PROTEIN INDUCED SUPPRESSION OF FOOD INTAKE VIA CCKA RECEPTORS

Mark A. Cochi

A thesis submitted in conformity with the requirements for the degree of M.&.

Graduate Department of Nutritional Sciences University of Toronto

@Copyright by Mark A. Cochi ZOO1

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PROTEIN INDUCED SUPPRESSION OF FOOD INTAKE VIA CCKA RECEPTORS

Mristers of Science, 2001 Mark A. Cochi

Griduate Department of Nutritional Sciences Univenity of Toronoto

The objective of this thesis was to test the hypothesis that in rats, the effkct of dietary

proteins on food intake is dependent on the time and duration of interaction between the

protein source and CC& receptors. Devazepide (a CC& receptor anatagonist) was given

intraperitoneally to block CC& receptors and was given 30, 60 and 90 minutes before rats

were presented with their food nips. Casein, soy and albumin was given intragastncally either

in their intact fom or as hydrolysates 30 minutes prior to the introduction of the food cups.

Food intake was measured during 0-1 h, 1 -2h and 2-3h.

The results showed b t devazepide blocked the suppression of food intake caused by

the protein. But, this effea of devazepide showed a time dependency that varied with the

protein source. These results support the hypothesis that the eEect of dietary proteins on food

intake suppression is dependent on the time and duration of interaction between protein source

and CCKA receptors.

ACKNOWLEDGEMENTS

1 would like to thank my supervisor, Dr. G. H Anderson, for giving me the opportunity

in experiencing first hmd the field of research Dr. Andenon's guidance, leadership and

patience motivated me to do my very best, and for that 1 am forever grateful.

1 would also like to thank Dr. Rao and Dr. Heim, for taking time out of their busy

schedules to lend their ean and give advice toward my work.

To Dr. Trigazis and Dr. Cho4 you provided me with the knowledge and encouragement

to p u m e my goal and accomplish it, and for that I thank-you.

To my lab members, thanks for the mernories and great times we shared. Special

thanks go out to the Double Pump Posse @ou know who you are), because it was you guys

who provided the f in (Yea, Yea. . . ).

Finally, 1 would like to thank my parents and siaer for their continual support

throughout this endeavor and to my closest ftiends thanks for being there when I needed you

most,

This project was funded by the Natural Science and Engineering Research Council of Canada

(NSERC). Persod financial support was provided by the University of Toronto Open

Fellowship and the Ontario Graduate Scholarship in Science and Technology.

TABLE OF CONTENTS

CHAPTER 1. INTRODUCTION 1

2.2. Regulation of Food Intake 2.2.1. Energy M e and Balance 2.2.2. Macronutrient Intake and Balance 2.2.3. Protein, Amino Acids and Peptides in Food Intake

2.2.3. i Protein and Food hake

2.3. Metabolic Regdaton of Protein-Induced Satiety 7 2.3.1. Post Absorptive Signais 7

2.3.1.1 .Plasma and Bmin Amino Acids-The Aminostatic Theory 8 2.3.1.2Brain Neurotransmitter Hypothesis 8

2.3 2. Pre-Absorptive Signais 10 2.3.2.1 Mechanoreceptom, Osmoreceptors and Chemorecepton 10 2.3.2.2.Gastrointestinal Hormones I I

2.4. Cholecystokinin 2.4.1. Physiological Action of CCK 2.4.2. Moleculas Characteriration of Cholecyaokinin

2.4.2.1 .Cholecystokinin Types 2.4.2.2.Synthesis. Release and Degradation 2.4.2.3 .Anatomical Distribution 2.4.2.4.CCK Receptors 2.4.2.5.CCK Receptor Antagonists

2.4.3. Mechanisms of Cholecystokinin Action in Food htake 2.4.3.1Proposed Peripherai CCK Mechanisims of Food htake

Regulation 2.4.3.2.CentraI Mechanisms 2.4.3.3 Receptor Antagonists and Feeding

3.1. Hypothesis 28

3.2. Objectives 28

Outline of Work 3.2.1. Introduction 3.2.2. Methods and Materials

3.2.2.1 .Anirnals 3.2.2.2Diets 3.2.2.3 m g s 3.2.2.4.Protein Pre-loads 3.2.2.5.Treatment Repararion 3.2.2.6 hceàures and Experimental Designs 3.2.2.7.Statistics

CHAPTER 4. EXPERLMENTAL RESULTS 41

4.1. Part 1-The effect of devwpide on food htake suppression caused by casein and soy proteins and their hydrolysates 42 4.1.1. Introduction 42 4.1.2. Results 43 4.1.3. Discussion 53

4.2. Part &The effixt of tirne of administration of devazepide on the feeding respoase to proteins and their hydrolysates 56 4.2.1. Introduction 56 4.2.2. Results 56 4.2.3. Discussion 70

4.3. Part III-To examine the eflect of quantity of protein and time of devazepide administration on the suppression of food intake 75 4.3.1. Introduction 75 4.3 2. Results 75 4.3.3. Discussion 79

CELWEIR 5. GENERAL DISCUSSION 84

5.1. General Discussion 85

5.2. Future Directions 94

CHAPTER 6. SUMlMAIPY AND CONCLUSION

6.2. Conclusion

LIST OF TABLES

Table 3.1.

Table 3.2.

Table la.

Table lb.

Ta bie 2a.

Table 2b.

Table 3b.

Table 4a.

Table 4b.

Table Sa.

Table Sb.

Table 6.

Table 7.

Table 8.

Summary of experiments

Composition of protein sources

Effkct of coadministration of casein and devazepide on food intake

Summary of main and interactive effkcts of casein and devazepide (2-way ANOVA)

Effect of coadministration of casein hydrolysate and devazepide on food intake

Sumrnary of main and interactive eEects of casein hydrolysate and devazepide (2-way ANOVA)

Effect of coadministration of soy and devazepide on food intake

Summary of main and interactive effects of soy and devazepide (2-way N O V A )

Effect of coadministration of soy hydrolysate and devazepide on food intake

Summary of main and interactive effects of soy hydrolysate and devazepide (2-way ANOVA)

EEect of devazepide given 15 minutes prior to casein hydrolysate gavage on food intake

Summary of main and interactive eEects of casein hydrolysate and devazepide (2-way ANOVA) 52

Administration of devazepide 30,60 and 90 minutes pnor to food cup introduction

The effect of devazepide on aibumin induced food intake suppression wheu given 30,60 and 90 minutes prior to food cup introduction 59

The effixt of devazepide on albumin hydrolysate induced food intake suppression when given 30,60 and 90 minutes prior to food cup Introduction 61

Table 9.

Table 10.

Table I l ,

Table 12,

Table 13.

Table 14.

Table 15.

Table 16.

The effect of devazepide on casein induceci food intake suppression when &en 3O76û and 90 minutes pnor to food cup introduction

The effect of devazepide on casein hydrolysate induced food intake suppression when given 3û, 60 aud 90 minutes prior to food cup introduction 65

The e f f ' of devazepide on soy induced food intake suppression when given 3q60 and 90 minutes pnor to fmd cup introduction 67

The effect of devazepide on soy hydrolysate inducd food intake suppression when given 30, 60 and 90 minutes pnor to food cup introduction 69

Range of interaction between protein and the CCKAreceptor 73

The effect of devazepide on albumin (0.5g/4ml) induced food intake suppression when given 30,60 and 90 minutes prior to f'd cup introduction 76

The effect of devazepide on albumin (1.0g/4rnl) induced food intake suppression when given 30, 60 and 90 minutes prior to food cup introduction 78

Range of interaction between albumin and the CC& receptor 82

LIST OF FIGURES

Figure 1.

Fipre 2.

Figure 3.

Figure 4.

Figure 5.

Figure 6.

Figure 7.

Figure 8.

Structure of human cholecystokinin-39 13

Representation ofthe human CCKAreceptor 18

Biochemicd structure of devazpide 21

Experimental design for experiment one or" Part II 39

Experimental design for Parts II and III 40

Duration of time over whkh protein interacts with the CC& receptor 74

Duration of time over which albumin interacts with the CCKA receptor 83

Relationship amoag dietary proteins, CC& recepton and food intake 87

C W T E R 1. INTRODUCTION

1. Introduction

Obesity affects 35% of the North Amencan population and this number continues

to grow every year. Obesity is known to increase the risk of diseases such as diabetes,

cancer and cardiovascdar disease. Thus, an understanding of the mechanisms involved

with the food intake regulatory process can aid researchers to decrease the incidence of

obesity and its adverse heaith affms.

There are numerous physiological and psychological signals that are sent to the

centrai nervous syaem which regulate food intake. These signals help animals reguiate

food intake according to their energy and nutrient requirements. Energy is derived fiom

the three macronutrients - protein, carbohydrates, and fat. Much work on food intake

regulatory mechaaisms has b e n focused on the role of these macronutrients and their

metabolites. Although dl macronutrients have the ability to suppress food intake, protein

suppresses food intake more than the other macronutrients, and beyond that which can be

accounted for by its energy content alone. Therefore the mechanisrns accounting for

protein induced satiety are of interest.

Of the three macronutrients, protein is the most potent stimulator of the gut satiety

hormone cholecystokinin (CCK) in the rat. CCK is released from endocrine cells within

the mucosa layers of the proximal srna11 intestine when stimulated by food. CCK binds

to receptors on the vagus within the gut. This discovery has prompted researchers to

determine if CCK plays a role in protein-induced satiety.

CCK has been shown to decrease food intake and increase satiety in many

species, including humans. Much of the research to date focuses on CCK as a general

inhibitor of total food intake. Howwer, littie research has foaised on the role of CCK as

a mediator of satiety induced by protein

To date only albumin (1,2), beef gelatin (3) and soy hydrolysate (4) have been

shown to suppress food intake via CC& receptors. Whether or not other proteins have

the ability to interact with CCKArecepton leading to a suppression in food intake has not

been investigated. As well the relationship between the time of administration and

duration of devazepide exposure and protein induced suppression of food intake has not

b e n described. Hence, the hypothesis of the thesis was that the effect of dietary proteiw

on food intake is dependent on the tirne and duration of interaction between the protein

source and CC& recepton.

The hypothesis was tested by measuring food intake in rats after alburnin, casein

and soy proteins and their hydrolysates were given by gavage and when devazepide, a

CC& receptor antagonist, was adrninistered 30, 60 and 90 minutes p i o r to the

introduction of food cups.

CHAPTER 2. LITERATURE REVIEW

2. Literature Review

2.1. Introduction

This literature review begins with a general discussion of the regulation of food

intake and the roles played by protein and its assuciated hydrolysates. An examination of

the pre and post-absorptive metabolic regulaton of protein induced satiety, with focus on

the pre-absorptive signals and the role of gut hormones follows. Finally, the role of CCK

and its rnolecular characteristics, physiological actions and effects on food intake is

examined.

2.2 Regdation of Food htake

Food intake regulation is a complex process involving both physiological and

psychological signals to the central nervous system. These signals which arise &om

perception, ingestion, digestion, absorption and metabolism of nutrients, initiate and

terminate feeding (5,6,7). Physiological control of feeding can be studied more readily in

laboratory animais, such as rats, because their regdatory mechanisms are not obscured by

non-physiological variables to the ment that they are in humans (8).

2.2.1 Energy Intake and Balance

Animals eat to meet energy and nutrient requirements. When energy

requirements are changed, food intake is appropriately adjusted. For example, exercise

(9). density of diet (IO), or food availability (1 1) al1 bring about quantitative adjustrnents

of food intake in rats.

2.2.2. Macronutrient Intake and Balance

There is evidence to suggest that animals not only regulate food intake accordhg

to their energy rquirementq but also to mamonutrient requirements (12,13,14). Food

seleaion experiments in animals have mggesteci that there are specific regdatory

mechanism for the intake and balance of the three macronutrients (15). Because the

content of this thesis foaises on the effects of protein induced food intake suppression,

the emphasis of the following sections wilI examine the specific regdatory mechanisms

that control protein intake and balance.

2.2.3. Protein, Amino Acids and Peptides in Food Intake

Proteins are essential to the body because of their constituent amino acids. These

amino acids allow the body to synthesize its own proteins and nitrogen bekng

molecules. Proteins are stnicturally determined by their primary, secondary, tertiary and

quatemary organization. Amino acids which are the building blocks of proteins account

for the proteins primary structure. The amino acids that form the primary stmcture are

held together by peptide bonds resulting in a polypeptide chah. Polypeptide chains can

be hydrolyzed, thereby producing shotter peptide chains. Hydrolysate composed of short

peptide fragments, ranghg fiom 2 4 amino acids, can be absorbed by the srnall intestine

independent fiom the amino acid uptake and transport system (1 6,17). At present, linle

knowledge is known about the distinct roies of intact protein, hydrolyzed protein and

amino acids in food intake regulation.

2.2.3.1 Protein and Food lntake

Animals given a choice of diets will select among thern to obtain a protein intake

that meet their requirement (13,18,19,). Anirnals such as rats not only regulate protein

intake on a day-today bas& but dso âom meai-to-meai (20). For aüunple, rats fed a

carbohydrate meal will prefer more protein and less carbohydrate in the next meal.

Conversely, if given a protein meal they select more carbohydrate and less protein in the

next meal. The ability of rats to reguiate for meal seleaion, suggests that food intake is

regdated by rnechanisms that adjust for cdoric or rnacronutrient needs (18,2 1).

Protein suppresses food intake more thm carbohydrate and fat (1,22,23,24,25,26).

Not only do protein preloads have the strongest satiating effects of the three

macronutrients, but its effects are seen for a longer duration of time compared to fat and

carbohydrate preloads (1). The suppression of food intake caused by protein is beyond

that which can be accounted for by its energy content alone (22,24,27).

2.3 Metabolic Regulators of Protein-induced Satiety

The mechanisms that elicit satiety responses to protein consumption have not

been defined. However, it is thought that satiety is achieved after ingestion of protein by

pre or pst-absorptive mechanisms working together via a series of signals and feedback

responses. Most of the midies reported in literature have focused on post-absorptive

signals

2.3.1 Post Absorptive Signals

Post-absorptive signals &se after nutnents have been absorbed in the small

intestine. Protein once digested into its constituent peptides and amino acids enters the

bloodstrearn via the portal circulation and alters plasma aïid brain amino acid

concentrations. Two popular theones of how protein modulates food intake regdation

are discussed beiow.

2.3.1.1 Plasma and Brain Amino Acids - The Aminostatic Theory

The aminostatic theory states that fluctuations in plasma amino acid patterns are

monitored by the brain to mediate feeding behaviour (28,29). It is postuiated thai f d m g

centers regulate amino acid concentratons in the brain within a specific range by

controllhg protein consumption (30,3 1).

It is uniiiely, however that changes in brain arnino acid concentrations provide an

explmation for the initial satiety signals arking fkom protein ingestion (7). Rats gavaged

with an albumin or amino acid mixture show no temporal association between the effect

of the gavages on plasma and whole brain amino acid concentrations and their food

intake. Appetite suppression occurrs prior to change in plasma or whole brain amino acid

concentrations. Similarly, microdialysis studies show that fiee amino acid concentrations

change in several brain regions but not until twenty to forty minutes afler rats begin to

feed. The time course of these changes suggest that flee arnino acids in brain regions

may serve as intermediary signais in the satiety cascade, but are too late to be the primary

signals that develop afler the rat eats protein (32)

2.3.1.2. Brain Neurotransmitter Hypothesis

How fiee arnino acids signal regdatory systems in the brain has not been

established. One popular hypothesis is that some amino acids do so through their action

as preairsors for neurotransmitters or as neurotransrnitters themselves. Tsrptophan,

tyrosine, phenylalanine and histidine are precurson to the neurotransmitten serotonin ,

catecholaniines and histirnine respectively, which are known to control feeding behaviour

(12,33).

Serotonin is a newotransmitter involved in the regulation of food intake. Since,

serotonin synthesis is partially under precumr control, dietary factor influencing

tryptophan availability affect serotoah synthesis (12,34,35). Increased serotonin activity

leads to food intake suppression (35,36). It aiso appears to be involved in the regulation

of food choice. Increased serotongenic activity leads to decreased preference for

carbohydrate in rats given dietary choice (12).

The catecholamines are known to modulate food choice and meal composition

primarily through a neuronal system coordinated by the hypothalamus. Tyrosine and

phenylalanine are the major constituents of the catecholamines, and fluctuations in these

amho acids affect the synthesis and tunover of the catecholamines in activated neurons

(37). Intrapentoneal injections of Tyr and Phe can suppress feeding in rats, but it has

not b e n established that this is because of there precursor role (3 8 , D ) .

The hypothalamus contains the highest concentrations of histidine and histamine

and increases in these concentrations have been associated with food intake suppression

(40). Intraperitoneal injection and/or infusion into the brain, of histidine or histamine

suppresses food intake (41,42,43). The action of histidine has been shown to be via

synthesis of histamine (42).

The role of individual amino acids arising h m protein ingestion in food intake

regulation in normal feeding is uncatain. Only large amounts of the single fke amino

acids suppress food intake (20). These large amowts are unrepresentative of amount of

amino acids acquired during a meal (38,39,44), suggesting that other metabolic events

must be occur in response to food intake in order for amino acids to play a iole.

It appears, therefore, that the post absorptive signals arising fiom amino acids are

important in food intake regdation, but their funaion may be in determining the i n t e d

between meals and in determining the composition of food selected at the next meal.

Howeva, thete is no evidence that they provide the kst satiety signais arising from

protein ingestion

2.3.2 Pre- Absorptive Signals

Pre-absorptive signais arise from the presence of food in the gastrointestinal (G.I.)

tract. These signals are most likely to be transmitted to the hypothaamic region of the

brain via the vagus nerve (45,46). Gastrointestinal mechanoreceptors, osmoreceptors and

chemoreceptors act to modulate food intake (20,47). Gastrointestinai homones are aiso

released upon the arrivai of food and play a role in regulating food intake (48,49,50,5 1).

