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Non-Invasive Measurement of Corticosterone
in Food Restricted Rats
by
Deborah Chava Cole
A thesis submitted in conformity with the requirements for the degree of Masters of Science
Graduate Department of Exercise Sciences University of Toronto
© Copyright by Deborah Chava Cole (2012)
II
Non-invasive Measurement of Corticosterone
in Food Restricted Rats
Deborah Chava Cole
Masters of Science
Graduate Department of Exercise Sciences University of Toronto
2012
Abstract !Blood CORT is commonly used to assess stress in rodents, but sampling can trigger a rapid
stress response. This study aims to identify whether faecal CORT metabolites (FCM) can reflect
changes in CORT induced by 7-day food restriction (FR) and an ACTH challenge. Blood and
24hr faecal samples were collected at baseline and Day 7 for control (n=8) and FR (n=10) rats.
On Day 8, after a baseline blood sample, an ACTH injection was administered and followed by
blood and fecal sampling. Results showed increased serum CORT and FCM in response to FR.
Increased adrenal sensitivity with FR was illustrated by a greater increase in serum CORT
compared to control in response to ACTH. Lastly, although it appeared that ACTH induced an
increase in FCM in FR and control, only the latter reached statistical significance. Thus FCM
might be better suited for quantifying chronic rather than acute changes in CORT.
III
Acknowledgments
It would not have been possible to write this Masters thesis without the help and
support of my supervisor, lab committee, lab partners, and my friends and family.
To my supervisor, Dr. Catherine Amara, thank you so much for your patience
and guidance in taking me through all the steps possible to make this thesis happen.
You have taught me so much about how to critique other papers and to create a story
from what may appear to the untrained eye as mere numbers. I would also like to thank
Dr. Carolyn Cummins and Dr. Jack Goodman for providing me with helpful suggestions
and positive feedback, and Dr. Rudy Boonstra for providing us with the equipment and
sharing his knowledge in the field.
I would like to thank the undergraduate and lab partners for their extra support.
Without my undergraduate volunteers as my second pair of hands in the data collection,
I would not have survived the long hours of data collection. In my daily work, I was
blessed with a friendly and cheerful group of fellow students who were always there for
support and assistance.
Finally, I would like to thank my family and friends for unconditionally being there
and encouraging me to complete my Thesis. Mom, Dad, Granddaddy, Avi, Jennifer,
Leora, and Opher, you have all provided personal support and great patience at all
times. Thank you for sitting through numbers of explanations and practice presentations
and attempting to understand what I have been doing for the past two years.
Table of Contents
ACKNOWLEDGEMENTS III
TABLE OF CONTENTS IV
LIST OF TABLES VII
LIST OF FIGURES VIII
LIST OF ABBREVIATIONS IX
LIST OF APPENDICES X
CHAPTER 1: INTRODUCTION 1
CHAPTER 2: REVIEW OF THE LITERATURE 4
2.1 The Stress Response 4
2.2 Blood Cortisol/Corticosterone Measurement 6
2.3 Importance and Application for a Non-Invasive Corticosterone 7
Measurement
2.4 Corticosterone Production 12
2.4.1 Steroidogenesis 12
2.4.2 Hypercortisolism 14
2.4.3 Hormone Regulating Factors: Leptin, Liver, and HDL 17 2.5 Coticosterone Metabolism 21 2.6 Corticosterone Measurement Techniques: RIA and EIA 23 2.7 Cortisol/Corticosterone Response to Food Restriction 25 2.8 Objectives 30
V
CHAPTER 3: METHODOLOGY 32 3.1 Data Analysis 32
3.2 Animals and Housing 34
3.3 Fecal Sampling 35
3.4 Fecal Corticosterone Extraction 35
3.5 Blood Sampling and ACTH Challenge 36
3.6 Blood Corticosterone Extraction 37
3.7 Food Restriction Protocol 38
3.8 Sample Size Justification 38
3.9 Statistical and Analytical Plan 39
CHAPTER 4: RESULTS 40
4.1 Body Composition 40
4.2 Effects of 7 Days of Food Restriction on Corticosterone Levels 41
4.2.1 Serum Corticosterone 41
4.2.2 Faecal Corticosterone Metabolites 42
4.3 Circadian Rhythms 43
4.4 ACTH Challenge 48
4.4.1 Serum 48
4.4.2 Faecal Corticosterone Metabolites 49
4.5 Sampling Effect 51
CHAPTER 5: DISCUSSION 52
5.1 Effect of 1-Week Food Restriction on Serum Corticosterone 52
5.2 Effect of 1-Week Food Restriction on FCM 55
VI
5.3 Underlying Mechanisms of Hypersecretion of Corticosterone 57
5.4 Circadian Rhythms 59
5.5 Adrenal Sensitivity 61
5.6 Limitations 62
5.7 Conclusion 64 References 66 Appendix 75
VII
List of Tables Table 1 - Change in Body Mass over 7 Days Measured 40
in Grams ± S.D.
Table 2 - Average Daily Food Intake over 8 Days in Control 40 and Food Restricted Animals Measured in Grams ± S.D
Table 3 – Speed of Sample Collection Measured in 41
Minutes ± S.D Table 4 - FCM in Ad Libitum and Food Restricted Animals 43 Table 5 – Number of Animals that Produced Samples at 45
a Given Time
VIII
List of Figures Figure 1 - Possible Explanation for Dissociation of ACTH and CORT 15 Figure 2 – Schematic of Rat Model Study Design 33 Figure 3 - Effect of Food Restriction on Serum Corticosterone. 42 Figure 4 - Effect of 7 Days of Food Restriction on Faecal Corticosterone 43
Metabolites. Figure 5 - Circadian Rhythm of Animals at Baseline and Day 7 with 46
the 20:00h-22:00h Time Omitted in Control (A), and FR (B). Figure 6 - Normalized FCM of Circadian Rhythm in (A) Control Group 47
and (B) FR animals.
Figure 7 - Serum CORT of Control and FR Animals After an ACTH 48 Challenge at Baseline 0, 30, 60, and 120 min Post Injection.
Figure 8 - 24 hr Average FCM Measurements at Day 7 and Day 8. 49 Figure 9 - ACTH Challenge over 32hrs in Control and FR Animals 50
After 8 Days Food Restriction Looking at FCM ng/g (A) and Normalized FCM (%) (B).
Figure 10 - Anticipatory Response of Serum CORT to the Sampling 51
Procedure in Control and Food Restricted Animals
IX
List of Appendices
Appendix 1
A. Animals Use Protocol Form / Amendment Request 75
Appendix 2
A. Parallelism and Recovery Test 76
Appendix 3: Raw Data
A. Date of Birth and Arrival of Animals 85
B. Animal Body Mass (g) and Faecal Corticosterone 86
Metabolites at Baseline, Day 7, and Day 8 ACTH
C. Average Mass of Food Consumed per Day Measured in Grams ± S.D. 86
Appendix 4
A. Circadian Rhythm of Control Animals at Baseline and Day 7 87
B. Circadian Rhythm of FR Animals at Baseline and Day 7 87
X
List of Abbreviations ABA = Activity Based Anorexia ACTH = Adrenocorticotropic hormone AgRP = Agouti-related Protein AN = anorexia Nervosa ARC = Arcuate Nucleus AVP = Argenine Vasopressin CBG = Corticosteroid Binding Globulin CON = Control CORT = Corticosterone CRH = Corticotropin-releasing Hormone CSF = Cerebral Spinal Fluid DMH = Dorsomedial Nucleus EIA = Enzyme Immunoassay FCM = Faecal Corticosterone Metabolites FEO = Food Entrainable Oscillator FR = Food Restriction GC = Glucocorticoid GR = Glucocorticoid Receptor HDL = High Density Lipoprotein HPA = Hypothalamic-pituitary-adrenal Axis HRP = Horseradish Peroxidase LC-NE = Locus Ceruleus Noradrenergic LDL = Low Density Lipoprotein LEW = Lewis Rats LHA = Lateral Hypothalamic Area LXR = Liver X Receptor MR = Mineral Corticoid Receptor NPY = Neruopeptide Y POMC = Pro-opeomelanocortin PVN = Paraventricular Nucleus RIA = Radioimmunoassay RF = Restricted Feeding SD = Sprague-Dawley SR-B1 = Scavenger Receptor Class B StAR = Steroidogenic Acute Regulatory Protein TMB = Tetramethylbenzidine VMH = Ventromedial Nucleus
1
Chapter 1 Introduction
Plasma corticosterone (CORT) is commonly measured to detect stress responses in rodents.
However, measuring CORT levels in circulation is complicated as it varies in both diurnal and
ultradian rhythms and the blood sampling procedure can trigger a rapid stress response. Most
human interactions with laboratory rats, including handling, restraint [1], and anesthesia [2]
result in an activation of the hypothalamic-pituitary-adrenal (HPA)-axis. The influence of
moving a rat in its cage from shelf to floor or table in the animal room was shown to increase
CORT within 5 minutes [3]. Vahl and colleagues [4] demonstrated that a stress response can
occur in as little as 3 minutes as shown by an increase in adrenocorticotropic hormone (ACTH).
However, when blood samples were collected in under 3 minutes, ACTH levels were consistent
with basal values. Similarly, Siswanto et al. [5] demonstrated that rats treated with 10ug/kg of
adrenocorticotropic hormone (ACTH) exhibited a significant increase in serum CORT compared
to basal levels between 3 and 90 minutes, suggesting that the time required from a stressful event
to a significant CORT increase is limited to a few minutes.
A number of investigators [5-7] have used faecal samples to determine the concentration of
faecal corticosterone metabolites (FCM) because it is noninvasive and samples can be collected
with minimal disturbance to the animal [8]. Faecal pellets provide an alternative method to
measuring CORT as rodents can defecate several pellets every 1-2 hrs [9], making faecal pellets
a convenient measuring tool for analysis of circadian rhythms of hormones. Analysis of FCM is
also effective for documenting changes in CORT production over a long period of time, since rat
2
faeces have relatively high concentrations of corticosterone metabolites [10]. In addition, faecal
sampling is preferable, because small rodents have limited blood volumes, thereby limiting the
frequency, volume, and /or duration with which blood samples can be collected for repeated
measures within a single animal. Jugular cannulation, which is typically used for repeated blood
sampling, has been associated with high failure and complication rates after a week [11]. The
cannulation may also act as an additional stressor to the animal thereby altering normal
glucocorticoid levels and secretion pattern [12].
Faecal measurements are reflective of plasma CORT, and this is supported by observations of a
diurnal pattern with low levels in the evening and higher values in the morning [6, 10]. Most
mammalian species experience a marked circadian rhythm in circulating glucocorticoid
concentrations, with peak circulating concentrations occurring just prior to the daily active
period or the end of the light period in nocturnal species [13]. This rhythm is asymmetrical in
many mammalian species, including rats, where circulating CORT concentrations rise rapidly at
the end of the inactive sleep period, peak just prior to the active period, and then levels slowly
decrease across the day to the nadir at the end of the active period [13-16].
Serum CORT increases in both short-term [17] and long-term [18] food restricted (FR) rats. A
prior study from the present paper’s lab observed the temporal changes in body composition,
IGF-1, leptin, and voluntary wheel running activity during 4 weeks of a FR protocol that brought
body mass down to 75% baseline [19]. No substantial increase in CORT levels were found in the
FR groups compared to the ad libitum fed animals. This unexpected finding might have been a
result of the protocol used to collect the faecal samples. Samples were collected once per day and
3
this sampling time may not have captured the peak CORT levels in FR animals as intended. FR
alters the way CORT is metabolized, and may have shifted the temporal relationship between
blood CORT and FCM such that the sampling time captured the peak CORT levels in ad libitum
fed animals (as intended), but missed the peak in the FR animals. Previous studies demonstrate
that a stressor can be measured in FCM 4-12 hours later in a number of species, including cats
and dogs [20], mice [21-22], and rats [5, 10]. Stressors included an intraperitoneal injection of
radioactive CORT [10], and an intravenous injection of CORT [6, 22]. This sampling window of
4-12 hours is large, thus sampling at one specific time point might not be sufficient to account
for inter-individual variability.
The overall goal of the current study was to determine if multiple sampling over a larger
sampling window would make it possible to non-invasively determine the CORT response to
food restriction in rats. Faecal sampling is a non-invasive measure that facilitates multiple
collections in a longitudinal design and will reflect CORT responses. If feasible, these
measurements could provide insight on how CORT may be linked with a number of symptoms
of Anorexia Nervosa (AN) including hyperactivity and fat patterning upon refeeding [23]. The
current study has three objectives: To identify the CORT response to food restriction in both
faecal and blood samples; to determine the impact of food restriction on adrenal sensitivity using
an ACTH challenge; and to determine the timing offset between blood and faecal CORT to an
ACTH challenge. Answering these objectives will help determine the correct window of time to
most accurately capture peak CORT levels in faecal matter in FR rats.
