated with different doses of GLP-2 for various durations, we can

answer the above questions in our future work

Another interesting question to ask is " What is the identity of these

proliferating and programmed death-inhibited cells ? Are most of

them stem cells or terminally differentiated cells ?" To address this

question, in situ hybridization of enterocyte DPP N gene transcripts

might be a good indicator since this gene is expressed in

differentiating cells 31.

Bristol et al reported that elevated plasma enteroglucagon alone fails

to alter distal colonic carcinogenesis in rats 18. This observation is

consistent with our experimental results using GLP-2. Colonic growth

was not signifiant, as colon weights from previous investigations

cmied out in the Drucker lab (unpu blished data) were not different

in GLP-2-treated animals. In addition, none of Our long term GLP-2-

treated animals developed colon cancer, nor did the older mice

(up to 2 years old which rnight be 90 years old in equivalent human

ages) .

In addition to apoptosis, immune surveillance may play a vew

important role in monitoring normal bowel growth. Scavenger cells

and phagocytic ceils exist in the stroma of villi and cytokines have

been reported to play an important role in maintaining normal

intestinal growth 127. The relationship between GLP-2 and the

immune response should be considered in future experiments 134.

5.4 Therapeutic role of GLP-2

5.4.1 Short bowel syndrome in rat model

MSBR in experimentd animals is an accepted model of intestinal

adaptation. Rats are more suitable animals than mice for MSBR in

that they are bigger and easier to operate on and develop less

intestinal obstruction. In classic MSBR experiments, the small

intestine is resected from a point 5 cm distal to the ligament of Tritz,

to a point 5 cm proximal to the ileocecal valve 150. The ileum distal to

the anastomosis undergoes rapid adaptive growth with increases in

crypt ce11 production rate (CCPR) and DNA synthesis occurring within

the first 2 days foiiowing surgery 34. Taylor et al reported glucagon

mRNA levels increased threefold as part of the adaptive response;

the increase is maximal at two days 14. PCDP's have long been

believed to play a major role In this adaptation. It will be

worthwhile to measure the plasma GLP-2 level in post-MSBR rats to

confirm the trophic correlation of GLP-2 and in testinal adaptation.

The adaptive role of PGDP's after massive resectfon was believed not

to result from increased L-ce11 numbers but from an increase in the

content of proglucagon transcripts per cell1soy 52. The glucagon mRNA

levels increased in adaptation can last for weeks as previously

observed 5 2 3 - 103. However, a compensatory increase in endogenous

peptide production is usually auto-regulated with time 159. To

address the importance of GLP-2 as a growth factor, one should be

able to demonstrate that in animals receiving GLP-2 treatment, the

initiation of adaptation will be more rapid and the clinical condition

can be improved with the administration of GLP-2.

Another model for CLP-2 treatment will be chernical- or cold-

induced epithelial injury of rodents which can serve as an animal

model for inflammatory bowel disease or celiac disease. If GLP-2

demonstrates experimental improvement in rodents, it can be

applied to treat human disease.

5.4.2 Human Studies

Surgically-induced short bowel syndrome, necro tizing enterocolitis

(NEC), congenital anomalies of infants, CeUac disease and

inflammatory bowel disease in adults al1 await new therapeutic

strategies to improve the presentiy avaiiable treatrnent. If the

experimental models mentioned above succeed, GLP-2 may be tested

in humans.

Before this becornes possible, we must study the safety profile,

clearance, and normal values of GLP-2 etc. in humans. Collecting

blood samples and measuring GLP-2 in normal people and patients

with a variety of diseases, for example, pre-and post operation,

patients with intestinal diseases, as well as in pre- and post dialysis

patients, will help to determine the dose to administer and the blooci

levels to achieve in humans.

Chapter Six Conclusion and Future Experiments

The data presented in this thesis have characterized the biological

activity of GLP-2 and revealed possible mechanisms for GLP-2-

induced bowel growth. The optimal administration was found at 2.5

ug SC bid over 14 days using PBS as vehlcle. GLP-2 works in male

and femaIe mice of different ages (4 weeks to 2 years old).

Continuous administration of GLP-2 for up to 3 months resulted in

intestinal growth withou t causing histological abnormalities or

tumorigenesis. The GLP-2 effect on bowel growth regresses 10 days

following cessation of administration except in older mfce. 23 GLP-2

analogues were screened, and more potent GLP-2 analogues (than

the natural peptide) were found. Candidate antagonists were

identified and await further characterization. With GLP-2

administration. the crypt cell proliferation rate increases and

apoptosis rate decreases.

Future experiments include: 1. Analysis of plasma GLP-2 levels in

humans to identiw normal values, and hints about its clearance.

These data are crucial for human studies. 2. Functional assays of the

GLP-2-treated bowel by analyzing enzyme and RNA expression

patterns. 3. Establishing models of intestinal diseases such as short

-bowel syndrome in rodents and demonstrating efficacy of GLP-2.

Only if one can demonstrate that this peptide really helps in disease

States in these models, can the effect of GLP-2 induced-bowel groMh

be considered for human studies. 4. Continue analogue screening,

combining the strategies that proved to be helpful from the result of

recent testing. Design the stablest GLP-2 structure and test in vivo

for safety and efficacy. 5. Idenw the GLP-2 receptor, which wiii be

very helpful for in vitro studies and further characterization of this

peptide.

Taken together, the results derived from these studies and future

experiments as described s hould provide better charac terization of

the physioiogical and pharmacological aspects of GLP-2 and facilitate

the understanding of intestinal growth and adaptation mechanisms.

The long term goal is to develop G U - 2 as a novel treatment rnodality

for intestinal diseases.

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University of Toronto T-Space · 2020. 4. 7. · Characterization of the trophic properties of GLP-2, a novel intestinal growth factor. Thesis MSc 1996. Anne Chun-Hui Tsai, Institute