2.3 -2.1 Mechanorecepton, Osmoreceptors and Chemorecepton

One way that satiety is thought to be si@d pre-absorptively is by distention of

the gut via mechanoreceptors that line the stomach wdl and use the vagus to send a

signai to the brain leadhg to a suppression in food intake (52). However, animal midies

have shown that gastric distention does not necessarily inhibit food intake (53,54,55,56).

Sirnilady in humans, expanding a bdloon in the stomach reduces food consumption (57),

but this effect diminishes as the stomach adapts (58). Because satiety can l a s

approximately four hours afler a moderate meal in humans, it is clear that gastnc

distention done does not detemine satiety (59).

Gastric osmotic recepton detect osmotic pressire changes in the gut. Food is a

source of osmotic particles that can exert osrnotic pressure. It has been proposed that the

osmotic gradient causesi by food particles can draw water &om the body into the gut, thus

reducing the water content in the plasma Because hypertonie solutions suppress short-

t am feeding, there is evidence that osmotic receptors signai satiety (60).

Pmcessed cornponents of macronuments are able to provide satiety sipals to the

brain via the vagus aerve through chemorecepton in the wall of the small intestine (61).

These chemoreceptors are specific to amino acids as shown by increased finng rates in

the vagus after perfusion of the gut with amino acids (62).

2.3.2.2. Gastroint estinal Hormones

Many G.I. hormones are released fiom various endoaine, neiirocrine and

exocrine glands upon the entry of food into the small intestine. Such peptide hormones

as cholecy stokinin, bom besin, gastrin, secretin, glucagon, insulin, somatostatin,

neurotensin and pancreatic polypeptides, contribute to the process of satiation

(51,61,63,64).

Cholecystokinin (CCK) is the most hidied gut hormone and has been show to

modulate food intake. CCK is a peptide hormone released from the mucosa layen of the

duodenum by the adval of food. Endogenous and exogenous CCK reduce food intake in

anirnals including rabbits (65), mice (66), rats (67) and humans (68). Food in the small

intestine causes the release of CCK which acts upon peripherai CCK recepton (CCK3

on the vagus nerve to provide sensory information to the brain and contribute to rneal

termination. The following section discusses this hormone in greater detail.

2.4 Cholecystokinin

This profile on CCK will focus on the physiological actions of CCK, the

molecular characterization of CCK and its mechanisms of action. Finally, CCK' s role in

food intake via its receptors, specifically with protein-induced satiq, will be looked at in

detail.

2.4.1 Physiological Action of CCK

Ivy and Oldberg in 1928 discovered CCK based on the ability of intestinal

extracts to stimulate gailbladder contraction when ulfused into dogs (69). Harper and

Raper in 1943, saw that using similar intestinal extraas also stimulated pancreatic

enzyme srnetion and proposed the name pancreozymin (70). In 1968, it was discovered

by Mutt and Jones that CCK and pancreozymin where the same.

In addition to stimulating gallbladder contraction and pancreatic enzyme

secretion, CCK has other important digestive activities. CCK delays the rate of gastrîc

emptybg. It does so, by relaxing the proximal part of the stomach and by constriction of

the pyloric sphincter (71 J2). Endogenous CCK regulates postprandial gastrin secretion

(73), and it relaxes the lower esophageai sphincter (74), and the sphincter of Oddi in

humans (75). In the intestine CCK stimulates motor activity (76) and decreases intestinal

transit tirne (77). CCK has also been suspectecl in increasing blood ffow to the intestine

(78).

CCK functions as a satiety signal during a meal (79). urtially the discovery was

based of the observation that systemic injections of CCK reduced food intake in

normally-fed (79) and sham-fed rats (80). Subsequently it was shown that microgram

how a inimher a anfapnist dmgs have been deveioped as toois to investigate the mle of

CCK

2.4.2- 1 Choiecystokinîn Types

There are many C a typa. These Merences are a result of enymatic cleavage

on the prrprochoiecystokbh peptide as showri in Fig. 1(87). . . . -

C- terminus * - - - . - --

CCK was first isolated in porcine intestine as a 33 amino acid peptide (88). in addition to

the above peptide (CCK-33), there exists two larger forms-CCK-39 (Fig. 1) and CCK-58

(89). There also exists intermediate sized peptides such as CCK-25, -22, - 18, -10, -9, -8,

-7, -5 and 4 (90,91,92). Among that list of intermediate CCK peptides, CCK-8 is the

most potent form in sheep (93), pigs (94), rats (95), and humans (96).

Not all of the CCK variants are found in al1 animais. CCK in humans and pigs

ranges Eom CCK-58 to CCK-4 (97) while rats only express CCK-22 and CCK-8 (98).

CCK-8 has a sulfated octapeptide sequence that is relatively conserved anoss

species and appears to be the minimum sequence needed for biological activity (Fig. 1) in

the periphery (99). The C-terminus of CCK4 bas structurai homology to the

neuropeptide gastrin (100). Due to the stnictural sirnilarity between gastrin and CCK-5,

gastrin has slight CCK like activity and CCK has slight gastrin like activity. The

smictural homology shared between these two hormones explains why antibodies raised

against CCK oflen cross reacts with gastnn (101). To effectively fundion as CCK and

specifically bind to CCK receptors, the CCK peptide mua be extended to seven amino

acids and the tyrosine residue at position seven ffom the C-terminus must be suifatecf

(1 02).

2.4.2.2 S ynthesis, Release and Degradation

CCK is synthesized f?om a 750 nucleotide cDNA It codes for a 1 15 amino acid

precursor for CCK called preprocholecystokinin, consisting of a 20 amino acid signal

peptide, a 25 amino acid spacer peptide, the sequence for CCK-58 and a 12 amino acid

extension at the carboxyl terminus (103). CCK mRNA is abundantly expressed in the

cerebrai cortex and the duodenum of many species includiig rats and humans. CCK

gaies in rats (104) and humaas (105) display remarkable homology, such that the second

and third exom f?om a three exon gene encode for preprocholecystokinin.

I;itestinal expression of CCK mRNA is modified by diet. Of the three

macronutrients, protein most effectively releases CCK CCK mRNA expression declines

in the intestine when rats are fasting or given low protein diets. In contra% when rats are

fed high protein diets, CCK mRNA expression Uicreases (106).

In the brai% CCK is released 50m cortical, mesolimbic and hypothalamic

neurons (107). In the small intestine, CCK is released from endocrine cells of the

proximal small intestinal mucosa layers which are concentrated in the duodenurn and

proximal jejunum (108). The endocrine cells which house CCK are oriented such that

their apical d a c e is directeci towards the lumen of the gut, so that the microvilli-like

stnictures can corne into contact with the lumenal contents. (109,110). These endocrine

cefls are hown as 1 cells and function to secrete CCK (1 1 1 ) . As components of food

(nutrients) enter the proximal small intestine, CCK is released ftom the basai d a c e of

the 1 cells into the blood.

In humans, protein, peptides, amino acids, fatty acids and long chah triglycerides

adrninistered intraduodenaily (97,112,113) stimulate CCK release. Overail, protein is the

strongest secretagogue of CCK release. Of the &ee amino acids tested in humans, Trp and

Phe are the most potent CCK secretagogues (1 14). Carbohydrate; in the form of glucose

and starch are weak Secfetagogues in humans (97).

In rats, dietary protein and to lesser extent fatty acids, release CCK whereas

carbohydrates and amino acids do not (1 15,s 1,116). This variation fiom humans may be

present because of the lack of a gall bladder in rats. Long-chah trigylceride dependent

CCK release has been postulated to decrease gastric emptying thereby ensuring adequate

fat absorption because of the ladr of emulsifjhg agents in rats. (1 IV.

Of the three macrollutrients, protein is the strongest stimulant of CCK release in

rats. CCK concentrations in plasma rose aiter casein coasumption by 6.3I0.6 PM, but

after Edt (intrali pid) and &hydrate (saccharose) consumption incrernents were onf y

2.7s. 5 and 1.7M .4 pM respectively (1 18). Immediate increases in CCK concentration

in plasma are observeci after protein administration. A five rnilliliter 18% casein solution

administered by orogastric tube increased CCK levels to 7.9il.gpM fiom a baseline level

of O.5M.2 pM after five minutes (51). A h , a duodenal infusion of casein (SOOmgh)

resulted in increased plasma CCK levels ftom 0.8M. 1 to 4. If0.8 pM within four minutes

(1 15).

In rats, intact protein stimulates a greater increase in plasma CCK compared with

protein hydrolysates and amino acids (1 19). Because the source of protein influences

CCK (5 l), it appears that protein specific peptide sequences are required for the release

of CCK (120,121,122). These specific peptide sequences could be the produa of

digesteci protein in the gut. Evidence to support bat the chemicai structure of protein is

important in its ability to release CCK is provided by observation that ingested heat

treated soy protein did not stimulate CCK release but raw soy protein did (123).

Intact protein can stimulates CCK release mon than protein hydrolysates (119).

It has been hypothesized that this ocairs because intact protein can stimulate CCK

release by proteaing CCK-releasing peptides (CCK-RP) f?om proteolytic inactivation in

the intestinai lumen (1 19). CCK-RP is secreted f?om CCK-RP cells into the proximal

srnail intestine and inactivateci by trypsin released âom the pancreas. Postprandially,

when food euters the duod- partiy digested protein ftom the stomach binds to

trypsin thereby cornpethg with CCK-W. This cornpetition between the two proteins

(dietary protein vs. CCK-RP), allows for CCK-RP to rmain in its active fom for a

longer period of t h e and therefore increases the likelihood of CCK ceils releasing CCK

into the blood stream (1 19,124). This concept may explain why intact dietary protein

stimulates CCK secraion in rats whereas carbohydrates, fat and amino acids do not (5 1).

Recently two putative CCK releasing factors have been characterized. Lumenal

cholecy sto kinin-releasing fkctor kom rat pancreatic juice (1 25) and di azep am-binding

inhibitor fiom porcine intestine (1 19). These two factors dong with adenyi cyclase and

phosphoinositide may be involved with the mechanism of CCK release (126,127,128).

The degradation and clearance of CCK bas been studied in detail. Plasma and

brain synaptic membranes are responsible for the clearance of CCK, while most of the

degradation takes place in the liver (129). Enzymatic activity in other tissues such as,

membrane bound aminopeptidases have been shown to degrade CCK-8. However, the

most active degrading enzyme known as enkephalinase, found in the brain, breaks the

bond between Trp and Met rendering CCK inactive (130,13 1).

2.4.2.3. Anatomical Distribution

Peripheraiiy, CCK is found in endocrine cells in the mucosal layers of the

duodenum and jejunum in humans (log), dogs and rats (1 10). CCK endocrine celfs have

dso ben found in pituitary cellq adrend medullary cells and in enteric nerves (132,133).

CCK was also localized in the male nproductive organs, with high levels in the testis,

seminiferous tubules and in human sperm (1 3 4 1 3 5).

CCK receptor binding is saturable and reversible, having a high afnnity with a

dissociation constant reported at -1nM (139,140). CCK binds to a portion of the aMno

acid sequence that composes the N-terminus of the CCK receptor located on the

extracellular side of the plasma membrane. Binding of CCK to the CCK receptor causes

a conformational change of the receptor, leading to the activation of the G-proteins that

are coupled to the receptor motiety, which subsequently phosphorylates (advates)

phospholipase C. Phosphoiipase C acts as a secondary messenger, stimulating CCK

related intracel1ular activities. Sulfated CCK-8, compo sed of P he, Asp, Met, Trp, GI y,

Met, Tyr and Asp, is the most effkctive in binding to the CCK receptor and activating

phospholipase C as compared to other CCK foms. nie Tyr residue of CCK-8 once

nilfated greatly enhances binding and stimulation of the CCK receptor as compared with

unsulfated CCK-8 (1 39, 140).

In the gastrointestinal tract CCK receptors have been found on the pancreas,

gaiib ladder, lower esophageal sphincter, stornach, ileum and colon (1 39,14 1). CCK

receptors are also abundant in the brain and are located on some peripheral nerves

(1 42,143).

There are two types of CCK receptors: CC& receptors and CCKB receptors. The

CC& receptors are found predominantly in the periphery: pancreas, gallbiadder, spinal

cord and vagus neme. This receptor is also found in the brain, specificaily in the

interpeduncular nucleus, area postrerna, the nucleus tractus and the hypothalamus

(67,133,144).

The CCKe receptor was origidly cloned fiom the brain. This receptor is more

prevalent in the brain than any CCKA receptor and less in the peripheral tissues. The B-

noeptor constitutes a major portion of the CCK receptors in the CNS. These receptors

are distnbuted in the brain especially in the Iimbic regions, cortex, paravenîricular

nucleus, and the ventrornediai hypothalamus (145,144). The CCKB receptor has aiso be

indentised in the lateral nucleus tractus solitarius of the rat, an area that receives and

processes information regardiig peripheral neurovegetative function, sensory input as

weli as related centrai signais (146). The B-receptor is identical to the gasain receptor in

the stornach and is now temed gastrin(CCKe receptor (147). Where CCKA receptors

have a high affinity for sulfated CCK, and a low atIinity for unsulfated CCK and gastrin,

CC& receptors do not difkentiate between gastrin and CCK, whether nilfated or not

(148).

CC& receptors appear to be the target site for paipherally released CCK It has

been suggested that peripherally released CCK couid act on central CC& recepton by

transversing the blood brain barrier at gaps like the median eminence. Since, CCK has a

much greater affinity for peripheral receptors, a central action of peripherally released

CCK is unlikely (149). However, this does not exclude the involvement of CCKB

receptors, but emphasizes the role of the peripheral receptors.

2.4.2.5. CCK Receptor Antagonists

Cornpetitive inhibitors of CCK have been used as tools to investigate the

physiological roles of CCK and its receptors, especially with regard to feeding behaviour.

The use of CCK antagonkts in food intake regdation was eocouraged by observations

that antibodies against CCK or animals autohunized to CCK inneased food intake

compared to controls (1 50,15 1).

Fs 3. B û x k m i d aOemn ofdcveepidt

This magonist ha9 ben uscd adeiisively in studying food imtaLe rcguiation mechanisms

b o t h 8 i ~ d h v i v U . ~ i d e h k n a e h w t e d g c d a o t h e m s t e f f e a i v e

CCKA receptor antagonist to date (152). Devazepide haeases food intake above control

in rats (l53,l), pigs (154) and mice (155,156).

2.4.3 Mechanisms of Cholecystokinin Action in Food Intake

Much of the research to date has been elucidating the rnechanisrn by which CCK

mediates food intake. Peripheral and central responses to CCK have been show to

decrease food intake, however the mechanisms appear to be separate (87).

2.4.3.1 Proposeci Peripheral CCK Mechanism of Food Intake Regulation

The penpheral satiety feeding system tenninates meal consumption by creating a

sensation of fùllness (157). Increased plasma levels of peripheral CCK following a meal,

is believed to aid in terminating meal consumption (97,158). ui the rat, peripheral CCK

released fiom the small intestine stimulates satiety by binding to CCK receptors. A

classic study done by Gibbs et al., (79) demonstrated that cerulein (a CCK like substance)

decreased food intake in a dose dependent manner and the behavioural responses usually

seen afker feeding in rats such as grooming, e x p l o ~ g and sleeping appeared (79).

It appears that for CCK to suppress food intake an intact vagus nerve is required.

Evidence for this mechanism anses âom the obsemation that a total abdominal vagotomy

or selective gastnc vagotomy, abolishes the satiety effect of intraperitoneal injections of

CCK (159). It has been discovered that CCK can be manufachired in primary vagal

afkent neurons and has the ability to travel by axoplasmic flow both back into the

submucosa and up to the central terminais (1 6O,l6 1).

The penpheral endocrine hypothesis and the peripheial neurocrine hypothesis are

two different proposed mechanisms for the effect of CCK on satiety. The penpheral

endocrine theory suggests that CCK is released by intestinal mucosai cells into the

ciradation and is returned to abdominal

sensory neurops to terminate feeding (46).

visera activating target receptors on vagal

Lf this hypothesis is c o r n then plasma CCK

levels during and afkr feeding must be sufficient to produce satiety. Evideace shows that

postprandial plasma levels of CCK in dog (153), humans (162) and rats (163) are an

orda of magnitude lower than those required to inhibit food intake. Moreovq

exogenous CCK binds to the low affinity type A receptor to inhibit feeding in rats (164),

whereas endogenous CCK binds to the high affinity type A receptors to stimulate

pancreatic enzyme secretion ( 165). Hence, postprandial plasma CCK levels are probably

too low to bind to the low afiinity type A receptor, and therefore do not induce

suppression via the endocrine pathway (1 63,164,165). Fuithemore, immunoneutralizing

of circulating CCK completely blocked pancreatic secretion when maximal doses of

CCK where given, but had no e f f ~ on food intake (46,166,167). These results illustrate

that pancreatic enzyme secretion seems to be controlled b y an endocrine rnechanism, but

that satiety is induced via a non-endocrine mec hanisrn.

The penpheral neurocrine hypothesis States that intestinal CCK acts within the

entenc nervous system. As nutrients enter the duodenum, CCK is released corn the

enteric neurons and is able to aîtach to its recepton found on the vagus nerve. These

receptors transmit a signal to the satiety centre of the brain via the vagus and

subsequently decrease food consumption (46). Binding of CCK to the CC& receptors

on the vagus nerve allaw high concentrations of the hormone to aime into contact with

the low f inity receptors that have been shown to associate with satiety signaling.

2.4.3.2 Central Mechanisms

CCK has k e n found in various regions of the brain whicb include the cerebral

cortex, hypothalamus and midbrain (168,169), but a role for centrai CCK in feeding has

not been &!%hed. CCK is less effeaive in decreasing food intake when injected

centdy as cornparad to periphdly in rats (159). Some studies have shown that

injections of CCK into the brain causes a decrease in food consumption compareù to the

control, while other studies show no effkct (170). It has aiso k e n suggested that high

central doses of CCK used in feeding midies may act periphdly to induce satiety (17 1).

Antagonia shidies support a role for central CCK in feeding. Proglumide, an

antagonist for both CCK A and B receptors, inneases food intake in rats when injected

into the paraventncular nucleus of the hypothalamus (172). ln dogs, ICV administration

of the CC& receptor antagonist L-365, 260, blocked suppression of sham feeding

induced by gastric distention but had no effect on suppression of sham feeding induced

by small bowel nutrient infusions (173).