4
Chapter 2 Review of the Literature
2.1: The Stress Response
Under normal conditions, the pancreatic beta-cell hormone insulin triggers the fast uptake and
oxidative catabolism of glucose in liver, muscle, and adipose tissue, and simultaneously inhibits
glycogenolysis and gluconeogenesis in liver during feeding [24]. Low plasma glucose levels
during fasting and exercise trigger a drop in insulin and an increase in the peptide hormone
glucagon from alpha-cells within the pancreatic islets and adrenal CORT is released into the
circulation [25]. Adverse situations trigger responses of the adrenals, which result in an increase
in CORT to help defend the organism against the stressful conditions. CORT counteracts
glucose-storing insulin and increases blood sugar through gluconeogenesis to provide immediate
energy for the body. During short-term stress, CORT facilitates the escape from life-threatening
situations and improves the energy availability by breaking down protein and fat to provide
metabolites that can be converted to glucose in the liver [26]. However, during severe chronic
stress, the long-term elevation in CORT can be hazardous to the body and may decrease
individual fitness by immunosuppression and atrophy of muscle tissues.
CORT is secreted with a strong diurnal rhythm where it normally peaks in the morning and
diminishes during the night [27]. Upon synthesis, it diffuses out of the adrenal cells into the
plasma, where most of it is transported by a carrier protein, corticosteroid-binding globulin
(CBG). The unbound hormone is free to diffuse into target cells. All nucleated cells of the body
5
have glucocorticoid receptors in their cytoplasm [27]. The hormone-receptor complex enters the
nucleus, binds to DNA with the aid of a hormone-response element, and alters gene expression,
transcription, and translation.
The hormonal pathway by which stress leads to glucocorticoid secretion is referred to as the
HPA axis. Briefly, the brain perceives a stressful situation, and the paraventricular nucleus
(PVN) located in the hypothalamus sends corticotropin-releasing hormone (CRH) to the
adenohypophysis, which secretes ACTH [27]. In turn, ACTH circulates to the adrenal cortex,
where it signals glucocorticoid secretion. When an animal encounters a stressor, the PVN is
stimulated, and CRH is released into the hypophyseal portal system that connects the
hypothalamus and the anterior pituitary. CRH and arginine vasopressin (AVP) stimulate the
anterior pituitary to convert pro-opiomelanocortin into ACTH. ACTH is released in the blood
stream where it stimulates the adrenal cortex to secrete glucocorticoids well above basal levels,
where it normally takes 3-5 minutes to result in measurable increases in plasma corticosterone
concentration [28]. Plasma corticosterone levels usually peak 15-30 minutes after a stressor and
return to basal levels within 60-90 minutes [29]. With an acute stressor, the feedback mechanism
operates efficiently and the system rapidly returns to normal. With a chronic stressor, feedback
signals are weak and the system remains activated for longer periods [28]. Boonstra [30]
provides an example of the difference between chronic and acute stress: an animal being chased
for a short duration will have an acute response; animals being chased for long durations
(occurring over many hours or weeks) will have a chronic response where chronically elevated
levels of CORT might impact the acute response, and thus, result in a diminished response to an
ACTH challenge, for example, in comparison with control animals.
6
Metabolic cages are often used in biomedical research and it is debated whether housing in these
cages is more stressful than single housing in standard rodent cages and how long rodents need
to acclimate to metabolic cages prior to a study. Housing rats on a grid floor compared with
housing rats in cages with saw dust bedding has been reported to be associated with higher
plasma CORT levels [31], and increases in blood pressure and heart rate [32]. However, duration
of time rats spend in metabolic cages has also been shown to affect CORT levels whereby 12
weeks resulted in notable increases in plasma CORT [31] but 3 days did not [33]. Eriksson et al.
[33] noted that when moved to a metabolic cage, rats showed signs of anxiety and stress
including reduced body weight gain and increased frequency of defecation. However, the stress
was not of sufficient magnitude or duration to result in a significant increase in CORT excretion,
indicating that housing for a few days in metabolic cages is not associated with major stress for
laboratory rats [33].
2.2 Blood Cortisol/Corticosterone Measurement
Corticosterone is the major glucocorticoid found in rats and is the analogue to cortisol in
humans. Methods of blood withdrawal and associated handling during sampling may stress
laboratory animals [34]. To reduce stress, blood sampling in laboratory animals is often
performed under anaesthesia or via cannulation. Taking blood under anaesthesia can be
problematic in itself, because most anesthetics lead to respiratory depression, cardiovascular
disturbances, and endocrine and metabolic changes [34]. There is an increasing demand for
techniques that allow easy, fast, reliable, repeated and non-invasive collection of CORT. Fluttert
et al. [35] discuss many issues involved with intravenously implanted catheters in the jugular
7
vein: When only one or two blood samples were needed, surgery was required and rats had to be
housed singly to prevent them gnawing off each other’s cannulae. Furthermore, the catheter can
be a source of infection and irritation [36], and can consequently disrupt CORT patterns [12].
Thus, longitudinal experimental designs over the course of months or years are not possible with
this technique.
A commonly used alternative is blood collection from a tail vein. An advantage of this technique
is that tail vein nicking does not require surgery and is therefore simpler and less invasive.
However, restraint of the animal is still required during this procedure, which acts as a stressor in
itself altering secretory patterns of circulating stress hormones. To avoid this confounding effect,
Vahl and colleagues [37] concluded that blood samples should be collected in <2-3 minutes so
that sampling is completed before activation of the HPA axis.
2.3 Importance and Application for a non-invasive Corticosterone Measurement
In contrast to the blood CORT measurements, which are influenced by the stressful sampling
procedures, and which reflect a momentary situation, the collection of faeces allows for the
monitoring of chronic hormone metabolite levels without handling animals. The determination of
metabolites in animal urine samples is hampered by the difficulty of obtaining the samples, and
most of the CORT metabolites in rats are excreted via the faeces [10]. Changes in FCM
concentrations have been shown to be a good indicator of changes in blood CORT
concentrations in captive, experimental, and free-ranging animals. Wasser et al. [38] found that
the transfer of a captive owl from her usual enclosure to a novel environment resulted in a
8
comparable response in both serum and faecal CORT levels. Mashburn and Atkinson [39] found
that Stellar sea lions exposed to an ACTH challenge had a 3-fold increase in serum CORT
concentrations and an 18-fold increase in FCM concentrations.
Previous studies using rats [5, 13] have found demonstrable circadian rhythms with well-defined
acrophases and nadirs in faecal corticoids. Results from a number of studies indicate that the
circadian FCM rhythm is shifted between 6-12 hours compared to circulating blood CORT, but
the temporal dynamics are very similar in both mice [40] and rats [12-13]. Royo et al. [12]
demonstrated that faecal CORT can be used as a marker of acute stress in rats, since faecal
CORT showed a similar pattern to serum CORT with a time delay between the presence of
elevated CORT levels in blood and in faeces being approximately 12 h. Thanos and colleagues
[8] examined the applicability of a non-invasive faecal CORT metabolite measure to assess the
circadian rhythm by comparing faecal CORT metabolite levels to circulating CORT levels. The
use of a minimally invasive rapid blood sampling procedure altered both faecal output rhythm
and faecal CORT metabolite levels demonstrating that even a blood collection done in under 3
minutes can trigger stress in an animal [8]. FCM circadian rhythm was time-shifted from the
plasma CORT rhythm by approximately 7-9 h and reflected circulating CORT levels. The
relationship between the two measurements was linear and strong where rats with high faecal
CORT output also had high circulating CORT levels.
FR is a long-term stressor, and therefore, faecal sampling might provide a preferred method for
measuring corticosterone over blood measurements. A previous study using FR mice, found a
significant increase in overall FCM levels at 12 weeks and 24 weeks of food restriction
9
compared to the ad libitum control group [21]. In their study, the FR mice were fed a 60% ad
libitum calorically restricted diet, where diets were nutritionally balanced for equivalent
micronutrient levels.
There are a variety of factors associated with variations in measurement of faecal glucocorticoids
and their metabolites including but not limited to diurnal and seasonal variation, reproductive
status, diet, habituation, sex, and species variation [41]. A study in mice [40] showed that both
sex and time of day impacted the metabolism and excretion of CORT such that males excreted
more FCMs than females (73% vs 53%), and the mouse wake cycle resulted in an increased 3H-
Corticosterone metabolite excretion rate compared to their sleep cycle. Furthermore, the diet of
an animal can affect FCM concentration as well. For instance, a diet high in fibre can decrease
gut transit time [42], which could result in less time for re-absorption of glucocorticoids [43]
and, therefore, increased FCM levels.
While the current study will use female rats, the results should also be applicable to male rats,
but with males having expectedly higher values [13]. Cavigelli et al. [13] looked at sex
differences between rats and found that females excreted less overall mass of corticoid
metabolites from blood into faeces than males, which might have been due to their plasma
corticosterone-binding capacity exceeding that of males and having a slower fractional clearance
rate. Thus, females may catabolize less corticosterone through the liver and / or excrete less mass
of corticosterone metabolites in faeces, and more in urine, than males. However, between-sex
similarities in the rhythms of faecal corticoid excretion (i.e. acrophase and nadir) were clear,
therefore, confirming the usefulness of corticosterone fecal measures in both sexes. Lepschy et
10
al. [6] found no sex differences concerning the route of excretion of GC metabolites.
Furthermore, most of the CMs were excreted via the faeces (75+/-9%) in both sexes. Using mice,
Touma et al [40] found similar results where most of the CORT metabolites were excreted via
the faeces in both sexes, but the amount of radioactivity recovered in the faeces of males was
higher than females. The time courses of 3H-Corticosterone metabolite excretion in urine and
faeces, however, did not differ between the sexes.
The basal activity of the HPA axis tends to increase with age in rats as revealed by increased
CORT [44]. Han et al. [45] observed the effects of 60% food restriction in male rats of three
different age groups. They found that when the rats were 9-months of age, afternoon plasma total
and free CORT concentrations of FR rats were significantly higher than those of ad libitum fed
rats. At 15- and 21-months of age, only afternoon free CORT levels in FR rats were significantly
higher than those in ad-libitum fed rats. With an ACTH injection, peak levels of total CORT
were significantly higher in FR compared with ad libitum rats at 10 and 16 months of age.
However, at 22 months of age, the difference disappeared between the two groups, but peak
levels of free CORT were maintained at higher levels in FR rats at all three ages. Their findings
indicate that the enhanced response of total CORT to ACTH in FR rats extends to 18 months of
age, and for free CORT, extends to 22 months of age. Thus, it can be expected that the FR rats in
the present study, which were 4 months of age, will show both increased total and free CORT
levels compared to ad libitum fed rats.
Although many strains of rats show increased CORT levels in response to stress, variability has
been observed between different strains. Using a Plexiglas restrainer, Dhabhar et al. [46] tested
11
stress responses of three different rat strains to 1h of acute restraint stress, once in the morning
and once in the late afternoon on two successive days. They found differences in CORT levels
between the three strains, with Fischer 344 (F344) rats showing consistently higher diurnal and
stress CORT levels than Sprague-Dawley (SD) and Lewis (LEW) rats. In the morning, basal
CORT levels of all three strains were low. In the evening, SD and F344 rats showed the diurnal
rise in CORT levels, while the LEW rats did not. The stress CORT response of F344 rats was
significantly higher than the SD and LEW rats in the morning and evening. When differences
were observed, LEW rats consistently showed lower levels of CORT than both SD and F244 rats
to the restraint test. In another study, following exposure to a novel environment, no differences
were observed between Sprague-Dawley (SD) and Wistar rats in CORT release, while basal
locomotor activity was higher in Wistar rats than SD [47]. Based on studies demonstrating
existing differences in CORT responses to stress between different rat strains, it is important to
note the strains used in similar studies when comparing values and results. Since Wistar rats
have been shown to be similar in the CORT response to SD, we can expect our Wistar rats to
show both lower diurnal and stress response levels of CORT than F344, but higher than LEW
rats.
Oitzl and colleagues [48] conducted a study looking at a series of differences in HPA-axis
regulators between LEW and Wistar rats. They found that LEW rats displayed an increased
capacity of mineralcorticoid receptors (MR)s in the hippocampus and hypothalamus and a
decreased capacity of glucocorticoid receptors (GR)s in the pituitary while the binding affinity
for MRs and GRs in the hippocampus was comparable. Lower concentrations of CRH mRNA
were detected in the PVN of the LEW hypothalamus. While adrenal weight was similar between
12
the two strains, LEW rats had about 30% less adrenocortical cells. Subjecting adrenocortical
cells to increasing doses of ACTH in vitro resulted in about a 60% smaller release of CORT in
LEW rats. LEW rats responded with lower ACTH and CORT levels than Wistar rats to a variety
of stimuli including a tail nick and restraint. Their results may offer explanations to the
variability seen between rat strains. The authors suggest that the shift of the central MR/GR
balance of LEW rats is the central regulating mechanism of the hyporeactive HPA axis in this rat
strain.
