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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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Canada
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.
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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.