The changing CCK levels in the brain after feeding and peripheral administration

of CCK, indicates that CCK may begin its actions in the periphery and relay sensory

information to various brain sites controlling food intake. For example, CCK

immunoreaaivity increased in hypothalamic tissue homogenates of food-deprived rats

that had been ailowed to feed (1 5 1,174).

2.4.3.3 Receptor Antagonists and Feeding

CCK receptor antagonists have ailowed researchers to investigate the role of CCK

as a physiological rnodulator of the feeding response. Since, CCK antagonists increase

food intake when given alone, it can be argued that endogenous CCK evokes satiety

(1 53).

Dwepide, when administered alooe, increases food intake in rats (1). rnice

(175), pigs (152) and monkeys (176) and increases hunger r a ~ g s in humans (177). In

rats, devazepide (0.1 and 1 .O mgkg) also prevents the reduction of sucrose intake (5%

sucrose solution) caused by CCK injection (8psn<g, i.p.). Other studies have show

similar resuits (153,156,178). It therefore appears that CC& receptors mediate nutrient

induced food intake suppression, but the role of the individual macronutrients has not

been fully resolved.

CC& receptor antagonists appear to reverse the effects of carbohydrate induced

suppression of feeding in some species. Devazepide, attenuated suppression of sham

feeding induced by maltotriose (179), maltose (180) and glucose in rats (1). However,

devazepide did not reverse the suppressive effects of glucose when administered into the

upper intestine of meal fed pigs (18 1).

CCK receptor antagonists did not block the satiety response elicited by fat fed to

meal rats. Devazepide (0.6mglk& i.p.) reversed the satiety response induced by oleic

acid in sham fed rats (179,180) but was not able to reverse the suppressive e f M s of oleic

acid in meal fed rats (182,183).

CCKA receptor antagonists are ineffective in modulating the satiety response of

individuai amino acids. L-phenylalanine infused intraintestUlally into rats suppressed

sham feeding and this effkt was not attenuated by the CCK receptor antagonist

devazepide (1,179,180).

CC& receptor antagonists block the reduction of food intake after aibumin

Rdministration.. OrttmaM (184) originally described a complete block of albumin

induced satiety by the CC& receptor antagonist. Similady, TfigaSs (1) found with

albumin when given in coojunction with devazepide 30 minutes prior to food cup

introduction to rats, aibumin induced satiety was r e v d whereas amino acids,

cosnstarch and corn oil induced satiety were not (1) .

Additional studies were done to investigate if other proteins besides ovalbumin

could elicit a similar satiety response via CCKA receptors. Morgan (4) tested soy, whey

and their respective hydrolysates and concludeci that only soy hydrolysate protein was

suppressing food intake via CC& receptors. Recently, Woltman (3) demonstrated that

bacto-peptone, a beef gelatin digest, suppressed food intake via CCKA receptors. An

explanation for the observation to date that only egg-albumin, soy hydrolayste and bacto

peptone function through CC& receptors rnay be due to the time of interaction between

devazepide binding to the receptor and the digestive release of active CCK like peptides.

Therefore, this study examined the role of time of devazepide administration. on the

interaction between protein source and CC& recepton

CHAPTER 3. EXPEFUMENTAL WORK

3. Experirnental Work

Hypothesis

The effect of dietary proteins on food intake is dependent on the time and duration

of interaction between the protein source and CCKArecepton.

3.2 Objectives

The overall objective of this thesis was to determine the time of interaction

between the protein source and CC& receptors on food intake suppression.

The specific objectives of this research were:

1. To describe the effect of blocking CC& recepton with devazepide on food intake

suppression caused by albumin, casein and soy proteins and their hydrolysates.

2. To examine the effect of time of administration of devazepide on the feedhg

response to these proteins and theu hydrolysates.

3. To examine the effect of quantity of protein and time of devazepide

administration on the suppression of food intake.

3.3 Outline of Work

3.3.1. Introduction

Three series of experiments (Table 3.1) were conducted.

Part 1: Five experiments were conducted. Experiments 1-4 were designed to detemine

the &ect of devazepide given with preloads of casein, casein hydrolysate, soy and soy

hydrolysate, respectively, on food intake suppression. The fifth experiment, was

conducted to determine the eEecî of devazepide given 15 minutes before a casein

hydrolysate preload on food intake

Part II: Seven experiments were conducted. The first experirnent exarnined the role of

t h e of devazepide administration aione on food intake. Experiments 2-7 examined

whether albumin, casein, soy and their respective hydrolysates suppress food intake via

CCKA receptors when devazepide is administered 30, 60 and 90 minutes prior to food

cup presentation.

Part III: Two experiments were conducted. Experiments one and two were designed to

determine the e f f ' of quantity of albumin preload and time of devazepide

administration on food intake suppression.

Table 3.1 Summary of Experiments

Part 1: The effect of blocking CCKA receptors with devazepide on ,uced food intake suppression 1 1 EEea of devazepide on casein hduced food intake suppression - 1

2 1 Effect ofdevazepide on casein hydrolysate induced food intake

3 4

Part 2: The effect of t h e of administration of devazepide on the feeding

suppression Effkct of devazepide on soy induced food intake suppression Effect of devazepide on soy hydrolysate induc;? food intake

5

proteins and their hydrolysates 1 1 Effects of devazepide alone on food intake

suppression Effect of devazepide given 15 minutes pnor to the preload on casein hydrolysate induced food intake suppression

- 1

2 1 Effkct of time of devazepide administration on albumin induced

3

4

food intake suppression Effkct of time of devazepide administration on aibumin hydrolysate induced food intake suppression Effect of time of devazepide administration on casein induced

5 food intake suppression EEkct of thne of devazepide administration on casein

6

1 induced food intake suppression

hydrolysate induced food intake suppression Effect of time of devazepide administration on soy induced food

7

Part 3: The effect of quantity of protein and time of devazepide

intake suppression Effect of time of devazepide administration on soy hydrolysate

administration on the suppression of food intake Exn.# 1 1 1 Effkct of 0.5g/4ml dose of albumin preload and time of

1 - 1 dwazeoide administration on food ktake su~~ression

-

2 devazepide administration on food intake suppression Effixt of 1.0g/4ml dose of albumin preload and time of

3.3.2. Methods andMaterials

3.3.2.1. Anirnals

Male Wistar rats (St. Charles Breeding Laboratones, St. Constanî, Quebec),

purchased at a body weight of 170+10g were used in ail experiments. The rats were

individually housed in wire mesh cages and maintained on a 12 hour Li@/ 12 hour dark

cycle (lights on at 0600h) with controlled room temperature (22I0C). Water was

provided ad libitum from an automated watenng system, but food was presented only at

the onset of the dark cycle (1800h). and removed at 0800h.

A new set of rats were used for each experiment, with the exception of

experiment 1 of Part II where those rats were also used in experiment II of Part El. The

sample size was detennined by prior work in this lab (1,184); the sarnple size used to

perfonn statistical analysis was at least n = 10 for al1 experiments, unless o t h d s e aated

(see Appendix 1 for the detailed calculation). The procedures for this expenment were

approved by the University's Animai Care Cornmittee

3.3.2.2. Diets

A 25% casein maintenance diet and a protein-fiee diet were used in al1

experiments. The 25% casein maintenance diet was used to provide rats with their daily

protein requirements, wMe the protein f k e diet was used to assess the effects of the

protein preloads. Using t!k protein fiee diet prevented other protein sources besides the

protein under investigation (such as casein in the 25% maintenance diet) Born

confounding the d y s i s in determinhg which protein is amai interacting with the

CC& receptor to nippress food imake.

The protein-fie. diet containecl 80.3% conistatch, IO0? fàt in the fonn of corn oil

(Mazola), 5% fiber (ceiiu1ose), 3.5% mineral (AIN76). 1% vitamins (AIN76A), and 0.2%

choline bitartrate. The 25% casein diet contiiiris similar ingredients but has less of the

cornstarch component because of the addition of casein. Cornstarch (Nacan Produas;

Toronto, Ontano) and corn oil (Mazola; Best Foods Canada) were obtaiaed Eom a local

supplier (AUied Foods, Toronto, Ontario) and dl other ingredients were obtained from

Teklad Test Diets (Madison, WI). The dia was presented in 250ml staidess steel food

cups.

The m e n t AIN93 diet was not utilized in this work for two reasons. First, in

order to make comp~sons with the previous work fiom this Iaboratory that investigated

the effects of protein induced food intake suppression (1,4,185,184) the same rnodified

AIN76 diet had to be used. Second, the AIN93 diet substituted soybean oil for corn oil to

increase the amount of linoleic acid. However, the present studies were short term

(approximately 2-3 weeks) and it would be unlikely that the rats would be at nsk of

developing essential fatty acids deficiencies during this penod îrom corn oil (The linoleic

content of corn oil is 1% compared to 7% for soybean oil) (186).

3.3.2.3. Drugs

The CC& receptor antagonist devazepide (L-364, 71 8) ((3s) (-)N-(2,3-dihydro-

1-methyl-2-0x0-5-phenyl- IH-1,4-benzodiazepine-3-y1)- IKindole-2-carboxamide} was

donated by ML Laboratones PLC (London, England). The antagonist was suspended in

a vehicle of methylcellulose bought &on BDH Inc. (Toronto, Ontario). The dose used in

all experiment was 0.25mg/kg of rat body weight.

3.3.2-4. Protein Pre-loads

The composition and amounts of the protein sources used in the experiments of

this thesis can been viewed in table 3.2. Albumin, albumin hydrolysate and casein

hydrolysate were adjusted to give each nit Ig of protein in a volume of 4mVrat in Parts I

and II. Intact casein and soy were adjusted to 0.5g/4ml in Parts 1 and II because of their

resistance to solubilization as compareci to the other proteins at their given dosage. Soy

hydrolysate was adjusted to give 0.62g/4ml because work done by Morgan showed this

quantity of protein in 4ml of water to suppress food intake via CCKA receptors (4).

Albumin in Part III was adjusted to 1.0g/4rnl and OSg/4d.

3.3.2.5. Treatment Preparation

In al1 experiments, Devazepide, was dispersed in a methocel solution. The

methocel solution was prepared by adding 0.2Sg of methyl cellulose powder to lOOg of

hot (80°C) deionized water. nie 0.25% methocel solution was stirred for one minute and

allowed to chill to SOC for 2-3 hours. Every haif hour the solution was stirred until it was

clear with no visible particles.

A glas homogenizer (Tissue Grinder, b e x Brand, No.7725; Thomas Scientific,

Swedesboro, NT) was used to prepare the dmg stock solution of devazepide (O.Srng/rnl).

To calculate the quantity of dmg stock solution required for each rat to receive a lm1

injection of O Z m g devasepiddg rat body weight, the following steps were taken:

Table 3.2 Composition of Protein Sources

Source

Ash

*62-88%

Total N

12% +

Arnino N/ Total N

Hyd. 85%

12.5% +

Fat

" Purchased form Sigma, St. Louis Missouri. Purchased fkom Flavow Force, Sarasota, Fiorida.

) Purcbased fkom ICN Biomedicals Inc., Aurora, Ohio. As determined by supplier

na. - Data not availabie

85%

15% +

n.a.

15%

12-14%

n.a.

0.5 8g4d

0.5g/4ml

13-14%

100% ?

1.17g14ml

l.Og/4ml

L

n.a

n.a

Amount Given Conc.

Eauivalent

n.a.

1.17gi4ml (85%)

1 .Og/4d

The weight of the rat in kilograms w u multiplied by 0.25mg/kg, in order to

detemine the quantity of devazepide aeeded.

The required amount of devazepide was then divided by the concentration of

the stock solution (OSmgM), to estab lish the volume needed in a 1 ml vial.

The calailated volume was multiplied by 1.5, because the total volume for

each rat was adjusted to 1Sml ushg 0.25%methocel, so that each treatment

via1 had an excess of solution.

Each rat received lm1 injection of their individuaiized dmg suspension.

Protein treatments were dissolved to a volume of 4ml in distilled water. The

protein solution was mixed with a magnetic stirrer to ensure a uniform consistency. The

4mi solution was given by gavage to each of the participating rats.

3.3 -2.6. Procedures and Experimental Designs

On amival, rats were given a week to adapt to the new environment, diet,

interperitoneal injection, and gavage procedure before any experimental studies were

conducteci. Two days before beginning behavioural studies, an adaptation test to the

gavage procedure was conducted to ver* that food intake kom baseline levels did not

Vary. One half of the rats rmived Aine injections and distilled water gavage (4mYrat)

on day one at 17304 while the other half received nothing. Rats that received the

injection and gavage on day 1 received nothing on day 2.

In Part I, the protein âee diet was presented to al1 rats at 1800h, 0% afker

injection and gavage for a two hour period, at which tune the 25% casein diet was

presented for the remainder of the feeding period (2000 to 0800h). Food consumption

(adjusted for spillage) was measured under red light to the nearest O. lg after 1,2 and 14 h

For Parts II and III, the protein k dia was provided for a tbree-hour period, and

food consumption was mea~u~ed under red light der 1,2 and 3 hours. The third hour

replaced the 2-14h and 0-14h food imake rneasurements beçause work done by Tngazis

(13, Morgan (4) and Part 1 of this thesis showed no treatment effects at these later times,

probably because the haif-life of devazepide is approximately 2-3h in duration (165). Ail

experiments began when food intake &a water gavage and saline injection treatment did

not differ fiom the food intake untreated rats.

Experiments 1-4 of Part 1 were designed to examine the e f f ' of devazepide,

when given concurrently with the preloads, on food intake suppression afier casein,

casein hydrolysate, soy and soy hydrolysate were given to rats. Each experiment in Part I

involved a new set of rats in which four treatments were randomfy administered, they are

as follows:

CONTROL (drug vehicle-methyl cellulose, i-p.; distilled water, i.g.)

PRO ( h g vehicle-methyl cellulose, i.p.; protein, i.g.)

DRUG (0.25 mgkg devazepide, i.p.; distilled water, i.g.)

PRO + DRUG (0.25mgkg devazepide, i.p.; protein, i.g.)

The treatments were given 30 minutes pnor to food cups being introduced to the cages.

Experiment 5 of Part 1 used the wune design as experiments 1-4, but the

devazepide injection @p.) was given at 1735 (protein pre-load was still given at 1730h).

This was done to assess if time of devazepide administration has any effect on food

intake suppression.

In Experirnent 1 of Part 9 the objective was to detennine the efféct of devazepide

on food intake when given 30, 60 and 90 minutes prior to food cup presentatioo. A

withui subject study design was used to assess the effect of demzepide alone on food

M e . Each of the times where devazepide was administaed (39 60 and 90 mimites

prior to food cup presentation) were tested separately. Fu* devazepide was

administered at 90 minutes prior to food cup introduction. Once the results for 90

minutes were obtained, then the test for devazepide given 60 minutes prior to food cup

presentation began, and so on for when devazepide was given 30 minutes before food

accessibiliîy. Between each testing penod a wash out day was given. The same fourteen

rats were used to test devazepide's effect alone at 30, 60 and 90 minutes prior to food cup

introduction, and the coatrol (methyl cellulose) or test (devazepide 0.25rngkg) treatments

were randordy adrninistered to rats for each test time (Fig. 4).

Experiments 3-7 each used a new set of fourteen rats, accept experiment 2 of Part

II which used the rats &om experiment 1 of Part II. Similar to experiment one of Part II,

the test times of devazepide administration were each tested separately starting with 90

minutes prier to food cup presentation, then going to 60 and 30 minutes prior to food cup

accessibility, in order to determine the effect of time of devazepide administration on

food intake suppression when given with the protein preload. The protein preload was

given 30 minutes prior to food cup presentation for each devazepide test time. Between

each testing period a wash out day was given, and the control (proteinj or test

(proteiirtdevazepide) treatments were randomly adrninistered to rats for each test time

(Fig. 5).

Experiments 1 and 2 of Pari RI each used a new set of 14 rats. Part III followed

the same experimental design as experiments 2-7 in Part II. However, part examined

the effkcts of albumin (1.0g/4ml and 0.5ghl) a - different doses and t h e of devazepide

admiai-tion on food intake suppression

3.3.2.7. Smktics

Data in Part I are expressed as means * standard emr of food intake and for Parts

II and III are expressed as the mean diffmnce of food intake * standard enor of test and

wntrol treatments. Food intake &ta at 42 and 14 hom in Part I was analyzed by a two-

way aaalysis of variance (ANOVA) with repeated measures. A posthoc Duncan's New

Multiple Range Test was used in comparing group means &er neatment difkrences

were declared by ANOVA

ui Part 1I and III, food intake measurements bom 1,2, and 3 hours was analyzed

by a Student's paired t-test. A statistical cornputer program (SAS 6.1, SAS Institute, Inc.,

Cary NC) was used to perform the anaiysis. A probability level of p < 0.05 was accepted

for the purpose of dedaring statistical significant.

20:00h F.I.

l

Fig. 4. Experimenial design for experiment one of Part II

CHAPTER 4. EXPERIMENTAL RESULTS

4. Experimental Results

4.1. Part 1 - The effect of devazepide on food intake suppression caused by casein and soy proteins and their hydrolysates

4.1.1. Introduction

The primary objective of this series of experiments was to investigate the eEect of

the CC& receptor antagonist, d a e p i d e , on food intake f ier preloads of casein, soy

and their respective hydrolysates were given to male Wistar rats

4.1.2. Results

Etpriment nt: Effect of devazepide on casein induced food intake suppression

Based on the two-way ANOVA the main effect of the casein preload was to

lower food intake during 0-lh t h e interval, and the main effect of devazepide was to

increase food intake during the &lh tirne interval (Table Ib). There was no significant

interaction seen during any t h e period (pX.05) indicating that the eEect of casein was

shown whether devazepide was present or not. For example, during 0-2h casein alone

suppressed food intake by 0.36g compareci to control, and when devazepide was used,

casein suppressed food intake by 0.16g indicating that the drug is not blocking casein

induced suppression of food intake. Aithough no significant interaction at 0-1 h was seen

in the two-way ANOVA, the results of the one-way ANOVA followed by a Duncan's t-

test suggest that devazepide blocked the action of we in . The net effea was that rats

given devazepide + casein ate less than after devazepide alone. However, they ate more

after the combined treatrnent than after casein alone. The increase in food intake

however, was primuily due to the effea of devazepide alone and not to its eEect in

blocking the action of casein hydrolysate as suggested by the one-way ANOVA post hoc

Duncan's test. Devazepide alone resulted in similar food wnmmption as the controi

group. Thus, devazepide did not prevent the reduction in food intake caused by casein

aione.