Wistar rats have previously been used in studies examining plasma CORT, faecal corticosterone,
and the correlation between food restriction and glucocorticoids [49-51], because similar to
humans, caloric restriction induces an increase in cortisol/corticosterone. Wistar rats have also
been used in activity based anorexia (ABA) studies [52] as they show similar hormonal patterns
to AN patients: decreased leptin levels co-occurring with weight loss and an increase in leptin
and insulin sensitivity in the hypothalamus of exercised rats in an IL-6 dependent manner [53].
Therefore, for this study, Wistar rats provide a good model to examine the effects of food
restriction on CORT levels.
2.4 Corticosterone production 2.4.1 Steroidogenesis
Corticosteroids are synthesized from cholesterol that is derived from receptor-mediated uptake of
lipoproteins, intracellular stores of esterified cholesterol or de novo synthesis from acetate [54].
The initial step in steroidogenesis is the conversion of cholesterol to the first steroid
pregnenolone. Pregnenolone is the precursor to every steroid hormone and constitutes a rate-
13
limiting and hormonally regulated step in steroidogenesis. The transfer of cholesterol from the
inner- to outer-mitochondria is rate-limiting, because the hydrophobic cholesterol cannot traverse
the aqueous intermembrane space of the mitochondria and reach the P450scc enzyme rapidly
enough by simple diffusion to support acute synthesis [55]. ACTH induces the expression of
StAR to increase the entry of cholesterol across the inner and outer mitochondrial membranes to
be metabolized by P450scc to pregnenolone [55].
Following ACTH stimulation of the cells, hormone sensitive lipase (HSL) mediates cholesterol
ester hydrolysis to free cholesterol, which is then released from lipid droplets to the mitochondria
[56]. The steroidogenic acute regulatory protein (StAR) is a transport protein that mediates the
delivery of cholesterol from the cytosol to the mitochondria to the site of its first enzymatic
conversion of cholesterol to pregnenolone by P450scc [56]. Inside the mitochondria, the action
of the cytochrome P450 side-chain cleavage (P450scc), converts the cholesterol to pregnenolone
[57-58]. Pregnenolone then exits the mitochondria and 3beta-hydroxysteroid dehydrogenase
(3beta-HSD) converts it to progesterone in the microsomal compartment [55].
There are reported cases of dissociation between CORT and ACTH, such as those observed in
depression, AN, and Alzheimer’s Disease [59-61]. Therefore, there must be other mechanisms
controlling CORT levels aside from the HPA axis. It appears that there are ACTH-independent
factors regulating the adrenal cortex including the immune system and neural input. Sympatho-
adrenal control may regulate both adrenal medullary secretion of catecholamines and also
cortical function by adrenal innervation [62].
14
2.4.2 Hypercortisolism
Several mechanisms have been postulated for the hypercortisolism documented in AN patients,
including decreases in cortisol clearance, affinity for cortisol binding globulin, and increased
glucocorticoid-receptor concentration [60]. However, defects in clearance or protein binding
alone cannot produce hypercortisolism in patients with normal hypothalamic and pituitary
responsiveness to the negative feedback of cortisol [60]. The dissociation between plasma ACTH
and CORT observed in some cases may result from changes in adrenal responsiveness to ACTH
induced by stress. ACTH is the only naturally occurring agonist for the ACTH receptor, which is
principally expressed in cells of the adrenal cortex [63]. This receptor signals the cells in the
zona fasciculata of the adrenal cortex to synthesize and secrete CORT. If adrenal cortical
secretion is controlled exclusively by plasma ACTH, then the changes in plasma CORT should
occur in parallel with changes in plasma ACTH [63]. However, as already discussed, there are
several reports of dissociation between changes in plasma ACTH and CORT, suggesting that
factors in addition to ACTH could contribute to adrenal CORT production.
15
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Figure 1 - Possible Explanation for Dissociation of ACTH and CORT
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16
Although decreased ACTH response to CRH observed in AN patients indicates the presence of
feedback at the pituitary level, inappropriately normal baseline ACTH levels and failure of
dexamethasone to fully suppress cortisol suggest possible impairment in feedback at a level
above the pituitary [64]. An elevated CRH level in the cerebral spinal fluid in AN has been
observed and implies a CRH-driven process [64].
In addition to the HPA-axis, the stress system also includes central activation of the sympathetic
neurons leading to activation of both the systemic sympathetic nervous system and, through the
splanchnic nerves, the adrenal medullae. ACTH-independent factors, including the immune
system and neural input, are other explanations for the dissociation between the HPA-axis and
the adrenal cortex. Sympatho-adrenal control may regulate both adrenal medullary secretion of
catecholamines and also cortical function by adrenal innervation [65]. Studies indicate that a
large number of neuropeptides, neurotransmitters, opioids, growth factors, cytokines, adipokines,
and bacterial ligands are capable of modulating adrenal CORT release independent of pituitary
ACTH [66]. Adrenocortical cells express a variety of receptors for these factors, enabling them
to act directly on CORT release [66].
Many studies indicate the prevalence of CORT release controlled at the level of the adrenals
themselves. The adrenal gland is composed of two glands: an inner medulla comprised mainly of
chromaffin cells and an outer cortex comprised of steroidogenic tissue, all of which is
surrounded by the adrenal capsule [63]. The adrenal cortex receives abundant innervation by
nerve fibers of both extrinsic and intrinsic origin. Along with the direct innervation of cortical
cells, the adrenal medulla itself may indirectly govern cortical function by secreting
17
catecholamines or neuropeptides. Cortical cells are interspersed within the adrenal medulla, and
clusters of chromaffin cells lie adjacent to cortical cells in the outer cortex [67]. Since secretion
by chromaffin cells is regulated predominantly by sympathetic preganglionic innervation,
sympathetic innervation of the adrenal could regulate secretion of medullary factors that act
locally to influence cortical function [68]. FR to a certain time of day has previously been shown
to affect steroidogenesis at the level of the adrenals by controlling StAR gene expression within
the adrenocortex [66].
2.4.3 Hormone Regulating Factors: Leptin, Liver, and HDL
Both AN and malnutrition are characterized by a marked decrease in circulating leptin
concentration and an increase in cerebral spinal fluid neuropeptide Y (NPY) concentration [69].
NPY was shown to both suppress and stimulate sympathetic activity depending on location in the
brain [70]. These hormone and protein levels may provide an explanation as to why the HPA
axis in these subjects is activated in the presence of a profoundly hypoactive locus ceruleus
noradrenergic (LC-NE)-sympathetic system [69]. Stress is involved in the regulation of appetite
by influencing the appetite-satiety centers in the hypothalamus. Fasting-stimulated increases in
NPY enhance CRH secretion, while they concomitantly inhibit the LC-NE-sympathetic system
and activate the parasympathetic system, thereby facilitating digestion and storage of nutrients
[69].
Leptin is a satiety-stimulating polypeptide secreted by the white adipose tissue. Leptin inhibits
hypothalamic NPY [69] and has been shown to also have an inhibitory effect on steroidogenesis
[69]. There is an inverse relationship between leptin and CORT observed during food restriction
18
and recovery. The decrease in leptin levels occurring during starvation may lead to increased
CORT due to lack of leptin’s inhibitory effects on StAR, P450scc, and 3betaHSD. Lin and
colleagues [69] demonstrated how leptin inhibits 8-bromo cAMP-stimulated progesterone
production in a concentration-dependent manner. Leptin also inhibits the expression of cAMP-
stimulated StAR protein [69]. A leptin-induced inhibition of expression of the steroidogenic
enzymes cytochrome P450 C21-hydroxylase (P450C21), side-chain cleavage (P450SCC), and C17
!-hydroxylase (P45017!) has been demonstrated in the bovine adrenal gland [70-71], in rat [71],
and human adrenocortical cell in vitro. Furthermore, in mice, the stimulation of CORT secretion
induced by starvation or restraint stress can be partially counteracted by concomitant
administration of leptin [73]. Cherradi et al. [72] showed that the physiological induction of
StAR protein by ACTH is significantly reduced by leptin treatment. Their data indicate that
leptin can counteract ACTH-stimulated steroidogenesis by preventing the hormone-induced
increase in StAR mRNA steady state levels. Leptin’s effect on CORT production is independent
of weight loss as demonstrated by Gairdner and Amara [19] who recently showed that the
reductions in leptin in response to severe food restriction was not correlated with body fat when
fat mass was low.
The liver is fundamental to the metabolism of biomolecules including carbohydrates and lipids.
Hypothalamic and midbrain nuclei are connected via vagal and splanchnic nerves to the liver,
allowing the organ to participate in the control of food intake by sensing and regulating the
energy status of the body. The liver is considered an important constituent of the food-
entrainable oscillator (FEO) [73]. Before food access, there is a prevalence of oxidized
cytoplasmic and mitochondrial redox states, an increase in adenine nucleotide levels, an
19
enhanced mitochondrial capacity to generate ATP, and a hypothyroidal-like condition that is not
systemic but exclusively hepatic [74]. However, after feeding, the hepatic redox state becomes
reduced in both cytoplasmic and mitochondrial compartments, the levels of ATP decline, and the
level of T3 within the liver increases. It has been found that imposing a restricted daylight
feeding time will uncouple the rat liver circadian activity from the SCN rhythmicity along with
inducing adaptations in the size, ultrastructure, and glycogen and triacylglycerol content in
hepatocytes [75]. Diaz-Munoz et al. [75] showed that the main adaptations caused by the
restricted feeding schedule occurred during the food anticipatory activity, and could be
accounted for as a cellular and metabolic anticipation by the liver in preparation for processing
more efficiently the ingested nutrients.
During chronic stress, there is a sustained import of cholesterol in the cell and mitochondria.
Cummins et al. [76] showed that the liver X receptors (LXRalpha and LXRbeta) prevent
accumulation of free cholesterol in mice adrenal glands by controlling expression of genes,
including StAR, involved in all aspects of cholesterol utilization. Under chronic dietary stress,
adrenal glands from LXR-alpha and LXR-beta deficient mice accumulated free cholesterol
implying that these liver receptors act as a protective mechanism to control elevated cholesterol
levels. Liver X receptors stimulate expression of genes that lower cholesterol. There are two
forms of identified LXR: LXR-alpha and LXR-beta. LXR-alpha is expressed at high levels in
liver but also at more modest levels in cells that are involved in cholesterol transport and
metabolism. LXR-beta are known to control the expression of genes involved in transport of
excess cholesterol from peripheral tissues to the liver and hepatic metabolism of this cholesterol
by cytochrome P450 7A1 to bile acids. The hyperproduction of CORT in the adrenal cortex is
20
associated with very high levels of cholesterol transport, lipoprotein receptors (LDL receptor and
the HDL receptor known as scavenger receptor-B1 (SR-B1), stored cholesterol esters, and
enzymes that metabolize cholesterol.
StAR has been shown to be up-regulated by cholesterol containing lipoproteins, including both
low density lipoproteins (LDL) and high density lipoproteins (HDL) [77-78]. HDL provides one
of the only ways to clear cholesterol from the body by returning cholesterol from the tissues back
to the liver [79]. Increased expression of StAR, together with increased cholesterol substrate
availability, enables adrenocortical cells to rapidly increase steroid hormone synthesis [78]. Both
HDL and LDL have been shown to increase expression of StAR mRNA 2-3-fold [78].
In general, FR lowers serum cholesterol and triglycerides relative to controls. Specifically, HDL
cholesterol levels, however, in rodents have been reported to be both lower [80] and higher [81]
in FR than in controls. Energy restriction leads to a significant decrease in cholesterol and
triglyceride levels, with an increase in levels of HDL2, particularly HDL2b cholesterol [82-83].
Verdery and colleagues [82] found monkeys restricted to 70% of ad libitum calorie intake for 6-7
years was accompanied by an increase in HDL sub-fractions HDL2b and HDL1+2b. The HDL
cholesterol has been reported as both unchanged [84] and higher in AN patients [85-87] with
normal lipid levels or increased [86-87] levels of LDL.
21
2.5 Corticosterone Metabolism
Circulating steroid hormones are metabolized by the liver and excreted as conjugates via the
kidneys into the urine or via the bile into the gut [88]. Steroids may enter the enterohepatic
circulation to be reabsorbed into the blood stream and are extensively metabolized by the
microbial flora, but the sterane skeletal structure is not degraded [89]. This allows for detection
of steroid metabolites in the faeces of mammals. As mentioned previously, a lag time occurs
between the instantaneous blood CORT levels and the faecal CORT metabolites. This lag time
depends mainly on the intestinal transit time from the duodenum to the rectum and is largely
species-specific [89]. Rather than the actual steroid concentration, faecal hormone metabolite
levels reflect the production rate or the cumulative secretion and elimination of hormones over
several hours [89].