Time

0- 1 h

1-2h

W h

2-14h

O- 14h

44

Experiment 1. Table l a mect of coadministration of casein and dwazepide on food intake

#Mcan food intake (g) I SEM n=20 a distilled water ig ; methocel i.p. P ~ . ~ g p r o t d 4 d distüled water (ig.) 'Devazepide 0.25mglLg (i.p.) Meam within a row with the samt superwipt are not sigdicaniiy different by one-way ANOVA followed by pst-hm D u n c a ~ ' ~ test

Experiment l. Table lb. Summary of main and interactive effects of casein and devazepide (2-way ANOVA)

Casein m3 Casein X Drug Tirne (h) F P F P F P

0-1 29.15 <0.0001 6.29 0.02 0.42 11s. 1-2 5.03 0.03 1.12 QS. 0.0 I ILS.

0-2 1.95 as. 2.14 ILS. O. 19 as. 2-14 0.2 I 11 S. 0.75 n S. 3.07 11s.

0-14 0.5 1 IL S. 1.36 ns. 3.24 ns. ns.=non-si&~cant, p0.05, df-1,19

Eqenment 2: Wéct of devazepide on casein hydrolysate induced food intake suppression

Based on the two-way ANOVA, the main effeçt of the casein hydrolysate preload

was to lower food intake during 0-14 0-2h and 0-144 and the main effect of devazepide

was to increase food intake during 1-2h and 0-2h (Table 2b). There was a close to

statistidly significant interaction term for the 1-2h tirne period @=0.06), but casein

hydrolysate suppressed food intake in the prexnce of devazepide. For example, during

the O-2h interval, casein hydrolysate alone suppressed food intake by 1.39g compared to

control; when devazepide was present, casein hydrolysate suppressed food intake by only

0.92g. Similarly as show by the one-way ANOVA and Duncan's t-test at the 0-2h

intervai (based on the signifiant effects of h g and casein hydrolysate via the two-way

ANOVA) the net effect was when the rats were given devazepide plus casein hydrolysate

they ate less than after devazepide alone (Table 2a). However, they ate more f ier the

combined treatment than after casein hydrolysate aione. The increase however, was

primarily due to the effect of devazepide alone and not to its effect in blocking the action

of the casein hydrolysate preload. Devazepide alone resulted in similar food

consumption as the control group. Thus, devazepide did not prevent the reduction in

food htake caused by casein hydrolysate alone.

Experiment 2. Table Za. Effect of coadministration of casein hydrolysate and devazepide on food intake

f i l ~ c a n food intalrc (g) * SEM n=20 " distilled water ig.; mahocel i. p. '1.0g pmteid4ml distilleci watcr (ig.) 'Devazepide 025mgkg &p.) Means within a row with the same superscript are not si&niscatitly Merenf by one-way ANOVA followed by post-hoc Duncan's test

Expenment 2. Table 2b. Summary of main and interactive effects of casein hydrolysate and devazepide (2-way ANOVA)

Casein Hyd D W Casein Hyd X Dnig Time (h) F P F P F P

O- I 85.35 4).000 1 1.87 ILS. O. 15 as. 1-2 2.57 as. 10.10 a .0 1 3.93 0.06 0-2 5 1.87 <0.0001 16.76 4 . 0 1 1.41 a S.

2- 14 2.08 QS. 0.16 QS. 0.28 ILS.

0-14 5.88 0.02 2.02 as. O. 10 n S. n.s.=non signifiant, pX.05, d e l , 19

&perhent 3: Effect of devazepide on soy induced food intake suppression

Baseci on the tweway ANOVA the main effect of the soy preload was to lower

food intake during Ql h and 0-24 and the main effect of devazepide was to increase food

intake during 1-24 0-2h and 0-14h (Table 3b). There was no sipficant interaction seen

during any time period (pM.05) indicathg that the effkct of soy was shown whether

devazepide was present or not. For example, during 0-2h soy alone suppressed food

intake by 0.86g wmpared to the control treatment, and when devazepide was used, soy

suppressed food intake by 0.818 hdicathg that the dmg is not blocking soy induced

suppression of food intake. Similady as shown by the one-way ANOVA and Duncan's t-

test the net effect at al1 times was that &er the devazepide plus soy treatment, rats ate the

same amount as after soy alone (Table 3a). Thus, devazepide did not prevent the

reduction in food intake caused by soy alone.

Experiment 3. Table 3a Effeçt of condministration of soy and devazepide on food intake

1

#Mean food intake (g) f SEM. n=21 " distilleci water ig.; methocel i. p. @O.sg proW4ml distilled water (ig.) 'Devazepide 0.25mgkg &p.) Means within a row with the same superçaipt are not significdntfy dinerat by one-way ANOVA foll~wed by post-hoc hin~an's test

T i e

W h

Expenment 3. Table 3b. Summary of main and interactive effects of soy and devazepide (2-way ANOVA)

Controla s0g ~evazcpide' SW + Oevazepide

1.46d. 14#' O S M . 16b 1.63e. 16' 0.79f0. 14b

s OY D W Soy X Drug Tb-e (h) F P F P F P

O- 1 42.43 <0.000 1 2.43 ILS. 0 . 0 3 ILS.

1-2 0.0 1 as. 5.20 0.03 0.00 as. 0-2 2 1.0 1 4.001 9.25 4 . 0 i 0.0 1 ns. 2-14 2.5 1 n S. 2.35 ILS. 0.00 as. 0-14 O. 16 ns. 4.36 0.04 0.00 ILS.

n.s.=non significant, pN.05, df=1,20

Qxnment 4: Effkct of devazepide on soy hydrolysate inmiced food intake suppression

Based on the two-way ANOVA, the main effea of the soy hydrolysate preload

lowered food intake the entire 14h period, and the main effect of devazepide was to

increase food intake duriog 1-2h and O-2h (Table 4b). There was no signifiant

interaction seen during any t h e pend (pN.05) indicating that the effect of soy

hydrolysate was shown whether devazepide was present or not. For example, during O-

2h soy hydrolysate done suppressed food intake by 0.82g cornpareci to the comrol

treatment, and when devazepide was use& soy hydrolysate suppressed food intake by

0.99g indicating that the h g is not blocking soy hydrolysate induced suppression of

food intake. Similarly as shown by the one-way ANOVA and Duncan's t-test the net

effect was that when rats were given devazepide plus soy hydrolysate, they ate less than

after devazepide aione (Table 4a). However, they ate more &er the combined treatment

than after soy hydrolysate alone. The increase in food intake however, was prirnarily due

to the effect of devazepide done and not to its effect in blocking the action of the soy

hydrolysate preload. The devazepide group resulted in greater food intake than the

control group. Thus devazepide did not prevent the reduaion in food intake caused by

soy hy droly sat e alone.

Experiment 4. Table 4a Wect of cuadminisûation of soy hydrolysate and devazepide on food * d e

I

#Mean food iatake (g) f S E M n=22 " W e d water ig; methocel i.p. @Log pmtein/4ml distîlled wakr (ig) 'Devazepide O.ZSmg/kg (Lp.) Means within a row with the same supetscript are not significanîiy dinerent, by one-way ANOVA foliowed by post-hoc Duncan's test

Experiment 4 Table 4b. Summary of main and interactive effeas of soy hydoiysate and devazepide (2-way ANOVA)

Soy Hyd D W Soy Hyd X Drug The (h) F P F P F P

0-1 74.30 a0001 4.17 0.05 0.93 ES. 1-2 11.62 G.0 1 6.66 0.01 0.21 ILS.

0-2 36.68 <O.OOOl 13.91 a.0 1 0.28 IL S.

2-14 6.52 0.0 1 0.02 IL S. 0.28 as. 0-14 28.6 1 4).0001 3.56 0.07 0.0 1 11s.

n.s.=non signifiant, pW.05, dH,21

Ekpriment 5: mect of devazepide on casein hydrolysate induced food intake suppression when given 15 minutes prior to the protein preload

Based on the two-way ANOVA, the main eEect of the casein hydrolysate preIoad

was to lower food intake during 0-lh and 0-24 and the main e f f a of devazepide was to

increase food intake during the 0-2h tirne interval PTable 5b). There was no significant

interaction seen during any time period (pM.05) indicating that the effect of casein

hydrolysate was shown whether devazepide was presan: or not. For example, during O-

2h casein hydrolysate alone suppressed food intake by 1.15g compared to the control

group, and when devazepide was present, casein hydrolysate suppressed food intake by

1.65g indicating that the dnig is not blocking casein hydrolysate induced suppression of

food htake. Similarly as show by the one-way ANOVA and Duncan's t-test the net

effkct at the 0-2h interval was that rats ate same amounts afker either devazepide plus

casein hydrolysate veatment or casein hydrolysate alone (Table 5 a). Thus, devazep ide

did not prevent the reduction in food intake caused by casein hydrolysate alone.

Experiment 5. Table 5a EfFect of devazepide given 15 minutes prior to casein hydrolysate gavage on food intake

Time

0-lh

1-2h

0-2h

2w14h

0-14h

" W e d water ig; methocel i.p. Dl .0g proteinl4ml distüied water (tg) 'Devazpide 0.25mgkg &p.) Means within a row with the same supersQipt are not S@canîiy dinemt, by one-way ANOVA foîlowed by past-hoc Duncan's test

Experiment 5 . Table 5b. Summary of main and interactive effects of casein hydrolysate and devazepide (2-way ANOVA)

Casein Hyd Dm8 Casein Hyd X Drug T i c (h) F P F P F P

0-1 102.46 4l.0001 4.17 0.06 0.54 ns. 1-2 0.2 1 as. 2.36 ILS. 1.72 n S. 0-2 48.29 4.0001 5.17 0.03 1.77 ILS. 2- 14 0.85 ES. 0.7 1 ILS. 0.0 L ILS.

4.1.3. Discussion:

Experiments one through five showed that both the protein and devazepide

trertfments significady altered food intake. However, the combined treatment of protein

and devazepide, fded to show statistically s igni f ia interactions for casein, soy and

their hydrolysates. The importance of the interaction term determined via a 2-way

analysis of variance, was to assess if the combined treatment effkct simcantly reversed

protein induced food intake suppression by CCKA receptor blockade. Because the CCKA

receptor antagonist, increased food intake, proof of devazepide' s effect in blocking the

suppression of food intake by the nutrient resided in obtaining a significant interaction

(nutrient x h g ) between the main effects of the neatments. Examination of the

interaction between devazepide and nutrient neatment was critical to determine whether

the reversal of nutrient induced food intake suppression by CCKA receptor blockade

reflected either a causal relationship between the receptor antagonist blocking the action

of the nutrient at the same receptor site, or reflected independent, opposing effects of the

CC& receptor antagonists increasing food intake and the nuuient suppressing food

intake.

Only experiment two yielded a close to significant interaction te- whereby the

F-value was 3.93 at the 1-2h time point. This result approached statistical significance

w . 0 6 ) and therefore experimem five was conducted. Experiment five used a design

very similar to experiments one through four. However, devazepide administration was

given 15 minutes prior to the casein hydrolysate preload. The time at which devazepide

was given was moditied to examine if time of h g administration would e f k t the

outcorne on food intake suppression. This was done bccause other antagonists nich as

Losarton, an antagonist for the Angiotensin II type receptor, can excite wnnecting

neurons differentiy when administered at different t h e points (195). Therefore,

devazepide was given 15 minutes earlier than in previous experiments.

Mer conducting experiment five, no interactions between treatments at any time

points were observed. However, of interest was that devazepide given 15 minutes prior

to the casein hydrolysate preload failed to Uicrease food intake at any of the times, in

contrast to its effect in the previous four expenments. This observation suggests that time

of devazepide administration may be a factor in its effects on not only food intake, but

also the ~ppressive action of the protein preloads.

In addition to the time of administration, failure to define an effect of devazepide

on the food intake suppressive action of the protein preloads rnay have been due to the

study design. Trigazis (1,2) and Morgan (4) reported that a statistical significant

interaction between drug and protein treatment indicates that the reversal of protein

induced food intake suppression by CC& receptor blockage, was due to the receptor

antagoaist blocking the action of the protein at the receptor site. When albumin was used

as the preload an interaction has been consistently observed. It is dificult to explain why

this interaction between albumin and devazepide was easily found and reproduced. But,

as suggested f?om the results of experiment 5, time of devazepide administration may be

a factor to consider. However, by introducing time in a repeated measures design as the

third variable, to examine the effect of protein, devazepide and time would require

analysis by a threeway ANOVA This cornplex design would require many repeat

meames on the same rat and over several weeks, introciucing a great deal of variability

in food intake. Therefore, a decision was made to simplify the study design.

In Part II, a witbin subject study design in which each rat received only the

protein or pmtein plus devazepide treatment was used to determine the eEect of

devazepide when administered at three times (3960 and 90 minutes) in wnjunction with

a protein preload a! 30 minutes prior to introduction of the food cup. To examine the

effkct of devazepide alone on food intake at the feeding times of Wh, 1-2h and 2-3h,

devazepide was given at 30,60 and 90 minutes prior to food cup introduction.

4.2. Part II - The effect of time of administrati . 'on of devazepide on the

feeding response to proteins and their hydrolysates

4.2.1. Introduction

The objective of this series of experiments was to examine the role of tirne of

devazepide administration on albumin, albumin hydrolysate, casein, casein hydrolysate,

soy and soy hydrolysate induced food M e suppression.

The objective of the fint experiment was to examine the effect of devazepide

alone on food intake when given 30, 60 and 90 minutes prior to food cup introduction at

the feeding intervals of 0-1 h, 1-2h and 2-3h. Experiments two through seven were

designeci to determine the effect of devazepide when administered 90,60 and 30 minutes

before food cup presentation and in conjunction with a protein preload 30 minutes pnor

to food cup introduction.

4.2.2. Results

Experiment 1: Effect of devazepide when given done 30,60 and 90 minutes prior to food cup introduction.

Devuepide did not signincantly increase food intake during any of the one-hour

intemals of 0-lh, 1-2h and 2-3h when given at 30, 60 and 90 minutes prior to food cup

introduction (Table 6). Statistical signîficance was obtained for the increase in

cumulative food intake fiom 0-2h and 0-34 but only when devazepide was given 60

minutes prior to food cup presentation (Table 5).

Experiment 1. Table 6. Administration of devazepide cup introduction

30,60 and 90 minutes pnor to food

Treatmcnt 1.38M.24 1.32130.22 1.06f0.24

Coatrol 1.00f0.2 1 0.88I0.22 1 . 1 0 . 1 4

MDS 0.37H.28 0.43M.22 -0.04f0.32

Tr eatment 2.19I0.26 1.75I0.18 1.76I0.39

2Jh ContxoI 1.97I0.27 1.85H. 18 1.70I0.29

MDS 0.2 1f0.4 1 4 . lW.26 0.06f0.47

O-2h Control 2.97M.36 2.50I0.28 3.20I0.33

MDS 0.4 lH.36 **0.7M.22 0.36f0.42

03 h Control 4.95f0.44 4.36f0.3 1 4.90f0.42

MDS 0.62f0.48 *0.65a.29 0.42f0.5 1

Mean food intake OfSEM; n=l4 ' m i & 0.ZSgkg @p.) f 25% Methocel (ip.) %îD&~ean Dinérence Score (Trcarmem-Control) m.05, *Sp<0.01

&miment 2. The &ect of devazepide on albumin induced food intake suppression when adminis td 3 9 6 0 and 90 minutes pnor to food cup #on

Devazepide administaed 90 minutes prior to food aip introduction and 60

minutes before the albumin preload signüicamly increased food intake during 0-1 4 O-2h

a d 0-34 compared with the e f f ' of the albumin preload alone (Table 7). When

administered 60 minutes pnor to food aip intduction (and 30 minutes pnor to the

albumin preload) devazepide increased food intake in the feeding interval of 0-lh, 1-2h,

Q2h and 0-3h as compared with the albumin preload Fable 7). Devazepide given with

the albumin preload 30 minutes prior to food cup presentation significantly increased

food intake at times 1-24 0-2h and 0-3h as compared to cuntrol (Table 7).

Experiment 2. Table 7. The effect of devazqide suppression when given 30,60 and 90 minutes

on albumin induced food intake prior to food aip introduction

Trcatmtnt 1 ./3Al.28 1.53-tO. 19 1.5W.22

Contr~l 0.4M. 1 5 0 .7M. 18 1.33I0.15

MDS *10.9M.22 *0.77I0.25 0.2W.29

Tmtment 1.2W. 14 1.53M.20 1.6W. 18

Controt 1.3W.20 1.63U.20 1.39I0.22

MDS 4.04M.27 -0.10I0.3 1 0.29I0.36

T~~ 3.67M.34 4.67I0.26 4.97M.28

Conml 2.4W.34 3.lW.33 3.74M.29

MDS ** 1.1W.30 **1.4M.24 1.23a.52

Mean faod intakt OfSEM; n= 14 'Devazepide 0.25g/kg (LppAlbumin (I.Og/Jml) %.ZN Methoal (ip.)tAibumin ' M D s = M ~ ~ ~ Mcrmce Score flreamient-Coonol) tp4).05, *-.O 1

Eiperiment 3. The &kt of devanpide on albumin hydrolysate induced food intake suppression when administaed 30,m and 90 minutes prior to food cup presentaîion

When devazqide was administered 90 minutes pnor to food cup introduction and

60 minutes before the albumin hydrolysate prdoaû, w significant &ects on food intake

wmpared with the albumin hydrolysate preload alone was observe. at any of the feeding

intervals (Table 8). When administered 60 minutes prior to food cup introduction and 30

minutes before the protein preload, devazepide significantly increased food intake during

1-2h, 0-2h and 0-3h as compared with the albumin hydrolysate preload (Table 8).