Corticosterone is metabolized by the liver prior to excretion both through the urine and the
faeces via the bile [90]. Only free CORT that is not bound to CBG is degraded by the liver.
CORT is normally tightly bound to the carrier protein, CBG, and only 5-10% of CORT is in the
free form, unbound and biologically active [91-92]. Since only the free CORT fraction from the
blood is available for metabolism and excretion, FCM concentrations more accurately reflect the
biologically active portion of free CORT [90, 92-93].
Radiometabolism studies have allowed us to understand the metabolism and excretion of
corticosterone including the route, the time course of excretion, and the types of metabolites
formed [6, 40]. Radiometabolism studies have been conducted on a variety of animals including
mice [40], rats [94], and snowshoes hares [7]. The route and delay of excretion as well as the
22
metabolites formed with faecal glucocorticoids differ largely between species [40, 95-96]. Mice
were found to have a lag time of 10 hours, whereas rats can have a lag time of 6-9 h following an
injection of 3H-corticosterone [13]. Time of day of administration is shown to influence the
excretion rate of CORT metabolites due to physical activity levels [39]. Touma et al. [40]
demonstrated that concentrations of 3H-corticosterone metabolites were already recorded after 4h
post-injection. Mice injected in the morning at 9am dispalyed peak CORT levels 10h post
injection and those injected in the evening at 9pm displayed peak CORT 4h post injection thus
proving an effect of the time of day. Therefore, when measuring faecal CORT, it is important to
recognize that increased activity levels can speed the gut passage time, and thus the excretion
time.
Interpretations of faecal assays are based on the assumptions that FCM reflect free, biologically
active, CORT levels in the plasma, and that differences in FCM levels are an accurate reflection
of an animal’s physiological state and thus of its ability to respond to a stressor [7]. Sheriff et al.
[7] verified these assumptions in a population of free-ranging snowshoe hares. Plasma free
CORT levels mirrored FCM levels, but plasma total CORT levels did not. Differences in FCM
concentrations among hares predicted their response to a hormonal challenge where hares with
higher FCM concentrations showed a greater resistance to the suppression of their free plasma
CORT following a dexamethasone injection. They also showed a marked increase of free plasma
CORT and FCM concentrations following an ACTH injection.
23
2.6 Corticosterone Measurement Techniques: RIA and EIA
There is a large variation between species in the kind of glucocorticoid metabolites that are
excreted. Therefore, selection of the proper antibody for use in an immunoassay test is a crucial
step in validation of the assay [97]. Many antibodies have cross-reactivity for other steroids or
steroid metabolites in the sample. Thus, it is important to use an antibody with little cross-
reactivity to prevent measuring additional corticosteroids and metabolites, which can lead to
false interpretations [41].
Immunoassays, including radioimmunoassay (RIA) and enzyme immunoassay (EIA or ELISA),
are commonly used for analyzing CORT levels. Both RIA and EIA can be competitive binding
assays and are highly sensitive. Competitive binding assays require an antibody directed against
certain parts of the steroid molecule of interest. RIAs rely on a radioactive isotope to generate a
radioactive signal to quantify CORT levels. RIAs have the disadvantage of using radioisotopes,
and the disposal of radioactive material can be difficult [28]. CORT determination from blood
samples can be obtained using either plasma or serum since both give the same result [98]. The
EIA corticosterone antiserum that will be used in the current study (CJM006) has previously
been used for both serum and faecal CORT measurement in ferrets [99] and rhinoceroses [100].
Physiological validation of FCM measurements can involve pharmacologically inducing
physiological changes in circulating glucocorticoid levels and evaluating whether these changes
are reflected in measured concentrations of FCMs afterward [89]. A number of strategies can be
used to determine whether CORT is responding in the expected physiological manner. These
24
validation methods include injecting animals with radioactive cortisol to recover radioactive
metabolites (as previously discussed), investigating diurnal rhythms, and exposing animals to a
known stressor.
Measuring the naturally occurring diurnal variation of CORT from faecal samples in a given
species can indicate biological relevance [12, 89]. Bamberg et al. [10] were unable to
demonstrate that changes in adrenocortical activity were well reflected by concentrations of
FCMs measured by their applied in-house corticosterone immunoassay and found their CORT
EIA to be unsuitable. The strong diurnal rhythm of the FCM observed might be an explanation
for the findings of that experiment since their attempt to detect higher CM concentrations caused
by ACTH stimulation might have been masked by the diurnal variation [6, 10]. Without
investigating the natural diurnal variation via a rigorous sampling regime, it might not be
possible to distinguish between the diurnal variation peak of CMs and a peak caused by an
ACTH injection or a stressor [6].
An ACTH challenge can be used to validate the CORT assay and to assess adrenal sensitivity.
Faecal samples should be ideally collected frequently before and after the injection of ACTH,
and they should reflect the sharp increasing and decreasing CORT levels after a certain lag time.
Out of 140 articles published in peer-reviewed journals dealing with faecal CORT in more than
70 species of mammals and birds, Touma and Palme [89] only found convincing physiological
and biological validation experiments in a few studies dealing with FCMs. An ACTH challenge
has previously been used in the literature to examine CORT in blood and faeces in ad libitum fed
rats [5]. It was shown that CORT rapidly increased in blood after ACTH stimulation, and an
25
increase in CORT excretion was detected in faeces 8 hours after ACTH injection. In contrast,
Bamberg et al. [10] demonstrated the presence of a diurnal variation (DV) and the suppression of
FCM as expected after a dexamethasone suppression test in rats. However, they could not find
any increase in the metabolites following an ACTH stimulation test. Therefore with the
antibodies used in their study, it would not be possible to monitor any stressor which acts for
only a short period of time.
An ACTH challenge has previously been used in FR rats to analyze adrenal sensitivity [101].
Han and colleagues [101] found that both in vitro and in vivo adrenal responsiveness to ACTH
was higher in FR than ad libitum rats. Garcia-Belenguer et al. [102] showed that a slight food
restriction to 85% of the ad libitum intake was sufficient to increase the pituitary reactivity to
exogenous CRF with the diurnal rise of CORT starting earlier in FR animals. Thus, it would be
expected to observe an increase in FCM in both FR and ad libitum fed animals to an ACTH
challenge but with FR rats displaying higher FCM levels than the ad libitum group. The ACTH
stimulation test has also been performed on AN patients resulting in an increased CORT
response than average weight participants [103]. These results further confirm that the adrenal
gland overproduces CORT in AN compared to healthy weight individuals.
2.7 Cortisol/Corticosterone Response to Food Restriction !Some animals have prolonged natural fasting to adapt to environments where food is either
unavailable or where feeding would disrupt activities of greater importance (e.g. hibernation,
incubation). The ability to suppress the stress response may permit fasting animals to utilize fat
26
stores and spare protein by preventing the catabolic, protein-mobilizing effects of glucocorticoids
[30]. Fasting is characterized by three phases: During phases I and II, glycogen stores and then
lipid stores, respectively, are the sources for energy with concentrations of corticosterone,
insulin, and thyroid hormone remaining low. If the fast continues until lipid stores reach some
critical lower limit, the body enters phase III and utilizes protein for its energy source.
Corticosterone and glucagon concentrations now increase markedly, and because CORT
promotes protein mobilization, protein now becomes the main energy source [30]. The net result
is muscle wasting.
It has previously been shown that fasting hypercorticosteronemia is caused predominantly by a
reduction in hormone clearance from the plasma, and that this is related to a reduced capacity for
hepatic metabolism of the hormone [94]. After a 48 h fast, rats were anaesthetized using
halothane and CORT was injected into the femoral vein. The tail artery was immediately
cannulated and blood samples of approximately 200 ul were taken into heparinized tubes at 5,
10, and 15 minutes after injection. After 20 min, a final blood sample was taken by cardiac
puncture and the animals were sacrificed. Woodward et al. [94] demonstrated that both sexes of
fasted rats had reductions in metabolic clearance rate (the volume of plasma cleared of steroid
per unit time), increases in plasma CORT concentration, and no change in plasma secretion rate.
They concluded that the increased plasma steroid levels are probably due to a reduction in the
rate of removal of hormone from the plasma, with no change in the rate of secretion from the
adrenal gland [94]. More recently, however, it has been demonstrated that the basis for the
hyperadrenocorticism in caloric restricted rats resides in the adrenal cortex as the consequence of
an enhanced sensitivity of adrenal cells to ACTH [101]. This is supported by studies showing
27
increased plasma CORT levels in the presence of decreased ACTH in FR rats [50]. Under
caloric restriction, there are elevated levels of plasma corticosterone [49-51] and it has been
demonstrated that CORT levels increase parallel to the amount of caloric restriction [51].
Most, if not all, daily rhythms are generated by an endogenous circadian oscillator, which, in
mammals, is located in the suprachiasmatic nucleus of the hypothalamus (SCN) [104]. The SCN
controls the rhythm of CORT secretion via direct and indirect neural control of CRH release and
subsequently ACTH [105], and through autonomic innervation of the adrenal gland [106]. Meal
time is a powerful external cue that can alter CORT diurnal rhythm when restricting food
availability to under a few hours per day leading to an anticipatory peak of CORT release 1-2 h
before the availability of food [107]. The daily schedule of food availability induces a feeding-
associated increase in locomotor activity prior to the expected time of feeding [108-109]. In food
restricted rats, there are two distinct peaks in plasma CORT: the light-dark (LD)-associated
component and the feeding-associated one [110-112]. This diurnal peak anticipating the time of
feeding has been correlated to a prefeeding release of norepinephrine in the paraventricular
nuclei of the hypothalamus where corticotropin-releasing hormone is produced and leads to an
activation of the HPA axis [113].
Restricted daily feeding of rats (being fed 50% of ad libitum daily food intake 2 h after the onset
of light) does not appear to affect the phase of the nocturnal peak of plasma CORT as it appears
to remain insensitive to feeding conditions [49]. After one month of restricted daily feeding, the
CORT anticipatory feeding peak was measured at 60 ng/ml compared to the nocturnal CORT
peak of 130 ng/ml [49]. But 2 months of restricted daily feeding resulted in the CORT
28
anticipatory feeding peak to be higher than the nocturnal peak (115 ng/ml vs 95 ng/ml). Thus,
one can expect both the CORT peaks to change over duration. Girotti and colleagues [107]
demonstrated that when rats are food restricted to three hours per day for 24 days, there is an
anticipatory peak in CORT secretion, but not ACTH 1h before daytime feeding.
Time of feeding may impact the ability to observe a difference between ad libitum and FR serum
CORT. The time of food availability has been shown to affect the CORT diurnal rhythm as
shown when food restriction to 2 hr [114], 3h [107], or 4h [111, 115] of the light period shifts the
CORT peak to the onset of the eating period. A CORT peak is not detected when food
availability is extended to 6 h or more [111, 116] in this condition, probably because the rats are
able to consume the amount of food normally ingested in 24h. Rats provided with food an hour
before dark phase onset also did not demonstrate a shift in peak CORT levels from the beginning
of the dark phase to time of feeding [51].
Restricting feeding to a certain time of day also affected StAR gene expression within the
adrenocortex, suggesting that timed FR resets the timing of peak steroidogenic activity of the
adrenal gland. The adrenal oscillator was affected by RF, reversing the expression profile of each
clock gene examined. Girotti et al. [107] suggest that the FR-dependent shift in CORT peak
secretion may result from both an altered response to changes in an extrinsic signal and the
resetting of the adrenocortex functional state, probably as a result of altered clock gene
expression. It is important to note that hypocaloric and not normocaloric timed FR has been
shown to change the oscillatory pattern of the master clock [117]. Therefore, it is important to
administer food during the wake cycle when comparing FR rats to ad libitum fed rats. One would
29
also expect to find a similar nocturnal peak of plasma CORT with an additional anticipatory
CORT peak prior to the onset of food.
Previous literature indicates an increase in CORT production during stress and food restriction,
with the ability to measure this hormone through blood and faecal samples. Serum and plasma
CORT measurements have shown increased levels of CORT production in FR animals. Blood
measurements are an invasive technique and the handling involved can result in increased stress.
FCMs are a non-invasive tool of collecting CORT samples and have previously been used in rats
to observe a stress response. However, it remains unclear as to whether this hormone can be
measured through FCM in FR rats. Therefore, the current study examined the possibility of
measuring FCMs in FR rats in order to find a non-invasive technique to measure CORT
production.
30
2.8 Objectives !1. To determine faecal CORT response in a rat model of food restriction
To the best of my knowledge, there is currently no research analyzing faecal CORT in food
restricted rats. Therefore, the first objective is to identify a CORT response to food restriction via
FCM. If CORT levels are increased in FR rats, and if it is possible measure CORT metabolites in
faecal matter, then it should be possible to identify a CORT response to food restriction in faeces
if sampled over a sufficient time frame. It is hypothesized that both peak and 24-hr CORT levels
will be higher in the FR group at the end of the one-week FR intervention period.