Devazepide given in conjunction with the aibumin hydrolysate preload 30 minutes prior

to food cup presentation, significantly increased food intake oniy fiom 0-3 h as compared

to control (Table 8).

Experiment 3. Table 8. The effect of devazepide on albumin hydrolysate induced food intake suppression when given 30, 60 and 90 minutes prior to food cup introduction

Treatment 1.1W.25 1.33îû.25 1.3M. 19

Conml 1.19M. 19 0.64I0.16 1.07I0.19

MDS 4.0 1S.24 YL68I0.27 0.3 1M.27

T R % ~ I E ~ ~ 1.6W.27 1.37Io. 18 1.53M. 12

Cantrof 1.0W.13 1.2 1M.2 1 L.38s0.12

MDS 0.56f0.26 0.1W.28 O. 1 4 a . 16

Treatmeat 1.7M.27 1.56a0.26 1 S W . 2 1

Control 1.53I0.22 0.79I0.17 1.33I0.23

MDS O. 1W.27 *0.7&t0.27 0.23I0.3 1

Treamnt 3.3at0.08 2.93M.23 3 . 1 W . 18

Control 2.6M. 19 2.0W.25 2.71I0.24

MDS *0.73a.26 **0.93f0.18 0.38IQ.26

fur- food intalie (g)fSU: n=14 Daazepide 0.2Sg/kg (i.p.)+AIbumin Hydrolysate (1 .OgMml)

'0.25% Mabavl (ip.)+Albumin Hydrolysatc m S = ~ e a n Différence Score (Tre&n~-Coatro1) *p4).05, *+p4).01

Iikperiment 4. The &ect of dwazepide on cssein induced food imake suppression when administered 30.60 and 90 minutes prior to fwd cup presentation

Devazepide administered 90 minuta prior to food cup inaoduction and 60

minutes before the casein preload, sipnificantly increased food at times 0-lh and 0-2h

cumpared with the effect of the casein preload alone (Table 9). When administered 60

minutes prior to food cup introduction (and 30 minutes prior to the protein preload),

devuepide significant1y increased food intake during the intervals of 0-1 h, 0-2h and 0-3 h

as ~ m p a r e d with the casein preload (Table 9). Devazepide given in conjundon with the

preload 30 minutes prior to food cup presenîation simcady increased food i d e at

times 0-14 1-24 0-2h and 0-3h as compared to control (Table 9).

Experiment 4. Table 9. The effkct of derazepide on casein induced food intake suppression when given 30,60 and 90 minutes prior to fwd aip introduction

Treatment 1.24I0.27 1.65I0.25 L.33IO. 19

2-3h Control 1.4W.2 1 1.3W. 16 l.51I0.13

MDS -0.24I0.38 0.34I0.27 4.17I0.26

Treatment 2.6W.33 2.JW.20 2.54M.20

O-2h Conml 1.NM.28 1.56a0.27 1.83Ml.22

MDS ** 1.2M.26 ** 0.9M.20 **0.7 l a . 19

Treaîment 3.93M.32 J.14a.3 1 3.8W.27

0-3 h Control 2.8W.3 1 2.87I0.32 3.34a.27

MDS ** 1.0Sû.29 ** 1.27I0.34 0.53M.35

Mean food intake (g)SEM; n= 14 ' Devazepide 0.25gkg (i.p.)+Casein (O.Sgl4ml) %.ZN M e t h d (ip.)+Caseh %DS=~ean Merence Score flreatxnentCon00l) w . 0 5 , * ~ 0 . 0 1

m e n t 5. The a e c t of devazepide on casein hydrolysate induced food intake suppression when administered 3 9 60 and 90 minutes pnor to food cup presentation

Devazepide admiaisterd 90 minutes pnor to food cup introduction (and 60

minutes before the casein hydrolysate preload), significantly increased food intake at

times 0-14 0-2h and 0-3h compared with the enkt of the casein hydrolysate preload

alone (Table 10). When adminiaered 60 minutes pnor to food cup introduction and 30

minutes prior to the protein preload, devazepide significantly increased food intake at

tirne 1-24 as compared with the casein hydrolysate preload (Table 10). When given with

the protein preload 30 minutes pnor to food cup presentation, devazepide significantly

increased food intake only from 1-2h as compared to control (Table 10).

Experiment 5 . TablelO. The &kct of d e v i d e on casein hydrolysate induced food intake suppression when given 30,60 and 90 mimites prior to food cup introduction

Treamicnt 1.ûtW.29 1.83M. 16 1.4M. 19

ConÛol 1.27I0.23 1.11I0.23 0.89I0.19

MDS L0.6OM.22 **0.7W.21 0.53I0.29

Treaîmenî 1.50.29 0.93M. 19 1.61I0.21

Conîrol 1.77M.28 1.6ûM.27 1.64I0.23

MDS -0.2W.47 4 . W . 3 8 4.03I0.26

Treatment 4.02M.46 3.1M.39 3.62Al.34

ControI 3.8W.34 3 . 5 W . 3 3 2.6JM.30

MDS 0.22I0.45 4.03I0.50 **0.9W. 18

Mean food intake n= 14 'Demapide O .Z@g (i.p.)+Casein Hydrolysate (l.Ogl4ml) '0.25% Mahoal (ip.)+Cascin Hydrolysate %DS=~ean Dinania Score (Treatment-ContmI) v . 0 5 , @-.O 1

Expriment 6 The &ect ofdevazepide on soy induced food intake suppression when administered 30,60 and 90 minutes pnor to food aip pce~entation

Devazepide administered 90 minutes prior to food cup introduction and 60

minutes before the soy preload, significantly increased food intaLe during 1-24 0-2h and

0-3h campard with the effect of the soy preload alone (Table 11). When administered

60 minutes prior to food cup introduction and 30 minutes prior to the protein preload,

devaepide significantly increased food intake at times 2-3h and 0-3h as compared to the

soy preload (Table 11). Devazepide given in conjunction with the soy preload 30

minutes pnor to food cup presentation significantly increased food intake in the feeding

intervais of 0-lh and 0-2h as compared to control (Table 1 1).

Expairnent 6. Table 1 1. The Hkct of devazepide on soy induced food intake suppression when given 3Q 60 and 90 minutes prior to food cup introduction

Treatment 1.5W.21 1.0M.20 1.50I0.21

Control 1.53I0.23 1.lW.26 0.9 lI0.22

MDS O.OW.20 4.1oI0.32 *0.5M.27

Treabnent 1.69I0.33 2.2M.21 1.77a.22

ConÛol 1.9M.29 1.3 7I0.24 1.74M. 17

MDS -0.22iO.39 e0.8510.33 0.03kû.30

Treatment 2.70I0.27 1.7M.20 2 .1M.28

Control 2.2W.30 1.83a.32 1.3M.26

MDS **0.5W. 13 4.1W.25 *O. 87M.29

Treatment J . W . 2 6 3.9Sa.25 3.97M.25

Conîrol 4.1W.29 3.20I0.25 3.Mddl.23

MDS 0.27M.33 **0.7M.23 *0.9W.3 1

Mean f d inidce (g)ISEM; n=14 'Devazepide O.Zcig/kg (i.p.)+Sûy (OSg/4ml) %.u% Methocel (Lp.)+SOy % D ~ = ~ e a n Differenœ Score (Treatmetit-Conaol) m . 0 5 , *+p4).01

&wfnzinent 7. The &ed of devazepide on soy hydrolysate induced food intake suppression when administered 3460 and 90 minutes prior to food nip presentation

Devazepide administered 90 minutes prior to food cup presentation and 60

minutes before the soy hydrolysate preload, significantly incruised food intake during 2-

34 0-2h and 0-34 compared with the effea of the soy hydrolysate preload alone (Table

12). Devazepide administraton at 60 minutes (30 minutes before the protein preload) or

30 minutes (given with the protein preload) prior to food cup introduction showed no

signifiant increases compared to control at any food intake measurement time points

(Table 12).

Experiment 7. Table 12. The effect of devazepide on soy hydrolysate induced food intake suppression when given 3460 and 90 minutes prior to food cup introduction

Treatment 1.6M.25 L.lSû.23 1 S7M.22

10% Control 1.42M.20 1.23I0.28 1.08I0.20

MDS 0.2W.22 -O. 1W. 4 1 0.4W.24

Treatmenî O.W.22 1.71I0.33 1.65iû.2 1

2-3h Conml 1.33M.29 1.33M.24 0 . 9 M . 2 2

MDS -0.43I0.28 0.37M.46 *0.7W.27

Treatmem 2.53H.22 2.13M. 14 2.07M.23

O-2h Control 2.0M.20 1.9W.24 l . W . 2 3

MDS O.UM.2 i 0.1W.25 **0.47*. 15

MDS 0.0 110.30 0.52I0.42 **1.17I0.32

Mean food intake @)SEM; n= 14 'Devazepide O.ZSglLg (i.p.)+%y Hydrolysitc (1 .Og/4ml) %.s% Methocel (ip.)+Soy Hydrolysate %lDS=~ean Diffhna Score (Trtaîment-Control) w . 0 5 , *Lp<O.Ol

4.2.3. Discussion

The experiments coaduaed in Part II introduced time of devazepide

administration as being mother factor to consider when investigating its effect on protein

induced food intake suppression. Albumin, casein, soy and their respective hydrolysates

were used to examine the role of time of devazepide administration in protein induced

food intake suppression via CCKA recepton. The results show that ail proteins exarnined

suppressed food intake via CC& receptors, but the response was dependent on the time

at which devazepide was adrninistered.

Prior to conductlng experiments two through seven., experiment one was

performed to determine if the dose of devazepide used would significantly inaease food

intake. This was important to establish because devazepide is known to increase food

intake beyond control in rats. Idedly, the dose selected for the purpose of these

experiments should block CCKA receptors, but not significantiy increase food intake.

This is because an increase in food intake when devazepide is given with protein,

compared with the effect of protein alone, should reflect the interaction between protein

and the CCKA receptor and not the effkct of devazepide independent of protein.

The results from experiment one showed that devazepide (0.25mg/kg of rat body

weight) given at 30, 60 and 90 minutes prior to food cup introduction, did not

significantly increase food intake at any of the individual feeding intervals of 0- 1 h, 1-2h

and 2-3 h (Table 6). This data is important for experiments two through seven, because it

shows that devazepide blocked CC& receptors but did not significantly increase food

intake during each of the hourly feeding intervals. A statistically significant increase in

food intake was shown for the cumulative data of 0-2h and 0-3h reflecting the

accumulation of non-signifiant increases in food intake at each of the hourly time

intervals.

Albumin, casein, soy and their hydrolysates nippressed food intake via CCKA

receptors. However, the response was dependent on the time of devazepide

administration, suggesting that the time at which the peptide produas of digestion

interact with the CC& receptor varies with the protein source (Table 13, Fig.6).

To describe more fùlly the interaction of the treatments with the CC& receptor,

the total time of devazepide exposure was caimiated Fable 13). The total duration of

tirne the rats were exposed to devazepide was determined by adding the midpoint of each

hour of measurement of food intake to the time at which devazepide was injected. For

example, total exposure to devazepide included the time at which the CCK* receptor

antagonist was administered (e.g. 30 minutes pnor to food cup introduction) plus 30

minutes for the first hour of the food intake measurement, giving a total exposure time of

60 minutes (Table 13). Because of the uncertainty associated with when rats ate within

the hourly measurement, an assumption was made that rats ate at the rnid point of every

hom.

The effect of the proteia source on food intake depended on the duration of

interaction between each protein source and devazepide (Table 13). Although, the

assumed eating time was taken at the midpoint of each hourly meanirement, for the

calculation it must be recognized that the specific time that rats eat within the hour is

unknown. Therefore, to Mly represent the possible duration of the interaction between

protein and the CC& receptor, an extra 30 minutes was added to both end of the ranges

over which there appear to be an interaction between devazepide and the protein. As

shown in Figure 6, albumin intenias with the C a A receptor between 60-180 minutes to

reduce food intake. Albumin hydrolysate suppresses food imake through interacting with

the receptor between 120-1 80 minutes. Similarly casein suppresses food intake between

30- 1 50 minutes, while casein hydrolysate suppresses food intake f?om 90- 1 80 minutes.

The interaction of soy with the CC& receptor appears to be bmeen 30-240 minutes but

as shown in table 13, it is not as clearly continuous as for the other proteins. Soy

hydrolysate suppresses food intake by interacting with CC& receptors between 2 1 0-270

minutes.

Intact proteins suppressed food intake earlier than their hydrolysates (Fig.6). This

is hard to explain because hydroly sates are fragments of intact proteins which require less

digestion and therefore, theoretically, should induce satiety earlier. Xntaa albumin, casein

and soy showed variances in the duration of interaction with the CCKA receptor. But,

there may be some uncertainty in this observation because different protein

concentrations were in the protein preloads. The albumin preload (1.0g) was larger than

the casein and soy preloads (OSg) because of their low solubility. It is therefore possible

that the quantities of protein preloads determined the duration of the interaction between

protein and CC& receptor. Therefore, Part III examined the effect of quantity of protein

on the duration of interaction between albumin and the CCKaïeceptûr.

- -

Treatment Assumed Duration Time Eating of Devaz.

Time Ex~osure

- - - - - - - - -

Range of Effectiveness

Alb Albhyd Cas Cashyd SOY Soyhyd

Table 13. Range of interaction between protein and the CCKA receptor.

*Significantly different compared to control food intake values at given time treatments. Assumed eating time is taken at the mid point, but the range is O-6Ornin, 60- 120min, and 120-1 80min for the O- 1 h, 1 -2h and 2-3h feeding intervals respectively.

Tirne (min) after devazepide administration

Fig. 6. Duration of time over which proiein intetacts with the CCKA recepior.

4.3. Part III - To ewmine the effect of quantity of protein and tirne of devazepide administration on the suppressioood intake.

4.3.1. Introduction

The objective of the expniments in Part III was to examine the effect of protein

quantity on the duration of protein interaction with the CC& receptor. Albumia was the

test protein because it is readily soluble in water at both O. 5g/41d (experiment one) and

1.0g/4d (experiment two).

4.3.2. Results

Exprimeni 1. The effect of O.5g/4mi dose of albumin preload and time of devazepide administration on food intake suppression.

Devazepide administered 90 minutes pnor to food cup introduction and 60

minutes before the albumin preload, significantly increased food intake during, 0-2h and

0-3h compared with the effea of the albumin preload alone (Table 14). When

administered 60 minutes pnor to food cup introduction and 30 minutes pnor to the

protein preload, devazepide sipnificantly hcreased food intake at times 0-lh, 0-2h and

0-3h as compared to the albumin preload (Table 14). Devazepide given in conjunction

with the albumin preload 30 minutes pnor to food cup presentation significantly

increased food intake in the feeding intervals of 0-III, 2-3h, 0-2h and 0-3as cornpared to

control (Table 14).

Expriment 1. Table 14. The effect of devazepide on albumin (0.5d4ml) induced food intake suppression when given 3960 and 90 mioutes pnor to food cup introduction

Treatment 0.96I0.0.25 1.20I0.32 I.4M.30

Coatrol 0.57I0.20 0.8SkO.26 1.33I0.25

MDS 0.3W.2 1 0.33I0.22 O. IM.37

Treaûnenî 2.07dd.23 2.2W.22 1 -74st0.36

Control 1.2OM.23 1.75I0.26 1.74M.29

MDS *0.87ddl.3 1 0 .4M.3 1 O.ûWû.36

Treatment 2.87I0.27 3. t M . 4 2 3 S4M.26

Control 1.90I0.3 1 2.33M.42 2.9210.27

M D S **O.-.24 **0.79iû.25 W.62M.24

Treatment 494M.32 5.37M.47 5.2W.47

Control 3.1W.41 4.09I0.47 5.66I0.4 1

MDS ** 1.8M.26 ** 1.28I0.28 *0.62f0.24

Mean food intake ( g ) a n= 14 '~~r;tzepide 0.25gkg (i.p.)+Afbwnin (0.5gl4rni) %.u% Methoce1 (ip.)+Aibumin %DS=~ean Différence Score f l r c a t m d o m r ~ l ) w . 0 5 , a w . 0 1

Erpement 2. The &ect of 1 .Og/4mî dose of albumin preload ~5 time of devazepide administration on food intake suppression.

Devazepide administered 90 minutes prior to food aip introduction and 60

minutes before the albumin preload, s i g n i f i d y increaseâ food intake during, 0-lh,

O-2h and 0-3h wmpared with the effkct of the albumin preload alone (Table 15). When

admllüstered 60 minutes prior to food cup introduction and 30 minutes prior to the

protein preload, devazepide signincantly increased food Uitake at times 1-Zh, 0-2h and

0-3h as compared to the albumin preload (Table 15). Devazepide given in conjunction

with the aibumin preload 30 minutes prior to food cup presentation significantfy

increased food intake in the feeding intervals of 0-lh, 1-2h, O-2h and 0-3as compared to

control (Table 15).

Experiment 2. Table 15. The effect of devazepide on aibumin (1.0g/4d) induced food intake suppression when given 30,60 and 90 minutes prior to food cup introduction

MDS **O. 83f0.24 ** 1.10f0.29 0.20I0.29

Mean food intake (g)m n=l4 ' ~ e ~ z c p i d e 0.250g (i.p.)hUbumin (l.Og14ml) 20.25% M e t h d (ip.)+Aibumin % l D ~ = ~ e a n Dinerence Score (Treaimenî-Control) w . 0 5 , *Sp<O.Ol

4.3.3. Discussion

The r d t s of Part III show that the quantity of albumin given to rats affects the

strength and the duration of i n t d o n between protein source and the CC& receptor.