2. To evaluate the effect of FR on CORT adrenal sensitivity
The second objective is to determine the impact of food restriction on adrenal sensitivity by
comparing the CORT response to an ACTH challenge between FR and ad libitum fed animals. In
addition, the measurements at 30, 60, and 120 minutes post injection!will allow us to determine
the magnitude of the CORT response. We hypothesize that FR animals will show higher levels
of serum CORT to the ACTH challenge than Control animals as previously research indicates
that adrenal responsiveness to ACTH is higher in FR than ad libitum rats [101].
3. To compare the temporal pattern of faecal CM and serum CORT to determine whether
differences exist between ad libitum fed and FR animals
A third objective is to observe whether the FCM will show a diurnal rhythm with peak
CORT levels occurring at the beginning of the wake cycle and levels declining throughout the
day. It is hypothesized that faecal CORT will have around a 9h time lag from serum CORT in
31
the Control as reported in previous papers. This time lag will be even greater in the FR group
compared with the ad libitum, because the rate of metabolism will be slower. Similar patterns
between the two measurements are expected. It is hypothesized that if serum CORT levels
increase in FR rats, then faecal CORT will also be elevated compared to ad libitum fed controls.
Based on the knowledge that resting energy expenditure (REE) is significantly lower in AN
patients than in healthy volunteers [118], and that caloric restricted rats have slower metabolisms
[119], we postulate that faecal CORT metabolites will be time-shifted from the plasma CORT
rhythm in FR rats more than ad libitum fed rats (i.e., they will occur later).
If group differences in CORT exist between faecal and blood measurements, then it will
consequently be possible to recognize the correct window of time to most accurately capture
peak corticosterone levels in faecal matter in food restricted rats. This study will be sampling
faeces every two hours in the first 12 hours post ACTH injection and every 4 hours 24 hours
following. This ensures enough sampling times to most accurately capture peak corticosterone
levels in faecal matter in food restricted rats.
32
Chapter 3 Methodology
3.1 Data Analysis
A previous study in this lab used adolescent female Wister rats to mimic the caloric
deprivation seen in AN that mainly affects women [19]. That study was conducted over four
weeks, but demonstrated an ability to bring the animals’ body weight down to 88% of original
body mass by the end of 1 week while being fed 7g of chow per day or 75% of ad libitum fed
rats. In this current experiment, there were 12 adolescent female Wistar rats (Charles River,
Quebec, Canada) using the same degree of food restriction over one week. All animals were
housed in separate cages with food and water ad libitum. Constant temperature of 23-25°C was
maintained with a 12-h/12-h reverse light-dark cycle (lights off at 8:00 h, lights on at 20:00 h).
There was a 1-week acclimation period for animals to adjust to the opposite light/dark cycle and
to their new environments. During acclimation, animals were placed in a metabolic cage for 30
minutes everyday as these were the cages used during faecal sample collection. The rats were
given unrestricted access to water throughout the experiment and fed on standard laboratory
chow (Purina Standard Rat Chow).
After animals were acclimated for one week, an initial baseline measurement (Baseline 1) was
taken that included one blood sample from each rat and faecal samples every 2 hours for 24
hours. A second baseline measurement (Baseline 2) was taken the following morning to ensure
that the samples were true baselines. Animals were then divided into two groups, ad libitum fed
controls (CON) (n=8) and 1-week food restricted (FR) to 88% - 92% body mass of controls
33
(n=10), all singly housed. FR rats were fed 7g/day, and all rats were weighed on days 1, 3, and 7
to confirm the degree of weight loss and projection for Day 8. To assess the changes in CORT
levels, on Day 7, one blood sample from each rat and faecal samples every 2 hours for 24 hours
were acquired. On Day 8, a blood sample was drawn to determine the effect of FR on serum
CORT. The blood sample served as the baseline level prior to an intramuscular injection of
ACTH, which was followed by blood samples (300 ul) at 30, 60, and 120 minutes post injection.
On Day 8, faecal samples were obtained every 2 hours for 12 hours followed by collection every
4 hours until 32 hours post injection. Animals were decapitated as outlined in the animal use
protocol, at the end of collection in order to collect the trunk blood and organs including the
liver, adrenal glands, and heart. Future studies will be conducted to analyze the trunk blood and
organs to investigate the underlying mechanisms of increased CORT.
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34
3.2 Animals and Housing
Study rats were 4 months old upon arrival weighing between 264 and 341.2 grams. A study
conducted in the current study’s previous lab [19], showed FR rats fed 7g/day ate 75% of ad
libitum fed CON animals. Rats were housed in metabolic cages where excreta could drop
through the bars of the wire floor and be separate from urine. Fecal samples were collected
separately, stored at -20°C and time of sampling was documented. As per Bosson et al. [120], the
cages were polypropylene rodent housing cages (47cm X 26 cm X 20 cm) nested within a
polycarbonate rodent housing cage. There was a wire mesh cage that allowed for collection of
faecal samples without disturbing the animals. With this set up, faeces could pass through the
upper mesh, but not through the lower mesh, while urine was able to pass through both levels of
mesh to the bottom of the container. The cages were less comfortable for the rats, and they
therefore, were housed in standard rat cages with bedding except during sample collections.
During the week of acclimation, animals were handled by the same people each day and
familiarized with the wire mesh bottom cages for 30 minutes a day. The rats showed decreased
signs of stress from interacting with them, including less hyperactivity and noise. Animals did
not appear startled when removed from cages or upon entry into the room and were calm when
handled. Importantly, the same people who handled the animals during acclimation did all of the
handling on testing days. On Day 5, animals were transferred to the metabolic cages. Housing
for a few days in metabolic cages is not associated with major stress for laboratory rats [33].
35
3.3 Faecal Sampling
Faecal samples were collected at baseline and day 7 for 20 hours every 2 hours until 02:00h and
the final sample was collected with a 4hr window between 02:00h and 06:00h. Faecal samples
were collected in order to assess the level of change of CORT, to compare the FR group to the ad
libitum fed group, and to compare FCM to the blood samples. On Day 8, faecal samples were
collected every 2 hours for 22 hours and every 4 hours thereafter until 32 hours post initial
collection. Samples that were not contaminated with urine were collected and transferred to a 1.5
ml plastic snap cap, labeled, and immediately stored at -20°C.
3.4 Faecal Corticosterone Extraction
Faecal samples were sorted by date into 17x100 mm plastic snap cap test tubes. They were
lyophilized for 14-18 h to dry them out and to control for water content [92, 97] and
homogenized. 0.5g +/- 0.05g of faecal matter was weighed into the corresponding tube and
recorded for future calculations to convert values into ng/g faeces. When all samples were
weighed, 1.0 mL of 80% methanol in dH20 was added to each tube immediately using a repeater
pipette and the sample vial was sealed to avoid evaporation. Samples were mixed for 30 minutes
on a rotator/shaker. Samples were centrifuged at 1500 g for 15 minutes. Extracts were stored at -
20°C.
Prior to the current study, the in-house assay was biochemically validated with parallelism and
recovery tests. The sample followed a similar pattern to the standard reflecting the binding
36
characteristics of CORT (appendix 2). Based on these data, we used a 1:40 dilution for the
sample. For the CORT EIA, 41.7ul antibody stock (1:100, -20°C) was added to 5 ml coating
buffer (working dilution, 1:12000). Following that, 50ul of the mixed antibody stock with
coating buffer was added to each well using an Eppendorf repeater pipette. Plates were labeled,
tightly sealed, and incubated overnight at room temperature. High standard (25ng/ml) was
diluted serially 2-fold using 200ul standard and 200ul assay buffer to make 9 standards
consisting of 25, 12.5, 6.25, 3.12, 1.56, 0.78, 0.39, 0.195, and 0.09. Samples were diluted in
assay buffer to the appropriate dilution of 1:40, according to linearity test. 25ul HRP stock
(1:1000) was added to 5 ml assay buffer (working dilution, 1:200 000), plates were washed 5
times with wash solution and blotted on paper towel to remove excess wash solution. For plate
loading, 50 ul standard, sample or control were added to each well. Using Eppendorf repeater
pipette, 50 ul of diluted HRP were immediately added to each well. Plates were tightly covered
and incubated at room temperature for 2 hrs. For the substrate, we added 40 ul 0.5M H2O2 and
125 ul 40 mM ABTS to 12.5 ml substrate buffer and mixed well. 100 ul of substrate was added
to all wells. Substrate mixture was covered tightly and incubated at room temperature for 30-60
minutes with shaking. Plates were read at 405nm with maximum OD of 1.0. !Sensitivity of the
CORT assay was 0.09 ng/mL. The inter-assay coefficient of variance was between 3.15 and
6.19% and the intra-assay coefficient of variance was between 4.4 and 10.1%.
3.5 Blood Sampling and ACTH Challenge
Animals were swiftly removed from their cages and ~300ul of blood was taken from the tail by
making a small lateral incision with a number 11 scalpel blade (Magna Almedic, Montreal
Canada) and allowing the blood to drop directly into a 1.5ml snap cap. After the sample was
37
drawn, pressure was place on the incision point to prevent further bleeding. Animals were then
returned to their cages in the animal housing room. All samples were taken in under 3 minutes,
as this time has been shown to have little effect on CORT levels [4, 121]. Two baseline blood
measurements on consecutive mornings were taken following 7 days of acclimation. Blood
samples were taken again on Day 7 after a week of food restriction to assess the effects of food
restriction on CORT levels. On Day 8, another blood sample was taken and followed by an
intramuscular injection of 4IU/kg Adrenocorticotropic hormone (ACTH) (Synacthen Depot,
CIBA, Ontario, Canada) in the hind limb. The protocol was based on a procedure outlined in
Boonstra and McColl [121]. Briefly, after the baseline blood sample had been drawn, an
intramuscular injection (4 IU/kg) of synthetic ACTH was administered. Animals were returned
to their cages until further blood samples were taken at 30, 60, and 120 minutes post injection.
3.6 Blood Corticosterone Extraction
Approximately 300uL of blood was taken from the tail vein and stored in a sterile microtubule.
The blood was left for 1.5-2 hours to allow for clotting, and then spun in a centrifuge at 3000
rpm for 12-15 minutes. Once spun, serum components were obtained by pipetting the
supernatant into a 1.5ml plastic cap tube and samples were then stored at -80 for later EIA.
Serum CORT EIA extraction was done according to kit instructions (mouse/rat Corticosterone
ELISA; ALPCO Diagnostics, Salem, NH). Briefly, 10µl of each calibrator, sample and control
with new disposable tips were dispensed into appropriate wells. 100µl of incubation buffer was
dispensed into each well. 50µl enzyme conjugate was then added into each well. Wells were then
incubated for 2 hours at room temperature on a microplate mixer. Sensitivity of the serum CORT
assay was 4.1ng/mL The inter-assay coefficients of variation reported by the manufacturer
38
ranged between 4.8 and 12.4%, and between 2.8 and 8.3% for the intra-assay coefficient of
variation.
3.7 Food Restriction Protocol
10 animals were food restricted for one week with unlimited access to water. They were placed
on a diet which consisted of 7g/day until the goal weight of 88%-92% of control body mass was
reached. The animals were individually monitored daily to reach the goal weight, since it has
previously been reported that animals adapt to reduced food intake in an individualized manner
[122-123].
3.8 Sample Size Justification
Using 10 male ground squirrels, Boonstra et al. [121] obtained significant differences in CORT
levels between two groups of squirrels following an ACTH injection (175.9+/-15.5nM/L for
Arctic ground Squirrels vs 343.6+/-32.4nM/L for Red squirrels; p<0.0001). 12 rats (n=6) were
used in an experiment by Chacon et al. [50] observing the effects of anesthesia and blood
sampling techniques on plasma metabolites and CORT in Wistar rats. This sample size was large
enough to see significant differences across the study groups from baseline concentrations
(19.4+/-5.6 ng/ml) with sampling procedure CORT levels (F6,60=9.90, P<0.05) altering in
comparison to the control procedure (+525 and 353% vs. +13%, both Ps <0.05).
Based on the results from Levay et al. [51], sample size for the current study was calculated
using Sigma Plot (minimal detectable difference in means = 50; expected standard deviation of
residuals = 56.6; number of groups = 2; desired power = 0.8; alpha = 0.05). A minimum sample
39
size of 10 total (n=5/group) is required in order to obtain significance comparing serum CORT in
FR rats to ad libitum fed rats. Due to the small sample size, we used 12 animals (n=6/group).
However, after analysis, it appeared that inadequate power was responsible for the lack of
significant findings for the FCM response to FR on Day 8. Therefore, another four FR animals
and 2 more CON animals were used giving us a sample size of n=10 for FR and n=8 for CON.
3.9 Statistical and analytical plan
Data are presented as means ± S.E.M. The means and variances of all groups were compared
with mixed model analysis of variance (ANOVA), corrected when appropriate for repeated
measures. In case of a significant main effect or interaction, a posteriori comparison was
performed using the Bonferroni HSD test. Statistical significance was accepted at p < 0.05. For
blood analysis, a 3-way ANOVA was used using group, day, and time as variables. Between
groups comparisons were analyzed for the effect of food restriction, and within group
comparisons were used to observe changes in CORT occurring across sample times.
40
Chapter 4 Results
4.1 Body Mass and Caloric Intake
FR animals were between 86 and 92% of original body mass and controls had a 0.7 to 9% weight
gain over 7 days (Table 1). Ad libitum fed animals ate more than the rats from Gairder & Amara
[19] (24.58±3.66g vs 16.0±0.63g), but this diet still reduced animals to the anticipated goal of
88% of original body mass. CON animals consumed on average 24.6g/day, while FR animals
consumed on average 7g/day or 28.5% of CON (Table 2). Food was provided at 14:00h every
day to the FR animals and was consumed within an hour.
Table 1 Change in Body Mass over 7 Days Measured in Grams ± S.D.
Body Mass
Group Baseline (g) Day 7 (g) % of Baseline to Day 7
Control 296.75±20.53 307.84±15.16 104.78
Food Restricted 290.16±26.54 258.67±24.65 89.12
Table 2 Average Daily Food Intake over 8 Days in Control and Food Restricted Animals Measured in Grams ± S.D
Day Control Food Restricted
Average Daily Food intake (g)
1 22.95±3.72 7.13±0.17
2 22.84±5.07 7.13±0.21
3 25.01±0.14 7.27±0.33
41
4 26.93±2.62 7.32±0.35
5 21.34±0.86 7.35±0.27
6 24.58±5.03 7.69±0.34
7 27.10±10.44 7.5±0.25
8 25.88±1.42 7.67±0.45
Total Average 24.58±3.66 7.38±0.30
4.2 Effect of 7 Days Food Restriction on Corticosterone Levels 4.2.1 Serum Corticosterone
All blood measurements were collected in under 3 min (Table 3). Baseline 1 blood samples
(6:30-7:00h) for serum CORT were not different between the groups, however a significant
difference between the groups was observed on Day 7 (p=0.007) with FR animals having higher
serum CORT (Fig. 3). The increase from baseline to Day 7 for FR animals did not reach
statistical significance.
Table 3: Speed of Sample Collection Measured in Minutes ± S.D.
Group Baseline (min)
Day 7 (min)
Day 8 (Baseline)
Day 8: 30 min (min)
Day 8: 60 min (min)
Day 8: 120 min (min)
Control 02:31±0.02 02.39±0.02 02:29±0.02 02:07±0.03 02:05±0.02 02:29±0.02
Food Restricted
02.35±0.02 02:56±0.01 02:24±0.02 02:24±0.02 02:18±0.02 02:33±0.02
42
Baseline Day 70
20
40
60
80
100ControlFR
*S
erum
Cor
t (ng
/ml)
Fig 3. Effect of food restriction on serum corticosterone. Blood samples were taken between 6:30 and 7:30am before the beginning of their dark cycle at 8:00am. FR animals showed higher levels of serum CORT than CON animals on Day 7 (p=0.007). The increase from baseline to Day 7 for FR animals did not reach statistical significance. (CON n=8; FR n=10).
4.2.2 Faecal Corticosterone Metabolites
FCM was originally examined using a three-way ANOVA analyzing groups (FR, C) X Time
(using 2 h blocks over 24 h) X Day (day of study protocol), but no significance was found using
all three variables. However, with a two-way ANOVA comparing the groups and a 24hr average
of FCM for each day, FR animals demonstrated a significant increase in FCM from Baseline
(295.4 ±16.2 ng/g) to Day 7 (419.1±25.1 ng/g) (p=0.000), while CON animals showed no
significant change from Baseline (355.4±18.5 ng/g) to Day 7 (384.1±20.9 ng/g) (p=0.213) (Fig.
4). Table 4 shows the 24 hour mean values of FCM for each group at Baseline, Day 7, and Day 8
ACTH challenge. There was a significant increase in FCM for CON Day 7 (384.1±29.1ng/g) to
Day 8(521.5±26.3ng/g) (p=0.001), but not for FR animals.
43
Table 4 Faecal Corticosterone Metabolites in Ad Libitum and Food Restricted Animals Group Baseline FCM
(ng/g faeces) Day 7 FCM (ng/g faeces)
Day 8 ACTH FCM (ng/g faeces)
Control 355.4 ±128.4 384.1±133.7 496.6±256.1 ** Food Restricted
294.6±145.6 419.1±250.5* 523.8±405.2
* indicates significant increase from Baseline to Day 7 in FR animals (p=0.007). Data are presented as ng/g ±S.D. ** indicates significant difference from Day 7 to Day 8 over a 24hr sample collection for the CON animals.
Baseline 1 Day 70
100
200
300
400
500
ControlFR
*
FCM
(ng/
g)
Fig 4. Effect of 7 days of food restriction on faecal corticosterone metabolites. Measurements are averages of 24 hours collections. FCM increased with 7 days of food restriction in the FR animals. (CON n=8; FR Baseline n=10; FR Day 7 n=9). 4.3 Circadian Rhythm FCM concentration in both groups of rats displayed a diurnal rhythm with a peak occurring
between 14:00h to 16:00h for CON animals (Fig. 5A) and between 16:00h and 18:00h for FR
animals (Fig. 5B). Peak values were ~ 2.7 times higher than nadir values for control animals on
Day 7 (457.7±64.5ng/g vs 210.5±111.8ng/g) and ~1.8 times higher than nadir values for food
44
restricted animals (496.3±55.9ng/g vs 275.2±79.0ng/g). The nadir for CON animals occurred
between 08:00h and 10:00h at Baseline and 06:00h and 08:00h on Day 7 (Fig. 5A). Figures 5A
and 5B illustrate the circadian rhythms of the CON and FR animals at Baseline and Day 7 with
the 20:00h-22:00h time omitted due to an unexpected dip. This omitted time point had limited
samples for both CON (3) and for FR (2) compared to the average number of samples at all other
time points combined (5.27 for Control and 3.64 for FR) (Table 4). Table 5 shows the number of
animals that provided samples for each time point. For graphs with the 20:00h-22:00h time
included, refer to appendix 3A and 3B.
To better visualize temporal patterns in FCM throughout a 24h cycle, data were normalized for
each animal by dividing by the average hormone concentration across all time points for their
particular group and multiplying by 100 (figures 6A and 6B). A clear circadian rhythm was
observed in the CON group. Figure 6A shows a monophasic peak in CON animals with
increased FCM from nadir at 08:00-10:00h (63% of the mean) to peak FCM at 16:00-18:00h
(125%) (p=0.003) at Baseline. A diurnal pattern was seen on day 7 with a significant increase in
FCM from nadir at 06:00-08:00h (58%) to peak at 14:00-16:00h (126%) (p=0.003). In figure 6B,
FR animals demonstrate a significant increase from nadir (68.1%) at 06:00-08:00h to peak FCM
(121.0%) at 14:00-16:00h (p=0.022) at Baseline, but no significant difference could be observed
in FCM throughout a 24h period on Day 7.
45
Table 5: Number of Animals that Produced Samples at a Given Time
% /01%2% /01%9%
#*=-%@'*.)%A<B% "'.)('7% 4''5%
&-,)(*+)-5%
"'.)('7% 4''5%
&-,)(*+)-5%
CDECCFC:ECC% O! G% 8% C%
:ECCF2CECC% H% 2C% D% I%
2CECCF28ECC% :% G% D% 8%
28ECCF2IECC% :% G% H% 8%
2IECCF2DECC% 9% 2C% D% 8%
2DECCF2:ECC% :% 2C% 9% :%
2:ECCF8CECC% :% G% 9% G%
8CECCF88ECC% H% I% J% 8%
88ECCF8IECC% 9% :% D% J%
8IECCFC8ECC% H% :% I% I%
C8ECCFCDECC% 9% G% D% I%
46
0
200
400
600
Baseline1Day 7A
FCM
(ng/g
)
6:00-8:00
8:00-10:00
10:00-12:00
12:00-14:00
14:00-16:00
16:00-18:00
18:00-20:00
20:00-22:00
22:00-24:00
24:00-2:00
2:00-4:00
4:00-6:00
6:00-8:00
8:00-10:00
10:00-12:00
12:00-14:00
14:00-16:00
16:00-18:00
18:00-20:00
20:00-22:00
22:00-24:00
24:00-2:00
2:00-4:00
4:00-6:000
200
400
600
Baseline1Day 7
B
Time (hrs)
FCM
(ng/g)
Fig 5. Circadian rhythm of animals at Baseline and Day 7 with the 20:00h-22:00h Time Omitted in CON (A), and FR (B). Black bars on X-axis represent the 12hr dark cycle from 8:00-20:00hrs. The grey arrows indicate the time of blood sampling at 07:00h. Black arrows indicate time of feeding at 14:00hr. Data have been double-plotted for clarity. CON n=8; FR Baseline n=10; FR Day 7 n=9).
47
0
50
100
150 BaselineDay 7** *
A
Norm
alize
d FCM
(%)
6:00-8:00
8:00-10:00
10:00-12:00
12:00-14:00
14:00-16:00
16:00-18:00
18:00-20:00
20:00-22:00
22:00-24:00
24:00-2:00
2:00-4:00
4:00-6:00
6:00-8:00
8:00-10:00
10:00-12:00
12:00-14:00
14:00-16:00
16:00-18:00
18:00-20:00
20:00-22:00
22:00-24:00
24:00-2:00
2:00-4:00
4:00-6:00
-50
0
50
100
150
200 BaselineDay 7
*
B
Time (hrs)
Norm
alize
d FCM
(%)
Fig 6. Normalized FCM of Circadian Rhythm in (A) CON Group and (B) FR animals. Black bars represent the 12hrs of dark cycle from 8:00-20:00hrs. The grey arrows indicate the time of blood sampling at 7:00hrs. Data have been double-plotted for clarity. A) * represents significant increase from nadir at 08:00-10:00h (63%) to peak FCM at 16:00-18:00h (125%) (p=0.003) at Baseline. ** represents significant increase from nadir at 06:00-08:00h (57.7%) to peak FCM at 14:00-16:00h (126%) (p=0.003) on Day 7 (n=8). B) * indicates significant increase from nadir (68.1%) at 06:00-08:00h to peak FCM (121%) at 14:00-16:00h (p=0.022) at Baseline (n=10).
48
4.4 ACTH Challenge
4.4.1 Serum
Both groups responded to an ACTH challenge with increased serum CORT levels compared to
Baseline before the injection. CON animals demonstrated increased serum CORT from Baseline
to 30 min (p=0.000) with CORT levels also remaining significantly higher than Baseline at 60
min (p=0.000), and 120 min (p=0.001) post ACTH injection. FR animals also exhibited an
increase in serum CORT from Baseline to 30 min (p=0.000), with this elevation also remaining
significantly higher than Baseline levels at 60 minutes (p=0.000) post ACTH injection. The
increase in serum CORT in response to the ACTH challenge was significantly greater in FR
animals compared to CON at 30 min (p=0.001) and at 60 min (p=0.002) post injection (Fig. 7).
Peak serum CORT was observed at 60 minutes, with levels declining by 120 minutes post ACTH
injection.
0 30 60 1200
200
400
600
800 ControlFR
* !
* !
* **
Time (min)
Ser
um C
ort (
ng/m
l)
Fig 7. Serum CORT of CON and FR animals after an ACTH challenge at Baseline 0, 30, 60, and 120 min post injection. (*) Significant difference in CORT from Baseline. (") significant difference between groups. (CON n=8; FR n=10).
49
4.4.2 Faecal Corticosterone Metabolites
Increased mean FCM levels over a 24hr collection period were seen in both groups after an
ACTH challenge (Fig 8). CON 24hr FCM was higher on Day 8 (521.5±26.3) compared to Day 7
(384.1±29.1) (p=0.001). Although there was a trend for higher FCM in FR on Day 8 vs Day 7,
the increase in FCM did not reach statistical significance for FR animals. When time points were
normalized over the 32hr period (Fig. 9B) on Day 8, CON animals significantly increased FCM
levels from nadir at 08:00-10:00h (76.7%) to peak at 16:00-18:00h (176.4%) (p=0.000).
Day 7 Day 80
200
400
600ControlFood Restricted
*
FCM
(ng/
g)
Fig 8. 24 hr average FCM measurements at Day 7 and Day 8. Food restriction began after Baseline measurements. There was a significant increase in FCM for CON Day 7 (384.1±29.1ng/g) to Day 8(521.5±26.3ng/g) (p=0.001) (CON n=8; FR n=9).