Aibumin served as the test protein because it is readily soluble in water at both 1.0g and

0.5g amounts as cumpared to casein and soy protein The duration of interaction of 30-

180 minutes between the 1.0g/4ml dose of albumin and devazepide were somewhat

longer than that observation in experirnent two of Part iI, probabIy because of the

variability associated between different sets of rats. However, when aibumin was given

at 0.5gl4ml the interaction started 30 minutes &er devazepide was exposed to rats,

Ming continuously until 120 minutes, followed by a half hour penod where no

interaction was found and then followed by another hour of interaction (Table 16, Fig. 7).

Thus protein quantity effects the duration of interaction between protein source and the

CCKA receptor, but more importantly the degree of food intake suppression caused by the

aibumin load of 1.0g vernis 0.5g. There is a more robust (stronger natisticai

significance) in the reversal of food intake suppression by devazepide when given in

conjunction with a higher dose of protein.

Furthemore, the results in Part III show a decreasing trend in MDS in food intake

at the 1-2h, 2-3 h and 0-3h time intervals with the 0.5g of albumin (Table 14) as compared

to 1.0g (Table 15). The decrease in food intake at the OSg level is more pronounced

when devazepide is given 90 and 60 minutes before the presentation of the food cups.

This decreasing effect observed at these time with the 0.5g/4ml dose of albumin as

compared with the 1.0g/4ml dose, firstly suggests that the anorexic effect of protein is

modified depending on the quanti@ of given and this confms a report done by Anderson

et ai., (5). And secondy, that the degree of food intake suppression is greater and longer

lasting with a greater dose of protein.

Because the quantity of albumin given affkts both the strength and duration of

the interaction between protein and devazepide and thus food htake suppression, this

would suggest that quantity of protein mediates the Iength and the degree of the

suppressive respoase. The observed differences between experiments 1 and II can be

attributed to the prolonged presence of the protein at the level of the gut. The digestive

enzyrnatic reaction at the level of the gut required to breakdown protein to its active form

leading to the release of CCK and thus suppressing food intake, is prolonged and

sustained with inmeasing quantities of protein. Because more time is required (due to the

pater quantity of protein) to complete the above reaction, this results in producing more

degradative products like the active peptide, and therefore increases and prolongs the

peptides chances in stimulating the release of CCK in allowing interaction with CC&

recepton to continue and m e r increase the degree of food intake suppression.

It has recently been show that the amount of CCK released upon stimulation of 1-cells

can impact the degree of food intake suppression. Higher doses of CCK have the ability

to inhibit gastnc emptying and funaion via vagd afferent fibers as compared with low

doses which seem to only function through vagd afferent CCKA receptors to suppress

food intake (187). This suggests that higher doses of CCK suppress food intake to a

greater extent probably because of the combined effects of inhibiting gastric emptying

and stimulating CCKA teceptors. It is yet to be determined, but if 1.0g of albumin c m

stimulate the release of more CCK than OSg, then the more robust suppression in food

i d e associated with 1 g as compared with 0.5g may be ünked to the greater release of

CCK

Treatment Assumed Duration of Time Eating Time Devaz.

Albumin Albumin (0.5d4rnl) (1 .Od4ml)

Table 16. Range of interaction between albumin and the CCKA receptor.

* * 4 - - *

Exposiire

Wgnificantly different compared to control food intake values et yiven tirne treatmenis. Assumed eating time is taken at the niid point, but the r a i i p is O-60min, 60- IZOmin, and 120-180min for the 0-1 h, 1-2h and 2-3h feeding intervals respective) y.

4 - - -

. - - 30- 120, 30-180 150-2 10

I L

60 90 120

FIO-l 30 FIO-1 60 FIO-1 90

FI2-3 30 FI2-3 60 Fi2-3 90

30min 30min 30min

Range of Effectiveness

15Omin l5Omin l5Omin

180 210 240

CHAPTER 5. GENERAL DISCUSSION

5.1 General Discussion

The results of this research support the hypothesis that in the rat, the effect of

dietary proteins on food intake is dependent on the time and duration of interaction

between protein source and CC& receptors. Aibumin, casein, soy and their respective

hydrolysates aii suppressed food intake via CC& receptors. But the respoase was

dependent on the time at which devazepide was administered, suggesting that the time at

which the peptide products of digestion interac~ with CC& receptors varies with the

protein source.

The hypothesis of this thesis was tested by adrninistering devazepide at three

times (30, 60 and 90 minutes) in conjunction with a protein preload given 30 minutes

pnor to the introduction of the food cup. A within studies design was used to obtain the

data and a Student's paired t-test was used to analyze it. In these studies it is concluded

that the increase in food intake after devazepide was given with the protein preload could

be atîributed to a reversal of protein induced food intake suppression by CC& receptor

blockade. To ensure that this increase in food intake was not due to the effect of

devazepide aione, expenment 1 of Part Il was conducted. It showed that the dose of

devazepide of 0.25mg/kg when administered to rats at 30, 60 and 90 minutes pnor to

food cup introduction did not significantly increase food intake compared to control

during the 0-14 1-2h and 2-3h intervals, but did during the cumulative times of 0-2h and

0-3h, but only when devazepide was injected 60 minutes before food cup presentation.

The results of expenment one of Part 11 are consistent with those of Part 1. The same

dose of devazepide was not found to increase food intake over the 04 h and 1-2h intervais

when administered alone 30 minutes prior to food cup introduction. Thus, the eff- of

the dmg on increasing food intake above control in experirnents 2-7 of Part II at the 0- lb,

1-2h and 2-3h intervals when devatepide was administered at 30,60 and 90 minutes prior

to food aip introduction in conjunction with a protein preioad, was primarily due to the

reversai of protein induced food intake suppression

A weakxess of the design in Part II was that the time of devazepide administration

was not randomized to control for an order effect. The rats were exposed to devazepide

twice in random order in Part I but Part II had received two injection (at 90 and 60

minutes) prior to the 30 minute test treatment. However, the difference in food intake

between the protein treatment and the treatment consisting of protein given with

devazepide 30 minutes pnor to food cup presentation in Part II was similar to that

observeci between these two treatments in Part 1. For exampie, the difference produced in

food intake by the treatments of casein hydrol y sate+devazepide and casein hydrolysate

alone was 0.60g in Part II (Table 10) and 0 . 7 7 ~ in Part I (Table 2a) at the 1-2h interval.

Similarly, the difference in food intake by treatments of soy+devazepide and soy aione

was 0.50g in Part II (Table 1 1) and 0.5 1g in Part 1 (Table 3a) at the 0-2h interval. Thus,

the f'lure to randomize the order of treatment time in Part II does not appear to have had

any systematic influence on the treatment effect.

The experirnents that led to the hypothesis of this thesis were derived fiom the

work doue by a former graduate student of this lab. Trigazis (1) first described a

relationship among albumin, CC& recepton and food intake (Fig. 8). Specifically, he

found that devazepide reversed the suppression in food inrake caused by albumin, but not

by glucose or corn oil (1). The results of the present study provide evidence that many

proteins sources including casein, casein hydrolysate, soy, soy hydrolysate and albumin

Digestion (Time)

ProteidProtein Hydrolysa te

(source, quant it y)

Active Peptides (AA sequence; structure)

Food Intake

Fig. 8. Relationship among dietary proteins, CCKA receptors and food intake.

hydrolysate also mediate their satiq response via CCKA receptors. In addition, the

present research shows that both protein quantity and source influences the duration of

interaction between protein digestion products, presumably containhg the active peptide

sequence, and the CC& receptor (Fig. 8).

To explain the duration of time required for interaction of the protein source with

the CCKA receptor it would be logical to assume that the interaction is dependent upon

the rate of protein digestion. Thus the more time required to produce the peptide product

of digestion releasing CCK, the greater the delay before food intake is suppressed. This

does not seem to be the simple explanation because casein which is a more slowly

digested protein than albumin, begins to suppress food intake 30 minutes earlier.

Possibly active peptides are released earlier in the process of digestion of casein. Opioid

peptides are readily released fiom wein during digestion. They slow down

gastrointestinal motility, thus delaying gastric emptying by interacting directly wit h

opiate receptors in the gut (188). It may be that this delay in gastnc emptying provides

greater opportunity for digestive processes to produce the specific glycosylated peptide

hgment believed to release CCK and afExt food intake suppression via CC& receptors

(121). Aiso, it may be that only a few fragments of the specific glycosyiated peptide of

intact casein are required to release CCK as compared perhaps with a greater portion of

digested albumin products that may be needed. Perhaps before a suppressive response is

observeci with albumin, a build up of the active peptide may be required.

The active peptide believed to be a produa of protein digestion capable of

stimulating the release of CCK (Fig. 8). It is uncertain to the fodstnicture of the active

peptide, but it is thought to be an amino acid sequence or a protein of specifk structure.

nie minimum amino acid sequence order of any CCK type required to stimulate CC&

recepton is Phe, Asp, Met and Trp (1 39,140). If the process of protein digestion can

produce a peptide containing the minimum amino acid sequence that stimulates CCKA

receptor activity, then this would provide some evidmce that the active peptide may be of

primary structure. However, ligaad/receptor binding commonly requires the ligand to be

of some distinct form. A specific giycoysaited peptide of casein, produced by

fhctionating the protein, was the ody peptide shown to greatiy stimulate CC& receptor

activity (12 1).

Because peptides of distinct structure or of long chain arnino acids rarely cross

ce11 membranes, this would suggest that the active peptide is a using a mechanism that

does not require direct binding to the CC& receptor. Possibly the active peptides lead to

CCK release by interacting with I-cells that Line the intestinal wall. CCK once released is

capable of binding to vagal CC& recepton that surround the gut. One of the many

observed responses related to peripheml CCW CC& receptor binding is a suppression in

food intake.

The duration of interaction between the protein source and devazepide blockage

of the CC& receptor is dependent on the protein source. Soy when cornpared with the

other proteins interacts the longest with CC& receptor. This prolonged interaction

between soy and the CC& receptor rnay be attributed to the degree of CCK release.

CCK release is influenced by types and foms of protein and their ability to act as a

substrate for protease degradation (1 28,IZ2,l89,lgO). The greater the atfinity of the

protein as a substrate for pancreatic proteases, the less likely the proteases will amck the

mechanism which l a d s to the release of CCK in the gut epithelium Certain dietary

proteins rnay act as cornpetitive substrates for pancreaîic enzymes in the small intestine in

place of CCK releasing peptides. If these enzyme, like trypsia are occupied in degrading

dietary protein at the expense of CCK releasing peptides, then the CCK releasing

peptides do not get broken down to the same extent and thus are free to stimulate the

release of CCK fkom intmluminal 1 cells (1 19,128,191). Thus, variations in protein

indu& food intake suppression may be due to variations in its utilkation of the protease-

negative feedback mechanism controlling CCK release.

It is possible that the prolonged effect of soy on reducing food intake may be in

part due to its intrinsic ability to inhibit the action of trypsin. The soy protein isolate used

in Parts 1 and II containeci 4.9-7.3 mg of trypsin inhibitor for every gram of protein. If

trypsin is inactive then its ability to degrade CCK releasing peptides will be impaired,

leading to an iacreased stimulation and suaahed release of CCK. However, it is

unknown if the processing procedures used by the m a n u f ~ e to produce soy protein

isolates used in this study have rendered the tryspin inhibitor component of soy protein

inactive.

The dose of protein given determines the strength and duration of interaction

between source and the CC& receptor. Expenments one and two of Part III, using

albumin at 0.5g and 1.0g as the test protein (because albumin is readily soluble in water

at both quantities), showed that protein quantity effects the degree of food intake

suppression confirming a previous report by Anderson et al., (5). In addition the quantity

of protein mediates the length and degree of the suppressive response (Part a5). This

wodd be expected based on the t h e required for the digestive enzymes to breakdown

protein to its active fom l d i n g to the release of CCK Because more time is needed to

amplete digestion if the quantity is greater, more of the active peptide would be released

over a prolonged the .

The importance of quaithy of proteh is supporteci by the observation that the

amount of CCK released upon stimulation of 1-cells ain impact the degree of food intake

suppression (187). High doses of CCK suppress food intake by inhibiting gastrk

emptying and fùnctioning via the vagus. However, low doses seem to ody hvolve vagal

CCKA receptor in suppressing food intake. Possibly the larger quantity of protein

suppresses food intake to a greater extent because of the combined effects of delayed

gastric emptying and stimulation of CC& receptors.

The protein hydrolysates generaily suppressed food intake later than their

respective intact proteins. This is hard to explain because hydrolysates are fragments of

intact proteins which require less digestion and therefore theorectically should induce

satiety earlier. The hydrolysates were stated by the supplier to be composed primady of

amino acids a d o r small peptide fragments. For example the albumin hydrolysate (Table

3.2) was claimed by the manufacturer to be 100% ffee amino acids. However, a fiee

amino acid mixture formulated after albumin protein has no eRect on food intake

suppression via CCKA receptors (2,192). Therefore, the interaction between the protein

hydrolysate and CCKA receptor mst be explaineci by the presence of peptides. It is

ditficult to determine the amount of peptide present without analyzing each preparation,

but the manufacture has acknowledged the uncertainty of the composition of these

produas (Cochi, persunal comment).

The delay in interaction between the protein hydrolysates and devazepide

blockage of the CCKA receptor, may have been due to the lack of active peptides present

in the protein hydrolysate mixture. If the composition of the hydrolysates reporteci by the

m8Illlfactufes was accurate and albumin hydrolysate did contain approxïrnately 100%

amino acidq then the interaction between hydrolysates and CC& receptors rnay be an

indication of a central response. It is hown that Eee amino acids and di and tri peptides

are rapidly absorbed into the blood via the s d l intestine without digestive processing

(193). Because amino acids are readily absorbed, their exposure tirne in the smdl

intestine is very limited, thus minimally affecthg the degree of CCK release in the

periphery (1). It is also known that proceeding the absorption of amino acids or small

peptide fi-agments, fluctuations in whoie brain amino acids are obsewed after &y

miriutes and beyond (5) and does effect food intake consumption. The associated time

delay with albumin hydrolysate food intake suppression rnay be reflective of whole brain

arnino acids interacting with devazepide centdy, thus leading to a satiety response

M e r in time than its native protein.

Protein form rnay be key to the length of the ~ppressive response. The

concentration of soy hydrolysate given (0.62d4ml) was close to the concentration of soy

protein given (0.5g/4ml). Devazepide revened the suppression of food intake casued by

both foms of soy protein by close to equal amounts, but the interaction between

devazepide and protein induced food intake suppression was much more prolonged for

the soy protein isolate than for the soy hydrolysate. Dependhg on the quantity of fiee

versus peptide bound amino acids in the soy hydrolysate mixture, the amount of the

active peptide aven to rats was perhaps small. It is difficult to explain the effect, but

possibly the limited amount of active peptide present in the preload delayed the

stimulation of CCKA receptors. Although the soy hydrolysate mixture containeci 29%

carbohydrate, it is unükely that this amunteci for the decrease in food i d e and the

reversai observed with devazepide, because devazepide does not reverse carbohydrate

induced f d intake suppression (1).

It seems likely that CCK is released by proteins as a result of peripheral, rather

than centrai CCKA receptor sites. CCKA recepton are found predominantly in the

periphery and that CC& receptor c o n s t i ~ e the major portion of CCK receptors in the

brain (133,144,145). Morgan (4) showed using a potent CC& receptor antagonist that

albumins' satiating effect was not blocked. Because the CC& receptor antagonist failed

to block aibumin induced satiety, whereas the CCK A receptor antagonist did, it appears

that dbumin acts penpherally to regulate food intake. Funhermore, Trigazis (1) showed

that albumin induced food intake suppression was blocked by PD-140,158, a CCKA

receptor antagonist that functions ody in the periphery. It has been mggested that

devazepide possess the ability to cross the blood-brain-barrier and therefore rnay impact

centrai, as well as peripheral rnechanisms that atfect food intake regulation. Furthemore,

devazepide rnay be able to bind to non-CCK receptors in the brain such as the

benzodiazepine receptor that is known to stimulate feeding (Morley, 1987). However,

PD-140,548 is a water soluble complex that is unlikely to cross the blood-brain-bmier.

Because albumin induced food intake suppression was blocked by PD-140,548 (1), this

streugthens the predominance of the peripheral action of CCK on food intake regulation.

Finally, central injections of CCK are less effective in decreasing food intake than

peripheral injections, once again suggesting that peripheral CC& receptors are more

important than central ones in food Uitake regulaîion (194).

The discovery that albumin, casein and wy and th& respective hydrolysates

involve perïphaal CCK~receptors to suppress fiiod W e aod that quantity and fom

protein (excluding amino acids) mediates the suppnssive response in food intake, helps

supports the idea that a specinc peptide of protein digestion is key to the rrlcase of CCK

If such a peptide exists its isolation may provide to be usefid in treating food intake

regdatory disorders Ore obesity.

5.2. Future Directions

A To determine, using the same design as established in Part II of this thesis, the

effects of amino acids mixtures of soy, casein and albumin on food htake

suppression via CC&receptors. In doing so, it wodd determine the role of

amino acids and peptides âagments of protein digestion on food intake

suppression via php heral CC& receptor~.

B. To elucidate if non-protein (amino acidq carbohydrate and fat) induced food

intake suppression of feeding is mediated by CC& receptors using devazepide, as

it was doue in Part II of this thesis,

C. To investigate fbrther the mechanism of action of the CCKA receptor magonia,

the role of the vagus neme in the transmission of the h g ' s effed should be

determineci by testing the receptor antagonjst in vagotomhed rats or OLETF rats

(rats that lack CCKA receptors). Additiodly, the coadrninistration of devazepide

with a protein preload in vagotomized or OLETF rats would provide fiirther

information in detennining whether blockade of the protein induced satiety

respoase is a periphed or central mechanism.

D. To determine the active peptide sequence responsible for protein induced food

intake suppression via periphed CC& receptors.

CHAPTER 6. SUMMARY AND CONCLUSION

6.1. Summary

k Albumin, casein, wy and their respective hydrolysates suppressed food intake

via CC& recepton.

B. The suppression in food intake elicited by these proteins was dependent on the

tirne at wtiich devazepide was administered, suggesting that the tirne at which

the peptide products of digestion interact with the CC& recepton varies with

the protein source.