50
0
500
1000
1500
ControlFood Restricted
A
FCM
(ng/
g)
6:00-8:00
8:00-10:00
10:00-12:00
12:00-14:00
14:00-16:00
16:00-18:00
18:00-20:00
20:00-22:00
22:00-24:00
24:00-2:00
2:00-4:00
4:00-6:00
6:00-8:00
8:00-10:00
10:00-12:00
12:00-14:00
14:00-16:00
16:00-18:000
50
100
150
200
250
ControlFood Restricted*
Time (hrs)
Norm
alize
d FC
M (%
)
B
Fig 9. ACTH Challenge over 32hrs in Control and FR animals after 8 days food restriction looking at FCM ng/g (A) and Normalized FCM (%) (B). Black bars represent the 12hrs of dark cycle from 8:00-20:00hrs. The grey arrows indicate the time of blood sampling at 07:00hrs. Black arrows indicate time of feeding at 14:00hr. B)* represents significant increase in FCM from nadir at 08:00-10:00h (76.7%) to peak at 16:00-18:00h (176.4%) in CON animals (p=0.000) (CON n=8; FR n=9).
51
4.5 Sampling Effect There was a trend in the groups to show increased CORT levels on the second day of blood
sampling following a prior sampling the morning before (Fig. 10) but this increase did not reach
statistical significance. Mean CON serum CORT tended to be higher on Baseline 2 (73.3 ±
19.44ng/ml) than Baseline 1(40.21 ± 10.55ng/ml), and higher on Day 8 Baseline (56.76 ±
19.36ng/ml) than Day 7 (31.8 ± 14.62 ng/ml). In addition, there was a trend for mean serum
CORT for FR animals to be higher at Baseline 2 (69.59 ±16.84 ng/ml) than Baseline 1 (58.57 ±
9.13ng/ml), and higher on Day 8 Baseline (82.16 ± 16.764ng/ml) than Day 7 (80.16 ±
12.66ng/ml).
Baseline 1 Baseline 2 Day 7 Day 80
50
100
150
ControlFood Restricted
Ser
um C
ort (
ng/m
l)
Fig 10. Anticipatory Spike in Control and FR Animals Observed in Serum CORT. Two baseline measures were recorded each over 24hrs. Day 8 is based on the first 24hrs of collection beginning at 6:00-8:00h. (CON n=8; FR Baseline n=10; FR Day 7 and Day 8 n=9).
52
Chapter 5 Discussion
This study investigated whether FCMs could be used as a non-invasive measurement in
rats to evaluate the chronic effects of food restriction on CORT levels. Results showed
that FCM in CON animals demonstrates a diurnal pattern with peak levels occurring
around 8-10hrs after the initiation of the wake-cycle. FR animals appear to have a
flattened diurnal rhythm with no significant difference between peak and nadir CORT
levels. However, FR animals did have an overall increase in FCM compared to CON
after 7 days of food restriction. Only CON animals showed a significant increase in a
24hr collection of FCM in response to an ACTH challenge. 7 days of food restriction
resulted in increased serum CORT and elevated plasma CORT response to exogenous
ACTH. The current study characterizes CORT response in female food restricted rats and
provides a physiological validation of the in-house EIA for the determination of FCM
concentrations in both CON and FR rats. The EIA used in this paper was able to measure
FCM enabling a non-invasive monitoring of adrenocortical activity.
5.1 Effect of 1-Week Food Restriction on Serum CORT
One week of food restriction resulted in increased serum CORT levels as has been shown
by previous studies, but the change did not reach significance. The serum CORT levels
measured in the current study (40.21±10.55ng/ml for CON and 80.16±12.66ng/ml for
FR) are lower than expected based on values from prior research [51, 75, 124]. While
levels in this current paper range between 40.21±10.55ng/ml for CON and
53
80.16±12.66ng/ml for FR, former studies report ad libitum fed animals reaching peak
serum CORT levels of 300ng/ml and 48h food restricted animals reaching peak serum
CORT levels of 475ng/ml [124]. Diaz-Munoz et al. [75] recorded similar results with
control male Wistar rats obtaining peak plasma CORT levels at 250ng/ml and 3-week
food restricted rats at 375ng/ml. Levay and colleagues [51] demonstrated even lower
results than Djordjevic et al. [124] and Diaz-Munoz et al. [75] with CON animals
reaching 175ng/ml plasma CORT and FR rats between 250 and 315ng/ml plasma CORT
depending on the severity of food restriction. The range in blood CORT levels ranging
from 175 to 300ng/ml in CON and 250 to 475ng/ml in FR animals between studies may
be due to several factors including age of animals, length of food restriction, breed and
sex of rat, time of estrous cycle in female rats, speed of sample collection, and time of
blood sample.
Age, sex, and breed of animals do not explain the lower serum in CON CORT values
observed in the current study compared to similar literature. Females were expected to
have higher serum CORT levels than data recorded on male Wistar rats (250ng/ml) [72],
but the female rats in the present paper were much lower (40.21±10.55ng/ml for CON
and 80.16±12.66ng/ml for FR) than their results. Age also does not explain the
discrepancy in the current results as rats in the study by Levay and colleagues [51] were
also 4 months old.
Speed of blood collections affect CORT levels, where samples taken in over 3 minutes
can result in increased CORT due to handling stress [4-5]. Sample collections in the
54
current study were taken in under 3 minutes and animals were acclimated for 1-week to
handling and cage moving. The precautions taken to decrease stress in the animals still
may not, however, explain the lower than expected blood CORT results obtained in the
current paper as previous studies took similar safety measures [51, 75].
The likely explanation for the lower than expected serum results observed in the current
paper may be a combination of sample collection occurring during the estrous cycle and
the time of day samples were collected. CORT levels will fluctuate based on time of
estrous cycle in female rats where daily CORT means are lowest on the day of estrus and
rise progressively during metestrus, diestrus, and proestrus [13]. It is possible that serum
CORT was collected during the estrus phase resulting in lower values.
The blood samples in the current paper were measured an hour before the wake cycle as
previous data [125-127] demonstrate a circadian rhythm of plasma CORT with peak
levels occurring shortly before the onset of the dark cycle. However, this peak appears to
have a range as other groups have reported peak CORT levels occurring 30 to 60 min
after lights-off [49, 128]. Only one blood measurement was taken per animal on a given
day, and therefore, our samples may not have captured the animals’ peak CORT.
Attempting to obtain the peak CORT in the diurnal rhythm by sampling at one time point
for all animals might not be reliable, as there appears to be variability among animals.
Additionally, the varying time of peak CORT reported in previous investigations
highlights the importance of finding a more accurate tool for assessing stress in these
animals.
55
The FR animals may have lower serum CORT than expected because of the length of
treatment and the amount of food restriction. It appears that the severity of food
restriction will affect the suprachiasmatic nuclei of the hypothalamus in a graded manner:
mild metabolic challenge was shown to affect c-fos but not the master oscillator [107],
while a more severe metabolic challenge was shown to alter central clock function [49].
Our treatment was only 1-week in duration and FR rats were fed 30.0% of ad libitum
controls. Studies looking at short-term food restriction, referred to as acute food
deprivation (AFD), are usually under 72 hrs and involve complete food deprivation,
which may explain the higher levels of CORT compared to chronic FR studies.
5.2 Effect of 1-Week Food Restriction on FCM
7 days of food restriction lead to an increase in FCM with peak levels reaching 1.4 times
higher than nadir values, while CON peak CORT levels were 2.7 times higher than nadir.
Despite FR animals having a smaller difference in change between nadir and peak,
CORT remained elevated for a longer duration resulting in an overall greater total
amount of CORT over a 24hr period.
The increase in FCM levels in the FR animals was expected as previous studies have
demonstrated increased blood CORT in response to short and long-term food restriction.
Furthermore, studies have also shown that FCMs can be used to measure stress responses
to numerous stimuli in rodents including temperature, environmental changes, and food
restriction in mice [21]. The increase in CORT during FR stimulates several processes
56
that collectively serve to increase and maintain normal concentrations of blood glucose
levels. It is theorized that the hyperadrenocorticism associated with FR is a protective
mechanism that results in a rise in locomotor activity and may reflect an increased drive
for refeeding [23]. One paper looking at FCM in FR mice took faecal samples, which
were collected as a random subsample of all faecal matter deposited over the course of a
24h period [21]. Food consumption in the FR mice was lowered 10% per week from ad
libitum until 60% of ad libitum was reached. Faecal samples were collected 1 week
before the initiation of FR and at 12-week intervals thereafter. The study on FR mice did
not, however, examine whether a circadian rhythm could be detected in FR rodents. The
current study further supports the ability to measure FCMs in FR rodents and extends the
research by Harper and colleagues [21] by tracing the circadian rhythm of FCM in food
restricted animals.
In previously reported data [13], ad libitum rats demonstrated nadir FCM values of 100
ng/3hrs and peak values of 425ng/3 h. The FCM values in the current study are expected
to be less than those for the male rats seen in Cavigelli et al. [13] because females have
lower FCM values than males as shown in their results. Male rats in their study peaked at
512.6±31.4ng/3h and dipped at 74.5±9.0ng/3h. Female rats in their study demonstrated
lower peak CORT than the male rats, but these values varied in peak CORT levels
depending on the estrous cycle (peak levels reached ~75ng/ 3h for estrus phase,
~100ng/3h for metestrus phase, ~150ng/3h for diestrus phase, and ~200ng/3h for the
proestrus phase). Peak faecal CORT levels during proestrus were two or three times
higher than peaks during estrus or metestrus. These data are measured in different units,
57
and thus values are not comparable to those found in the current study. One study [92]
observing seasonal changes in free-ranging snowshoe hares, reported FCM values in ng/g
and their values ranged between 450 and 700ng/g, depending on the season. Even though
the present study looked at a different species, changes in FCM seem comparable as
CON rats had nadir values of 210.5±111.8ng/g and peak values of 457.7±64.5ng/g.
Faecal glucocorticoid metabolite assays reflect an average level of circulating
glucocorticoids over a time period, rather than a single point in time, and therefore may
provide a more accurate assessment of chronic glucocorticoid levels [21].
5.3 Underlying mechanisms of hypersecretion of CORT
CORT triggers its effects by binding to cytoplasmic glucocorticoid receptor (GR) and
functions as a ligand-dependent transcription factor. In the absence of CORT, GR is
found in cytoplasm as part of a large multiprotein complex, which contains heat shock
proteins (HSPs) 70 and 90 that allow the proper folding of the receptor and control
entrance into the nucleus [129]. After CORT binds to GR, conformational change
releases the receptor from the complex, and it translocates to the nucleus to bind
glucocorticoid-response elements at regulatory regions of target genes, to which
repression is the most common response [129]. Increased CORT in FR may be a result of
the increased GR activation also seen in FR [128, 130-131]. Ogias and colleagues [131]
found that fasting decreased GR in the cytoplasm concomitant with an increase GR at
transcriptional and protein levels in the nucleus. Increased CORT and lower CBG would
facilitate the binding of CORT to GR and its shuttling into the nucleus.
58
Many studies indicate increased CORT with no change in ACTH during food restriction
[50, 132-133]. This finding may reflect the presence of a normal pituitary corticotroph
cell caught in the balance between excessive endogenous CRH stimulation from above
and a hyperresponsive adrenal cortex from below [60]. The increased CORT levels
observed in FR rats may be due to increased StAR protein, which is the rate-limiting
factor in steroidogenesis and CORT production and increases with increasing CRH [134].
It is unclear what causes the increased StAR production in FR, but possibilities include
decreased leptin [69], increased liver X receptors [135], and increased HDL [82].
Leptin, a satiety-stimulating polypeptide secreted primarily by the white adipose tissue,
has been shown to have an inhibitory effect on steroidogenesis [136]. During food
restriction, there is a decrease in leptin correlated with weight loss. Gairdner & Amara
[19] demonstrated that 1-week of food restriction to 92.65% of original body mass
resulted in leptin decreasing to 27% of original measurements. The low levels of Leptin
during FR may aid in increased CORT production because they are no longer inhibiting
8-bromo cAMP-stimulated progesterone production and the expression of cAMP-
stimulated StAR protein [136]. In mice, the stimulation of CORT secretion induced by
starvation or restraint stress can be partially counteracted by concomitant administration
of leptin [73]. During FR, there may be an increase in HDL as seen in humans [87, 137],
monkeys [82], and aged male rats [138]. There is limited information on the effects of
chronic FR on HDL levels in young female rats. It would be interesting to see if HDL
levels increase along with increased StAR protein and CORT as HDL cholesterol levels
in rodents have been reported to be both lower [80] and higher [81] in FR than in
59
controls. The changes due to increases in HDL may occur via regulation of StAR by the
liver X receptors that are activated by increasing intracellular cholesterol. Liver X
receptors (LXRalpha and LXRbeta) increase StAR gene expression by preventing
accumulation of free cholesterol [135]. The liver X receptors controlling cholesterol
buildup may also be responsible for the increased CORT observed in the FR rats in the
present paper.
5.4 Circadian Rhythm
FCMs were excreted in a circadian rhythm with well-defined acrophases and nadirs in the
CON animals. 2-hr sampling intervals provided a high degree of temporal acuity for
characterizing the circadian CORT rhythm. Ad libitum fed rats excreted FCMs in a
diurnal pattern with a rapid rise in CORT excretion at the onset of the dark cycle
followed by a more gradual return to trough levels over the day. This asymmetrical
rhythm was similar to that previously identified in both plasma [125] and FCM [13]. The
time frames of peaks and nadirs are in accordance with previous data where FCM of both
male and female Sprague-Dawley rats fed ad libitum rose from lowest to highest values
within 9h, and took approximately 15 h for FCM to fall back to nadir [13].
One might expect a flattening of the CORT rhythm in FR animals due to the increased
CORT levels through out the day, and because FR has been shown to blunt other
hormone diurnal variations such as thyroid stimulating hormone [50]. Cano et al. [139]
demonstrated that a high fat-diet increased CORT and blunted the 24 hr secretory pattern.
Garcia-Belenguer et al. [102] demonstrated that when rats were fed 85% of the control
60
chow, there was a sharpening of the peak CORT. However, with a more severe FR of
50%, mean CORT levels were increased and the day-night difference in circulating
CORT levels was reduced due to high levels maintained throughout the day. Their study
was over 4-weeks, whereas the FR protocol in the current paper reduced body mass to
88-92% of original body mass in one week. FR animals in the present study demonstrated
a blunted circadian rhythm (after 1 week of FR) with no significant difference between
the nadir and peak values. There was, however, a trend in FR values where they appear to
peak 2hr after control occurring between 16:00 and 18:00hr instead of 14:00 and 16:00hr.
A time lag in peak CORT levels between CON and FR was expected as food restriction
causes a slowed metabolism in both animals [140] and humans [141].
There is a trend in FCM levels peaking again between 2:00 and 6:00hrs. Our FR animals
seem to have an anticipatory food peak, which is shown in the FCM 12-14 hrs after
feeding time. This time of anticipatory peak makes sense since their 24h peak FCM
occurred 10-12 hrs after the beginning of their wake-cycle. The anticipatory peak may be
a result of restricted feeding resetting the timing of peak steroidogenic activity of the
adrenal gland as previously observed by altering StAR gene expression within the
adrenocortex [66]. Restricted feeding has also been shown to alter functional (c-fos, crh,
pomc) and clock gene expression (per1, per2, and bmal) at all levels of the HPA axis
[66].
61
5.5 Adrenal Sensitivity
The use of exogenous ACTH to induce CORT secretion in rats is well established and
has previously been used to analyze both serum and faecal CORT responses [5]. In
addition to providing physiological validation for the current paper’s FCM
measurements, the aim of the ACTH injection in the present study was to investigate the
time required from ACTH administration to a detectable increase in CORT concentration
in the blood compared to an increase in CORT metabolite in the faeces and to assess
whether differences in the magnitude of response occurred between groups. Results
demonstrate serum CORT increased at 30 and 60 minutes post injection and decreased
thereafter in both groups. The FR animals reached significantly higher CORT levels than
the CON group at 30 and 60 minutes post injection as was expected based on previous
publications looking at both rats [43] and pigs [142]. It has previously been demonstrated
that peak levels of total CORT are significantly higher in 9- and 15-month chronic FR
rats compared to CON in response to an ACTH challenge [43]. Based on the enhanced
response of serum CORT to exogenous ACTH in the FR group compared to the ad
libitum animals, the current data indicate that chronic FR increases the sensitivity of the
adrenal gland to ACTH.
Faecal CORT levels displayed a diurnal rhythm after the ACTH administration. A return
to baseline levels after an induced response is important since maintaining low levels of
CORT with a flattening of the diurnal CORT rhythm may indicate a neuroendocrine
dysfunction such as that observed in depression [143]. CON animals peaked between
16:00 and 18:00hrs and FR animals peaked between 18:00 and 20:00 hrs. Following an
62
ACTH injection, Lepschy et al. [6] recorded a 4-12h increase in FCM concentrations
which remained elevated over a period of 4-19h. Similar to their study, both CON and FR
animals also peaked for 10 hrs.
There was little or no significant difference between group peak FCM values after an
ACTH challenge suggesting that FCM measurements may not be sensitive enough to
detect acute stress in chronically food restricted animals. The significant increase in
CORT observed in the serum is a reflection of an immediate biological response to a
stressor at a given time. FCM response to an ACTH challenge reflects a biological
response to a stressor over a cumulative time frame, which may have resulted in a less
dramatic increase in FCM compared to Serum CORT. These results are not surprising as
a prior study analyzing two diets found that one group of mice excreted significantly
higher levels of FCM compared to the other group throughout the experiment. However,
even though the effect of the ACTH challenge could readily be detected, it had a
considerably lesser impact when comparing FCM levels than did the difference in diet
[22]. FR rats did not show a higher peak CORT than the ad libitum fed rats in response to
an ACTH challenge, but they remained elevated for a longer duration resulting from an
overall greater total amount of CORT release.
5.6 Limitations
One limitation of this study was that the estrous cycle was not recorded in the rats. The
nadir and peak values of female rats vary with the estrous cycle: daily CORT means are
lowest on the day of estrus and rise progressively during metestrus, diestrus, and
63
proestrus [13]. Peak faecal CORT levels on prostrus were two or three times higher than
peaks during estrus or metestrus. Peak levels reached ~75ng/ 3h for estrus phase,
~100ng/3h for metestrus phase, ~150ng/3h for diestrus phase, and ~200ng/3h for the
proestrus phase. Not recording the estrous cycle could have affected values in the current
study, as serum CORT levels were lower than previously reported data possibly because
some animals were in their estrus phase.
Another limitation with this study was the fewer amount of samples produced in food
restricted animals. FR animals tended to excrete the most 2 to 4 hours after feeding time,
but at other collection times only around half the animals had samples. However, despite
having fewer samples compared to the CON animals, we were still able to find
significant increases in the overall CORT levels.
Blood sampling on consecutive days appears to have resulted in an anticipatory increase
in CON CORT levels whereby a sequential morning blood measurement resulted in
increased serum CORT. Baseline 2 was higher than Baseline 1, and Day 8 baseline
measurement was higher than Day 7. The days in between blood sampling may have
abolished the anticipatory stress as CORT levels returned to initial baseline levels.
Furthermore, while Day 8 Baseline CORT was higher than Day 7, the increase was not as
prominent as between Baseline 2 and Baseline 1. This is in line with repeated stress
studies, which indicate that the CORT response subdues with further exposure to
stress.[144-145].
64
Finally, the current study did not record the volume of water consumed by animals, and it
is possible that food restriction lead to decreased water intake as indicated by previous
data [146]. Kiss et al. [147] demonstrated that food and water deprivation can lead to
further increases in plasma CORT compared to food deprivation alone (9.2±2.2 vs
6.9±1.2ug/dl, respectively). The average CORT of the CON animals in that study was
1.8ug/dl. Thus, the addition of complete water deprivation only increased the CORT
response to food deprivation alone by 17%. While water intake was not quantified in the
current study, it was apparent that at least some water was being ingested since water had
to be replenished to fill the bottles on a daily basis. Thus, the effect of possible
dehydration in this study would likely be much less than that reported by Kiss et al.
[147]. Additionally, the observed difference between CORT in FR vs. CON animals in
the current study is 183% greater than could be expected from the influence of
dehydration.
5.7 Conclusion
The current paper was able to measure CORT non-invasively in FR rats. It was
demonstrated that 7 days FR results in increased serum CORT, and we also, for the first
time, showed that 7 days food restriction results in increased 24hr mean FCM. While FR
animals responded with significantly higher serum CORT values than CON to an ACTH
challenge, there was little or no significant difference between group peak FCM values.
The insignificant difference between the two groups suggests that FCM measurements
may not be sensitive enough to detect acute stress in chronically food restricted animals.
The in-house assay used in the current study was validated as measurements show a clear
65
circadian rhythm in CON animals, FCM values were higher in FR rats, and both groups
responded to an ACTH challenge with an elevated CORT response. Therefore, given the
non-invasive nature of the measurement, it is recommended that future studies measure
stress and /or CORT in FR rats via fecal sampling. One such application might be to
examine FCM in response to chronic food restriction to better understand the disease of
Anorexia Nervosa and its response to exercise and/or refeeding. In addition, future
studies can test how this hormone is correlated to other variables such as body mass,
leptin, and StAR protein. Unlike serum CORT measurements, FCM integrates both the
baseline and total free CORT released, and thus provides a powerful indicator of the
physiological state of the animals.
66
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3A Animal Body Mass (g) and Faecal Corticosterone Metabolites at Baseline, Day 7, and Day 8 ACTH. Animals Baseline
Weight (g)
Day 7 Weight (g)
Day 7 Body Mass as a % of original body weight
24hr Avg. Baseline FCM (ng/g)
24hr Avg. Day 7 FCM (ng/g)
24hr Avg. Day 8 FCM (ng/g)
C1 314.7 318.6 101.1 339.5 336.4 550.3
C2 305.3 317.36 103.9 530.4 456.1 550.9
C3 314.7 317 100.7 438.8 572.8 653.5
C4 308.7 321.7 104.2 268.5 249.3 624.3
C5 310.0 330.0.0 106.4 364.2 363.4 501.9
C6 287.6 305.6 106.3 321.4 348.1 813.4
C7 266.0 292.2 109.8 304.8 258.2 362.3
C8 267.0 282.4 105.8 276.7 n/a 238.5
FR1 341.2 292.0 85.6 197.2 n/a n/a
FR2 307.6 285.0 92.6 308.7 460.1 422.0
FR3 318.0 294.0 92.4 288.4 480.9 606.7
FR4 293.5 258.0 87.9 394.8 477.2 531.3
FR5 301.0 270.4 89.8 376.1 635.5 970.8
FR6 275.0 241.0 87.6 546.5 800.7 1225.7
FR7 268.7 238.8 88.9 272.857 209.1 211.4
FR8 264.0 234.0 88.6 198.1 179.4 327.8
FR9 268.8 237.5 88.3 252.2 286.0 286.3
FR10 263.8 236.0 89.5 185.3 265.2 317.4
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Weight is in grams. FCMs are averaged over 24 h for each animal. Control group (C) included animals fed ad libitum and food restricted (FR) group was fed 7g at 14:00 h everyday.
3B. Date of Birth and Arrival of Animals
Batch Date of Birth Date of Arrival
1 March 10 2012 July 11 2012
2 May 5 2012 September 19 2012
3C Average mass of food consumed per day measured in grams ± S.D.
Control FR
Day Food Mass (g)
1 28.42±5.40 7.12±.0.16
2 25.96±0.97 7.13±.0.22
3 22.37±10.29 7.27±0.33
4 21.48±11.17 7.31±0.35
5 22.95±3.72 7.35±0.27
6 22.85±5.07 7.69±0.34
7 25.01±0.14 7.5±0.25
8 26.93±2.61 7.66±0.45
9 21.34±0.86
10 24.58±50.30
11 27.10±10.44
12 25.88±1.42
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4A Circadian Rhythm of Control animals at Baseline and Day 7.
6:00-8:00
8:00-10:00
10:00-12:00
12:00-14:00
14:00-16:00
16:00-18:00
18:00-20:00
20:00-22:00
22:00-24:00
24:00-2:00
2:00-4:00
4:00-6:00
6:00-8:00
8:00-10:00
10:00-12:00
12:00-14:00
14:00-16:00
16:00-18:00
18:00-20:00
20:00-22:00
22:00-24:00
24:00-2:00
2:00-4:00
4:00-6:000
200
400
600Baseline1Day 7
Time (hrs)
FCM
(ng/
g)
Black bars on X-axis represent the 12hr dark cycle from 8:00-20:00hrs. The grey arrows indicate the time of blood sampling at 07:00h. Data have been double-plotted for clarity.
4B Circadian Rhythm of FR animals at Baseline and Day 7.
6:00-8:00
8:00-10:00
10:00-12:00
12:00-14:00
14:00-16:00
16:00-18:00
18:00-20:00
20:00-22:00
22:00-24:00
24:00-2:00
2:00-4:00
4:00-6:00
6:00-8:00
8:00-10:00
10:00-12:00
12:00-14:00
14:00-16:00
16:00-18:00
18:00-20:00
20:00-22:00
22:00-24:00
24:00-2:00
2:00-4:00
4:00-6:000
200
400
600
Baseline1Day 7
Time (hrs)
FCM
(ng/g
)
Black bars on X-axis represent the 12hr dark cycle from 8:00-20:00hrs. The grey arrows indicate the time of blood sampling at 07:00h. Data have been double-plotted for clarity. !
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