C. Quantity of protein and t h e of devazepide administration &èct the strength

and duration of interaction between protein source and the CC& receptor.

6.2. Conclusion

In rats, the effect of dietary proteins on food intake suppression is dependent on

the time and duration of interaction between the protein source and CCKA

receptors.

CHAPTER 7. REFERENCES

7. References

1. Trigazis L, Ommann A, Anderson GEL Effect of a cholecystokinul-A receptor

blocker on protein-induced food intake suppression in rats. Am. J. Physiol

1997;272:R1826-R1833.

2. Trigazis L, Vaccarino FJ, Greenwood CE, Anderson GH. CCK-A receptor

antagonists have se ldve effects on nutrient-induced food intake suppression in rats.

Am. J. Physiol. 1999;276:R323-R330.

3. Woltman T, Reidelberger R Role of cholecystokinin in the anorexia produces by

duodenal delivery of peptone in rats. Am. J. Physiol. lW9;276:RI7O bR1709.

4. Morgan G. The role of cholecystolanin-A receptors in protein hydrolysate-

induced suppression of food intake in rats. : Toronto, 1 998: 1 42.

5. Anderson GH, Li ETS, Anthony SP, Ng LT, Bialik R Dissociation between

plasma and brain amino acid profiles and short-term food intake in the rat. Am. J.

Physiol. 1994a;266:R1675-R1686.

6. Anderson GH, Luo S, Ng LT, Li ETS. Non-essential amino acids and short-terni

food intake of rats. Nutrition research 1994b; 14: 1 1 79- 1 189.

7. Anderson GH, Luo S, Trigazis L, Kubis G, Li ETS. The effects of essential amino

acids on food and water intake of rats. Can. J. Physiol. Pharmacol. 1994c;72:841-848.

8. Flatt J. McCoUum Award Lecture, 1995: Diet, Mestyle, and weight maintenance.

Amencan Journal of Clinical Nuirition l995;62: 820-836.

9. Collier G, Leshner AI, Squibb RL. Dietary self-seledon in active and non-active

rats. Physiology and Behaviour 1969;4:79-82.

10. Bossardt DK, Paul W, ODoherty K, Bames RH. The influence of caloric intake

on the growth utilization of dietary protein J. Nutr. l946;32:64 1-65 1.

11. Adolph EF. Urges to eat and drink in rats. Am. J. Physiol. 1947; 15 1: 110-125.

12. Li ETS, Anderson GH. A role for vagus nerve in regulation of protein and

carbohydrate intace. Am. J. Physiol. l984;24î:E8 1 SE82 1.

13. Osborne TB, Mendel LB. The choice between adequate and inadequate diets as

made by rats. J. Biol. Chem. 19 l 8 ; E 19-27.

14. Richter CP. Harvey Lecture 1943;38:63-103.

15. Anderson GH. Hunger, Appetite, and Food Intdce. Present Knowledge in

Nutrition. 7th ed. New York: ILSI, 1996:1-6.

16. Grirnble GK, Keohane PP, Higgins BE, Kaminski MV, Silk DB. Effect of peptide

chah length on amino acid and nitrogen absorption from two lactablurnin hydrolysates in

the normal human jejunum. Clin Sci (Colch) 1986; 7 1 :65-69.

17. Grirnble GK, Rees RG, Keohane PP, Cartwright T, Desreumaux M, Silk DB.

Effkct of peptide chain length on absoption of egg protein hydrolysates in the normal

human jejunum. Gastroenterology 1987;92: 136- 142.

18. Anderson GH, Li ETS. Protein and amino acids in the regulation of quantitative

and qualitative aspects of food intake. Xnt. J. Obesity. 1987; 1 1 (Suppl3):97- 1 08.

19. Musten B, Peace D, Anderson GH. Food intake regulation in the weanling rat:

self-selection of protein and energy. J. Nutr. 1974; 1 O4:563-572.

20. Li ETS, Anderson GH Amino acids in the regulation of food intake. Nutr. Abs.

Rev. Clin. Nutr. l983;U: 169- t 81.

21. Anderson GH. Metabolic regulation of food intake. 3rd ed. Philadelphia: Lea and

Febingex, 1988b.

22. Barkeling B, Roessna S, Bjoemell H EEects of a hi@-protein meal and a hi@-

carbohydrate meal on satiety measuted by automateci cornputerid monitoring of

subsequent food intake, motivation to eat and food preférences. Int. J. Obes.

1990; l4:743-75 1.

23. Booth DA Chase 4 Campbell AT. Relative effectiveness of protein in the late

stages of appetite suppression in man. Physiol. Behav. 1970;5: 1299-1302.

24. Geary N. Food intake and behavioral caloric compensation after protein repletion

in the rat. Physiol. and Behav. 1979;23 : 1089- 1098.

25. Hill AJ, Blundell JE. Macronutrients and satiety: The effects of a high-protein or

high-carbohydrate meal on subjective motivation to eat and food preferences. Nutr.

Behav. 1986;3:133-144.

26. Maggio CA Greenwood MRC, Vasseili IR The satiety effects of intragastric

macronutrient infusions in fatty lean mcker rats. Physiol. Behav. 1983;3 1 :367-372.

27. Li ETS, Anderson GH. Meal composition influences nibsequent food selection in

the rat. Physiol. Behav. 1982;29:799-783.

28. Meliinkoff SM, Frankland M, Boyle D, Greipel M. Relationship between serum

amino acid concentration and fluctuations in appetite. J. Appl. Phy siol. 19S6;8: 53 5-5 3 8.

29. Peng Y, Tews JK, Harper A . . Amino acid imbalance, protein intake, and changes

in rat brah and plasma amino acids. Amer. J. Physiol. 1972;222:3 14-32 1.

30. Harper A . , Benevenga NJ, Wohlheuter RU Effects of ingestion of

disproportiomte amounts of amino acids. Physiol. Rev. 1970;50:428-558.

3 1. Peters JC, Harper AE. Amte effects of dietary protein on food intake, tissue

amino acids, aod brai. serotonin. Am. J. Physiol. 1987;252:R902-914.

32. Choi Y-H, Chang N, Anderson GH An inaagastnc amino acid mixture influences

extmeiiular amino acid profles in the lateral hypothalamic area of fieely rnoving rats.

Can. J. Physiol. Pharm 1999a;in press.

33. Anderson GH. Diet, neuroûmsmitten and brain funaion. Br. Med. Bull.

1981;37:95-100.

34. Anderson GH, Bialik RI. Li ETS. Amino acids in the regdation of food intake

and selection Berlin: NATO AS1 Seris, S pringer-Verlag, 1 988a

35. Blundell JE, Hill M . Nutrition, serotonin and appetite: Case study in the evolution

of a scientifïc idea. Appetite l987;8: 183- 194.

36. Luo S, Li ETS. Food intake and selection pattern of rats treated with

deisenfluramine, fluoxetine and RY 24969. Brain res. Bull. l990;24: 729-73 3.

37. Wurtman RI, Hefti F, Melamed E. Precursor wntroi of neurotransmitter

synîhesis. Pharmacol. Rev. 1981;32:3 15-335.

38. Bialik RT, Li ETS, GeofFroy P, Anderson GH. Route of delivery of phenylalanine

ifluences its effeas on short-term food intake in adult male rats. J. Nue. 1989; 1 19: 15 19-

1527.

39. Moms P, Li ETS, MacMillan ML, Anderson GH. Food intake and seleaion after

peripheral tryptophan. Physiol. Behav. 1987;4O: 155-163.

40. Schwartz JC, Lampert C. Properties and regionai distribution of histidine

decarboxylase in rat brain. Journal of Neurochem 1970; 17: 152% 1534.

41. Cheschmidt BV, Lotti VJ. Histamine: Intraventndar injection suppresses

ingestive behavi0u.r of the nit Arch Int. PharmaCodp. 1973;206:288-298.

42. Sheiner JB, Moms P, G H, Anderson. Food imake suppression by histidine.

Pharmd. Biochem. Behav. 1985;î3:721-726.

43. VaPn P. Histidine regdation of food and water intake in rats. Nutritioruil

Sciences. Toronto: University of Toronto, 1995: 1 16.

44. Femstorm JD. Food-indud changes in brain serotonin synthesis: 1s there a

relationship to appetite for specific macronutrients? Appetite l987;8: 163- 1 82.

45. Reidelberger RD. Cholecystokinin and control of food intake. J. Nutr.

l994;124: l327S-I333S.

46. Mer RC, Brenner LA, Tamura CS. Endogenous CCK and the peripheral neural

substrates of intestinal satiety. Annals of the New York Academy of Science

l994;7l3 :255-267.

47. B lundell J. Phannacological approaches to appetite suppression. TiPS

l99I;U: 148-157.

48. Bray GA. Peripheml metabolic &ors in the regulation of feeding. Berlin:

Dahlem Koaferenzen, 1976.

49. Kissileff Itallie TBV. Physiology of the ccntrol of food intake. Am. Rev.

Nutr. 1982;2:371-418.

50. Lee MC, Schiffman SS, Pappas TN. Role of neuropeptides in the regulation of

feeding behavior: a review of cholecystokinin, bombesin, neuropeptide Y and galanin.

Neuroscience and Biobehavioral Reviews 1994; 18:3 13-323.

5 1. Liddle R& Green GM, Conrad CK, W~lliams IA Proteins but not amino acids,

carbohydrates, or fats stimulate cholecystokinin secretion in the rat. Am. J. Physiol.

1986;25 1 :G243-G248.

52. Mcholi CG, Polak JM, Bloom SR The hormonal regulation of food intake,

digestion and absorption. Am. Rev. Nutr. 1985;s :2 13-239.

53. Deutsch 14 Young WG, Kalogeris TJ. The stomach signals satiety. Science

l978;2Ol: 165-167.

54. Iggo A. Tension recepton in the aomach and the urinary bladder. J. Physiol.

1955; l28:593-607.

55. Jauowitz HD, Grossman MI. Some factors affécting the food intake of normal

dogs and dogs with esophagostomy and g a k c fistula Am. J. Physiol. 1949; 159: 143-

148.

56. Paintal AS. A study of g a h c stretch receptors. Their role in the penpheral

mechanism of dation of hunger and thint. J. Physioi. 1954; 19 1 : 13- 18.

57. Pasquali q Besteghi L. Mechanisms of the action of the intragastric balloon in

obesity: effects on hunger and satiety. Brain Research l99O;Z4 1 :33 5-340.

58. Ramhamadanay EM, Fowler J, Baird hl. Effects of gastnc balloon versus sham

produre on weight loss in obese abjects. Gut 1989;3O: 1054- 1057.

59. Malagelada JR Gastric, pancreatic and biliary responses to a meal. New York:

Raven Press, 1981.

60. H q e r AE, Boyle PC. Nutrients and food intake. Berlin: Dahlern Konferenzen,

1976a

61. Read N, French S, Cunningham K The d e of the gut in regulating food intake in

Nutrition Reviews 1 994;52: 1 - 1 0.

62. Jeamingros R Vaga unitary responses to intestinal amino acids infusions in the

tmaesthetised cat: A putative signal br protein-induced satiety. Physioi. Behav.

198S;S8:9-21.

63. Bray G. Peptides affect the intact of specific nutrients and the sympathetic

nervous system. American Journal of Clinical Nutrition 1992;5 5 :265 S-27 1 S.

64. Smith GP, Gibbs J. Brain gut peptidesand the coatrol of food intake. New York:

hven Press, 198 1.

65. Bernstein IL, Lotta EC, Zimmennan JC. Cholecystokinin-induced satiety in

weanling rats. Physiol. Behav. 1976; 1 7: 54 1-543.

66. Moran TH, McHugh PR Cholecyaokinin suppresses food intake by inhibiting

gastric emptying. Am. J. Physiol. 1982;242:R49 1 -R497.

67. Moran TH, Robinson PH. Two brain cholecystokinin receptors: implications for

behavioral actions. Brain Research l986;362: L 75- 179.

68. SmithG,GibbsJ.SatiatingeffectofCholecystokinin.AnndsNewYork

Academy of Sciences 1994;7 13 :Z3 6-24 1.

69. Ivy AC, Oldberg E. A hormone mechanism for gallbladder contraction and

evacuation. Am. J. Physiol. l928;86:599-6 13.

70. Harper Aq Raper HS. Pancreozymin, a stimulant of secretion of pancreatic

enzymes in extracts of the small intestine. J. Physiolo. Lond. 1943; 102: 1 15- 125.

7 1. Anuras S, Cooke AR An inhibitory innervation of the gastroduodenal junction.

Journal of Cl inical Investigation 1974;54: 529-53 5.

72. Yamagishi T, Debas HT. Cholecystokinin inhibits gastnc emptying by acting on

both proximal and stomach and pylonis. American J o d of Physiology

1978;234:E375-E378.

73. Beghger C, Hildebrand P. A physiologid role for cholecystokinin as regulator

of gastrin secretion. Gastroeatetology 19%; 103 :490495.

74. Resin H, Stem DEL Effect of the C-terminal octapeptide of cholecystokinin B-

gastrin receptors in the human stomach. Gastroenterology 1973;64:946-949.

75. Luman W, Williams AJK Ifluence of cholecyaectomy on sphincter of Oddi

motility. Gut 1997;41:371-374.

76. Dinoso VP, Meshkinpour H. Motor responses of the sigmoid colon and rectum to

acogenous cholecystokinin and secretin. Gastroenterology l973;65:43 8-444.

77. Parker JC, Beneventano TC. Acceleration of small bowel contrast by

cholecystokinin. Gastroenterogly 1976;58:679-684.

78. Thulin L, Sannegard H. Chlatory effects of gastrointestinal hormones and

related peptides. Acta Chir Scand 1978;482:73-74.

79. GibbsJ,YoungRC,SmithGP.ChoIecystokinindecreasesfoodUitakeinrats.J.

Comp. Physiol. Psychol. 1973a;84:488-495.

80. Gibbs J, Young RC, Smith GP. CCK elicits satiety in rats with open gastric

fishilas. Nature 1973b;245:323-325.

81. McLaughlin CL, Baile CA Decreases sensitivity of Zucker obese rats to the

putative satiety agent CCK Physiol. Behav. l980;26:433-437.

82. Crawley M, Hays SE. Cholecystokinin reduces exploratory behaviour in mice.

Physiol. Behav. 198 l;Z7:407-4ll.

83. Koopmans HS, Deutch JA The effect of cholecystokinin pancreozymin on

hunger and thirst in mice. Behav. Biol. 1972;7:441-444.

84. Falasco JD, Smith GP. Cholecystokiain suppresses sham feedhg in the rhesus

monkey. Physiol. Behav. l979;Z 2387-890.

85. Holt JJ, Antin J. Cholecystokinin does not produce bait shyness in rats. Physiol.

Behav. 1974; l2:497498.

86. Sturdevant RAL, Goetz K. Cholecystokinin both stimulates and inhibits food

intake. Nature l976;26l:7l3-7l5.

87. Baile CA, Laughlin CLM, Della-Fera MA Role of cholecystokinin and opiod

peptides in control of food intake. Physiol. Rev. l986;66: 172-23 5.

88. Mutt V, Jorpes JE. Phannacolgical examination of cholecystokinin (CCK-8)-

induced contractile activity in the rat isolated pylorus. Peptides l968;8: 127- 134.

89. Eysselein V, Reeve J. Partial structure of a large canine intestinal cholecyaokinin

(CCK58): Amino acid sequence. Peptides l982;3 :68769 1.

90. Cantor P, Rehfeld JF. The Molecular nature of cholecyaokinin in human plasma.

Clin. Chim. Acta 1987; 168: 153-158.

91. Eng J, Du BR Purification and sequencing of a rat intestinal 22 amino acid C-

terminal C CK hgmem. Peptides 1984;s: 1203- 1206.

92. Liddle RA. Cholecystokinin cells. Annual review of physiology l997;59:22 1-242.

93. Calam JE4 Dockray GH. Identification and measurement of rnolecular variants

of CCK in duodenai mucosa and plasma J. Cün Invest. l982;69:2 18.

94. h g J, Shüna Y. Pig brain contains cholecystokinin octapeptide and severai

cholecystokinin desooctapeptides. Proceeding of the National Academy of Science, USA

1983;80:6381-6385.

95. Mulla JE, Straus E. Cholecystokinin and its C O O H - t d d octapeptide in the

pig brain. PrOceedlllgs of the National Academy of Science, USA 1977;74:3035-3037.

96. Reeve J, Eysselein V. Isolation and charactérizâtion of biologically active and

inactive cholecystokinin-octapeptides f?om hurnan brain. Peptides 1984;5:959-966.

97. Liddle Rq Goldfhe ID, R o s a MS, Taplitz RA, Williams JA Cholecyaokinin

bioactivity in human plasma: Molecular fomq rwponses to feeding, and relationship to

gailbladder contractions. J. Ch. Invest. I98S;Z: 1 144- 1 152.

98. Liddle RA, Goldfine ID, Williams JA Bioassay of plasma cholecystokinin in rats:

Effects of food, trypsin, inhibitor, and alwhol. Gastroentaology 1984;87:542-549.

99. Crawley IN, St-Pierre S, Gaudreau P. Analysis of the behaviorai activity of C-

and N-temiinai fkagments of cholecystokinin octapeptide. J. Phannaco l. Exp. Ther.

1984;230:438-444.

100. Mutt V. Cholecystokinin: Isolation., structure and function. New York: Raven

Press, 1980.

101. Anderson GH, Glanville NT, Li ETS. Amino acids and regulation of food intake.

Boston: John Wright. PSG Inc., 1983.

102. Williams JA. Cholecystokinin: a hormone and a neurotransmitter. Biorned. Res.

1982;3: 207-121.

103. Deschenes RJ, Lorenz LJ. Cloning and sequence analysis of a cDNA encoding rat

preprocholecystokinin. Proceediogs of the Naîional Academy of Science, USA

l984;8 1 :726-730.

104. Deschenes RJ, Haua RS. A gene encoding rat cholecystokinin. Journal of

Biological Chemistry 1985;260: 1280-1286.

105. Takabashi Y, Kato K. Molecular cloning of the human cholecystokinin gene by

use of a synthetic probe containing deoxyinosine. Proceedings of National Academy of

Science, USA l985;82: 193 1-1935.

106. Kanayama S, Liddle RA Influence of food deprivation on intestinal

cholecystokinin and somatostatin. Gastroenterology 199 1 ; 1 OO:gO9-9 1 5.

107. Dockray GJ. Brain Peptides. New York: Wiley Interscience, 1983.

108. Docloay GJ, Sharkey KA. Function and Dysninction of the Smail Intestine.

Liverpool: Liverpool Unversity Press, 1984.

109. Buchan 4 Polak J. Electron immunohistochemical evidence for the human

intestinal I ceil as the source of CCK. Gut 1978; 19:403dO7.

1 10. Polak J, Pearse A Identification of cholecy stokinin-secreting cells. Lancet

W S ; S : 1016-1021.

1 1 1. Solcia E, Pearse AGE. Revised Wiesbaden classification of gut endocrine cells.

Rend. Gastroenterol l973;5: 13-16.

112. Isaacs PET, Ladas S. Cornparison of effects of ingested medium and long chah

tnglyceride on gallbladder volume and release of cholecystokinin and other gut peptides.

Dig. Dis. Sci. 1987;32:481-486.

113. Go VL, Hoffina~ AF. Pancre0qm.h bioassay in man based on pancreatic enzyme

secretion: potency of specific amino acids and 0 t h digestive products. Journal of

Clinical Investigation 1 97O;N: 1 5 5 8- 1 564.

114. Konhink SJ, Rad& T, Thor P, Dembinski A Release of cholecystokinin by

amino acids. P.S.EJ3.M 1973; 143 :305-309.

115. L,ewis LD, William JA Regulation of CCK secretion by food, homones, and

neural pathways in the rat. Am J. Physiol. 1 SWO;258:GS 12- 18.

116. Schneeman BO, Chang 1. Effect of dietary amino acids, casein and soybean

trypsin inhibitor on pancreatic protein secretion in rats. Journal of Nutrition

1977; 1 OW8 1-288.

117. Mabayo RT, Furuse M. Medium-chah triacyglycerols enhance release of

cholecystokinin in chicks. Journal of Nutrition 1992; 122: 1702- 1705.

1 18. Douglas BR, Wouterson Rq Jansen JBMJ, Long kTLd, Lamen CBHW. The

influence of dSerence nutrients on plasma cholecystokinin levels in the rat. Expenentia

1988;44:21-23.

1 19. Herzig IC, Schon 1. Diazepam binding inhibitor is a potent cholecystokinin-

releasing peptide in the intestine. Proceedings of the National Academy of Science

l996;93 : 7927-7932.

120. ThVnister PWL, Hopman WM, al. CEJSe. Role of intraduodenal proteases in

plasma cholecystokinin and pancreaticobiliary responses to protein and arnino acids.

Gastroenterology 1996; 1 10567-575.

121. Beucher S, Levenez F, Yvon M, Corring T. Effects of gastnc digestive produas

from casein on CCK release by intestinal cells in rats. J. Nutr. Biochem. 1994;5:578-584.

122. Shi& Y Shiratori K, Watariabe S, Takeuchi T, Chang T-M, Chey WY. Effect

of protein derivatives on pancreatic secretion and release of secretin and CCK in rats.

Am. J. Phy siol. 1 994;267:G50865 14.

123. Gumbmann MR, Dugan GM, Spangler WL, Baker EC, Rakis JJ. Pancreatic

response in rats and mice to -sin inhibitors 60om soy and potato after short- and long-

terni dietary exposure. J. Nutr. 1989; 1 19: 1 598- 1609.

124. Miyasaka K, Guan D. Feedback regulation by trypsin: evidence for intralumenal

CCK-releasing peptide. Arnerican Journal of Physiology 1989;2Sî:Gl7S-Gl8 1.

125. Spannagel AW, Green GE. Purification and characterization of a luminal

cholecystokinin-releasing factor fiom nit intestinal secretion. Proceedings of the National

Academy of Science, USA. l996;93 :44 1 5-4420.

126. Aucouturier S, Bernard C. Fundonal wupling between the cyclic adenosine

monophosphate pathway and cholecystokinin secretion in RlN cells. Biochen. Biophys.

Res. Commun. I994;ZOO: 13 82- 13 90.

127. Cuber JC, Vilas F. Bombesin and nutrients stimulate release of CCK through

distinct pathways in the rat. Arnaican Joumal of PhysioIogy 1989;256:G989-G996.

128. Sharara AI, Bouras EP, Misukonis Mq Liddle RA. Evidence for indirect dietary

regulation of cholecystokinin release in rats. Am. J. Physiol. l993;26S:Gl 07-G112.

129. Deschodt-Lanckrnan M, Bui N. Cholecystokinin octa-and tetrapeptide

degradation b y synaptic membranes. II. Solu bilization and separation of membrane-

bound CCK-8 cleaving enzymes. Peptides 1983;4:71-78.

13 0. McDmott J, Dodd P. Pathway of inactivation of cholecysto kinin octapeptide

(CCK-8) by synaptosomal âactions. Neurochem. Int. l983;5 :64 1-647.

13 1. Najdovski T. Pont JJHHMD, Tessa GI, Penke B, hbrhez J, Deschodt-

h c k m a n M. Degradation of cholecystokliin octapeptide by the neutral endopeptidase

EC 3.4.24.11 and design of proteolysis-resistant analogues of the peptide. Neurochem

Int. 1987; 10:459465.

132. Rehfeld IF, Hansen HF. Gastrin and cholecystokinin in pituitary neurons.

Proceedings on the National Academy of Science, USA l984;8 1 : 1 902- 1 905.

133. Crawley JN, Corwin RL. Biological actions of cholecystokinin. Peptides

1994;15:73 1-755.

134. Persson H, Ericsson A Detection of cholecystokinin in spematongenic cells.

Acta Physiol. Scand. 1988; 134:565-566.

135. Schalling nd, Persoon H. Expression and localkation of gastnn mRNA and

peptide in spermatogenic celis. Journal of Clinical Investigation 1990;86:660-669.

136. Liddle RA. Regdation of cholecystokinin syuthesis and secretion in rat intestine.

J. Nutr. 19%; 124: 1308s-13 14s.

137. Smith GP, Gibbs J. The satiety effm of cholecystokinin: Recent progress and

current problems. Annais of the New York Academy of Science. l985;448:4 17-423.

13 8. Larsson LI, Rehfeld JF. Localization and molecuiar heterogeneity of

cholecystokinin in the central and peripherai nemous system. Brain Research

1979;265:201-218.

139. Hays SE, Beinfeld MC, Jensen RT, Goodwin Paul SM. Demonstration of a

putative receptor site for cholecystokinin in rat brain. Neuropeptides 1980; 153-62.

140. Innis RB, Synder SH. C h o l e q s t o ~ receptor binding in brain and pmcrem:

regulation of panmatic binding by cyclic and acyciic guanine nucleotides. Eur. J.

Phannad. 1980;65: 123-124.

141. Jensen RT, Lemp GF, Gardner ID. Intaaction of cholecystokixsin with specific

membrane receptors on panaeatic acinar c d s . Roc. Nd. Acad. Sci. USA

l980;77:2079-2083.

142. Power SP, 1. Foo ea. Use of photoaffinity probes containing poly ethylene glycol

spacers for topographical mapping of the cholecy stokinin receptor cornplex.

Biochemistry 199 I;30:676-682.

143. Reubi JC, B. Waser ea. Localization of cholecystokinin A and cholecystokinin B-

gasüin recepton in the human stomach. Gastroenterology 1997; 1 12: 1 197- 1205.

144. Woodniff GN, Hughes J. Cholecystokinin antagonists. An. Rev. Pharmacol.

Toxicol. 199 l;3 1 :469-5O 1.

145. Silver Al, Morley JE. Role of CCK in regdation of food intake. Progress in

Neurobiology 199 1 ;36:23-34.

146. Harro J, Oreland L. Cholecystokinin recepton and memory: A radial maze study.

Pharmacol Biochem Behav 1993;44:509-5 17.

147. Shulkes 4 Baldwin G. Biology of gut cholecystokinin and gastnn receptors.

Clinical and Expen mental Pharmacology and Phy sioiogy l997;24:209-2 1 6.

148. Walsh JH. Gastria Gut Peptides: Biochemistry and Physiology. New York:

Raven Press, 1994.

149. Asin KE, Gore P 4 BEdnarz L, Holladay M, Naduun AM. E f f ~ of selective

cholecystokinin receptor antagonists on food intalre after centrai or peripheral

administration in rats. Brain Res. l992b;S7 1 : 169- 174.

150. Deiia-Fera C.A. Baile ea Cholecystokinin antibody injectai in cerebral

vcntncles stimulates feeding in sheep. Science 198 1;2 l2:68%689.

15 1. McLaughlin CL, Baile C 4 Della-Fera MA, Kasser TG. Meal-stimulated

increased concentratons of CCK in the hypothalamus of Zucker obese and lean rats.

Physiol . Behav. l985;3 5:2 15-220.

152. Ebenezer IS, Riva CDL, Baldwin BA. Effects of CCK-receptor antagonist MK-

329 on food intake in pigs. Physiol. Behav. 1990;47: 145- 148.

1 53. Reidelberger RD, ORourke MF. Potent cholecystokinin antagonist L3 64,7 18

stimulates food intake in rats. Am. J. Physiol. 1989;25?:R1512-R15 18.

154. Corwin R, J. Gibbs ea Increased food htake after type A but not type B

cholecystokinin receptor blockage. Phyisiology and Behavior 199 1 ;SO:255-258.

155. Dourish C, W. Rycrofk ea Postponenment of satiety by blockage of brain

cholecystoklliin (CCK-B) rexeptors. Science 1989;245: 150% 15 1 1.

L 56. Moran TH, Ameglio PJ, Schwartz GJ, McHugh PR Blockade of type 4 but not

type B, CCK receptors attenuates satiety actions of exogenous and endogenous CCK.

Am. J. Physiol. 1992;262:R46-R50.

157. Smith GP, Gibbs I. Postprandial satiety. Prog. Psychobiol. Physiol. Psychol.

1979;8: 17% 142.

158. Bymes DJ, Headerscn L, Borody T, Rehfeld IF. Radioimmunoassay of

cholecystokinlli in human plasma Clm. Chim. 198 1;Acta m:8 1-89.

159. Peikin SR Role of cholecystokinin in the control of food intake. Gastroeneteroi.

Clin. Nr. Amer. 1989; l8:757-775.

160. Moran TH, Smith GP, HostetIer AM, McWugh PR Tmport of cholecystokinio

(CCK) binding sites in subdiaphragmatic vagal branches. Brah Res. l987;4 1 5 : 149- 152.

161. Zarbin Mq Wamsley JK, Inais RB, h h a r W. C h o l e c y s t o ~ receptors:

Preseuce and axonal flow in the rat vagus nave. Life Sci. 198 1 ;Z9:697-705.

162. Schick RR, V. Schusdnarra ea EEea of CCK on food intake in man:

Physiologicai or pharmaCologid effect? 2. Gastroenterology 199 1;29: 53-58.

163. Reidelberger RD, Solomaa TE. Comparative effects of CCK-8 on feeding, sham

feeding, and exocrine pancreatic secretion in rats. Amencan Journal of Physiology

I986;Z 1 :W7-R 105.

164. Weatherford SC, Laughton WB, Salabania J, et al. CCK satiety is diflerentidly

rnediated by high- and Iow-aainity CCK reçepton in mice and rats. -Am. I. Physiol.

1993;264:R244-R249.

165. Blewis GTJ, William JA ATP induces two cholecystokinin binding afEnity

states in penneabilized rat pancreatic acini. Am. J. Physiol. 1992;263 :G44-G5 1 .

166. Asin KE, Bednarz L. DEerential effeas of CCK-JMV- 180 on food intake in rats

and mice. Pharmocology Biochemistry and Behavior 1992a;42:29 1-295.

167. Reidelberger RD. Cholecystokinin and control of food intake. I. Nutr.

l9%;124: l327S-1333S.

168. Vanderhaeghen JJ, Lotsaa F7 May JD, Gilles C. Immunohistochemical

localization of choleçtstokinin- and gamin-like peptides in the brain and hypophysis of

the rat. Froc. Nd. Acad. SQ. USA 1980;77: 1 190-1 194.

169. Beideld MC. Cholecystokinin in the central nemous system: A minireview.

Neuropeptides 1983;3:411-427.

170. Kulkosky PI, Breckenridge C, Krinsky & Woods SC. Satiety elicited by the C-

terminal octapeptide of cholecy stokhh-pancreo e n in normal and VMH-lesioned rat S.

Behav. Biol. 1976; l8:227-234.

171. Passaro E, Debas H, oldendorf W, Yamada T. Rapid appearance of

intraventricularl y administ ered neuropeptides in the peri p heral circulation. B rain. Res .

1982;241:335-340.

172. Schwartz DH, Dorfriian DB, Hernandez L, Hoebel BG. Cholecystokinin: 1 . CCK

autagonists in the PVN induce feeding, 2. Effects of CCK in the nucleus accumbens on

exttaceiiular dopamine turnover. In: Wang RV, Schoedeld R, eds. Cholecystokinin

antagonists. New York: Aian R Liss, Inc., 1988:285-305.

173. Cheng DY, Deutsch JA, Gintalez MF, Gu Y. The induction and suppression of c-

fos expression in the rat brain by cholecystokinin and its antagonist L364,718. Neurosci.

L&t. 19%; l49:91-94.

174. Della-Fera Coleman BD, Doubek C 4 et al. Cholecystokinin concentration

in specific brain areas of rats fed during the light or dark phase of the circadian cycle.

Physiology and Behavior 1 989;45 :8O 1-807.

175. Dourish CT, Coughlan J, Hawley D, Clark M, Iversen SD. Blockade of CCK-

induced hypophagia and prevention of morphine tolerence by the CCK antagonist L-

364,718. New York: Alan R Lisq 1988.

176. Hewson G, Leighton GE, Hill RG, Hughes J. The cholecystokinin receptor

antagonist L 364,718 inmases food M e in the rat by attenuation of the action of

endogenous cholecystokinin Br. J. Pharmacol. 1988;93 :79-84.

177. Wokowitz OM, Gertz B, Weingartner H, Beccaria L, Thompson K., Liddle R

Hunger in humans induced by MK-329, a specific periphd-type cholecyaokinin

receptor autagonid. Biol. Psychiatry l990;28: 169- 173.

178. Miesner J, Smith GP, Gibbs J, Tyrka A Intravenous infusion of CCK-A receptor

antagonist inmeases food intake in rats. Am. J. Physiol. l992;262:R2 I6-lUl9.

179. Brenner LA, Ritter RC. Type A CCK recepton mediate satiety effects of

intestinal nutrients. Pharrnacology Biochemistry and Behavior 1996;54:62543 1.

180. Yox DP, Brenner L, Ritter K. CCK-receptor antagonists attenuate suppression of

sham feeding by intestinal nutrients. Am. J. Physiol. l992;26Z:RSWR561.

18 1. Gregory PC, McFaden M, Rayner DV. Duodenal infiision of fat, cholecystokinin

secretion and satiety in the pig. Physiol. Behav. 1989;45: 102 1- 1024.

182. Woltman T& Reidelberger RD. Effect of CCK-A receptor antagonists devazepide

on inhibition of feeding by duodenal infusion of oleic acid in rats. FASEB J.

1993;7:A205 (abs.).

183. Woltman T, Castelanos D, Reidelberger R Role of cholecystokinin in the

morexia produceci by duodenal delivery of oleic acid in rats. Am. J. Physiol.

I99S;Z69:Rl42O-R1433.

184. Orttmana A Involvement of cholecy stokinin in protein-induced satiety in rats. .

Toronto, Canada: University of Toronto, 1992.

185. Anthony SP. The eEects of protein, essential and non-essential amino acids on

food intake and seleaion in rats. . Tomnto, Canada: University of Toronto, 1991.

186. Dupont J, White PJ, Feldman EB. Saturateci and hydrogemted fats in food in

relation to health. J. Am COU. Nutr. 1991 ; 1 O:577-592.

187. Moran TH. Cholecystokinin and Satiety: Curremt Perspectives. Nutrition

2000; 16: 858.865.

1 88. Fruhbeck G. Slow and fkst dietary proteins. Nature l998;N 1 :843 -845.

189. Fushiki T, H. Kajiura et al. Evidence for an intraiuminal rnediator in rat pancreatic

enzyme secretion: Reconsitution of the pmcreatic response with dietary protein, trypsin

and the monitor peptide. Journal of Nutrition 1989; 119:662-627.

190. Guan D, Green G. Significance of peptic digestion in rat pancreatic secretory

response to dietary protein. Amencan Journal of Physiology 1 W6;U 1 :G42-G47.

191. Cuber JC, G. Bernard et al. Luminal CCK releasing factors in the isolated

vascularly perfused rat duodenojejunum. Am I Physiol lWO;259:Gl9 1 -G197.

192. Pupovac J. Unpublished. . Toronto: University of Toronto, 200 1 .

193. Baro L, E. Guadix et al. Serum amino acid concentrations in growing rats fed

intact protein versus enzymatic protein hydrolysate based diets. Biology of the Neonate

l995;68:55-61.

194. Schick a Harty GJ, Yaksh TL, Go VL. Sites in the brain at which

cholecystokinin octapeptide (CCK-8) acts to suppress feeding in rats: a mapping study.

Neuropharmaîology 1990;29: 109-1 18.

195. Lienard F, SN Thornton, FP Martial, MC Mousseau, S Nidaidis. Angiotensin II

CHAPTER 8. APPENDIX

8.1. Appendix 1 - Sample Size Calcuiation

Sample size estimation when testing for the mean of a normal distribution (two sided

aiternative). For a within subject design, the eguation is:

a = 0.05 (21-0.025 = 1.96) B = 0.20 (Z0.80 = 0.84 a = 1.27 (SEM = 0.3; n = 16 A = 1.1

Values were taken fiom Orttmmq 1992. A represents the minimal difference in food

intake observed between controi and treatment.