University of Joensuu, PhD Dissertations in Biology
No:18
Seasonality, photoperiod
and nutritional status
in the control of
endocrinological weight-regulation
by
Anne-Mari Mustonen
Joensuu
2003
Whenever a new discovery is reported to the scientific
world, they say first, “It is probably not true.” Thereafter,
when the truth of the new proposition has been
demonstrated beyond question, they say, “Yes, it may be
true, but it is not important.” Finally, when sufficient time
has elapsed to fully evidence its importance, they say,
“Yes, surely it is important, but it is no longer new.”
Michel de Montaigne (1533-1592)
Mustonen, Anne-Mari
Seasonality, photoperiod and nutritional status in the control of endocrinological weightregulation. – University of Joensuu, 2003, 211 pp.
University of Joensuu, PhD Dissertations in Biology, n:o 18. ISSN 1457-2486
ISBN 952-458-318-6
Keywords: energy metabolism, ghrelin, growth hormone, leptin, Lota lota, melatonin,
Nyctereutes procyonoides, Rattus norvegicus, seasonality, weight-regulation, winter sleep
Leptin and ghrelin are novel weight-regulatory peptides whose functions have been mainly
studied in laboratory rodents and humans. Exogenous leptin decreases their appetite and body
mass, whereas ghrelin is an orexigenic hormone stimulating food intake and fat gain. The
darkness hormone melatonin times seasonal rhythms and controls energy metabolism. As these
weight-regulatory hormones have evolved to improve the adaptations of animals to their
environments it is important to investigate their functions and interactions also in wild animals.
The aim of this thesis was to investigate the effects of seasonality, photoperiod and
nutritional status on the endocrinological weight-regulation of vertebrates. The possible
interactions of leptin and ghrelin with the seasonal rhythms of body weight, fat content, appetite
and reproduction were studied in the raccoon dog (Nyctereutes procyonoides) and the burbot
(Lota lota) exhibiting excessive but nonpathological accumulation of lipids in their white
adipose tissue or liver. The characteristics of the endocrine response to fasting of the raccoon
dog were determined during a two-month winter sleep induced by food deprivation. The effects
of a lack or an excess of melatonin on weight-regulation were investigated in the seasonal
raccoon dog and in the nonseasonal laboratory rat (Rattus norvegicus).
The autumnal leptin and growth hormone concentrations of the raccoon dog plasma were
low but increasing, whereas their ghrelin levels were high, probably inducing hyperphagia and
fat gain. The high leptin and growth hormone concentrations together with the low ghrelin
levels during the mid-winter could have induced lipolysis, protein conservation and sleepiness.
Long-term wintertime fasting decreased the plasma insulin levels and the activities of the
thyroid gland and the adrenal cortex, enabling efficient lipid mobilization and protein sparing.
Melatonin treatment advanced the seasonal rhythms of leptin, ghrelin and growth hormone,
whereas the fasting-induced winter sleep did not affect their concentrations.
Exogenous melatonin or constant light had no influence on the plasma leptin or growth
hormone concentrations of the nonseasonal rat, but melatonin decreased their ghrelin levels.
Constant illumination stimulated the carbohydrate metabolism of the rat liver, whereas
melatonin elevated the utilization of liver carbohydrates but suppressed the mobilization of fat.
Both constant light and exogenous melatonin stimulated the renal utilization of carbohydrates.
The levels of leptin- and ghrelin-like immunoreactivities in the burbot plasma increased after
spawning. The burbot liver contained high concentrations of leptin-like immunoreactivity,
suggesting that it could be one production site for the leptin-like peptide in teleost fish.
Anne-Mari Mustonen, Department of Biology, University of Joensuu, P.O. Box 111, FIN-80101,
Joensuu, Finland.
CONTENTS
LIST OF ORIGINAL PUBLICATIONS
ABBREVIATIONS
1
2
INTRODUCTION ........................................................................................................13
REVIEW OF THE LITERATURE ...............................................................................13
2.1 Melatonin................................................................................................................13
2.1.1 Background......................................................................................................13
2.1.2 The pineal gland ...............................................................................................14
2.1.3 Melatonin synthesis and catabolism...................................................................14
2.1.4 Melatonin binding sites .....................................................................................15
2.1.5 Innervation of the pineal gland ..........................................................................15
2.1.6 Control of the diurnal rhythm of melatonin synthesis .........................................16
2.1.7 Seasonality of melatonin secretion ....................................................................17
2.1.8 Role of melatonin in seasonal reproduction .......................................................17
2.1.9 Role of melatonin in thermoregulation ..............................................................18
2.1.10
Melatonin as a weight-regulatory hormone ....................................................19
2.1.11
Evolution of the pineal gland and the functions of melatonin ..........................20
2.2 Leptin ..................................................................................................................... 21
2.2.1 Background......................................................................................................21
2.2.2 Structure and expression of the leptin protein and leptin receptor......................22
2.2.3 Regulation of leptin secretion............................................................................23
2.2.4 Metabolic effects of leptin.................................................................................23
2.2.4.1 Anorectic influence.....................................................................................23
2.2.4.2 Effects of leptin on carbohydrate metabolism..............................................24
2.2.4.3 Effects of leptin on lipid metabolism ...........................................................24
2.2.5 Role of leptin in reproduction ...........................................................................25
2.2.6 Physiological significance of leptin....................................................................26
2.2.6.1 Seasonal starvation and obesity ..................................................................26
2.2.6.2 Leptin in seasonal mammals........................................................................27
2.2.7 Molecular evolution of leptin ............................................................................29
2.2.8 Leptin of nonmammalian vertebrates.................................................................30
2.2.8.1 Aves...........................................................................................................30
2.2.8.2 Reptilia and Amphibia ................................................................................30
2.2.8.3 Osteichthyes & Cephalaspidomorphi ..........................................................30
2.3 Ghrelin....................................................................................................................31
2.3.1 Background......................................................................................................31
2.3.2 Structure of ghrelin...........................................................................................31
2.3.3 Ghrelin and GHS-R expression .........................................................................32
2.3.4 Functions of ghrelin ..........................................................................................33
2.3.5 Regulation of ghrelin secretion..........................................................................34
2.4 Growth hormone.....................................................................................................34
2.4.1 Regulation of GH secretion...............................................................................34
2.4.2 GH receptor and IGF-I .....................................................................................35
2.4.3 Metabolic effects of GH....................................................................................36
2.4.3.1 Effects of GH on appetite...........................................................................36
2.4.3.2 GH and protein metabolism........................................................................36
2.4.3.3 GH and carbohydrate metabolism...............................................................36
2.4.3.4 GH and lipid metabolism ............................................................................37
2.4.4 GH in the regulation of sleep ............................................................................37
2.4.5 Seasonality of GH secretion..............................................................................38
2.4.6 Interactions of GH with leptin and melatonin ....................................................38
2.4.7 Evolution of GH...............................................................................................38
2.5 The experimental species of the thesis .....................................................................39
2.5.1 The raccoon dog (I-III) ....................................................................................39
2.5.1.1 Origin of the raccoon dog...........................................................................39
2.5.1.2 Ecology of the raccoon dog........................................................................40
2.5.1.3 Seasonal physiology of the raccoon dog .....................................................41
2.5.1.3.1 Thermoregulatory adaptations ...............................................................41
2.5.1.3.2 Seasonal BM cycle ................................................................................41
2.5.1.3.3 Winter sleep ..........................................................................................42
2.5.1.3.4 Reproductive processes .........................................................................42
2.5.1.3.5 Seasonal endocrinology .........................................................................43
2.5.1.3.5.1 Sex steroids.....................................................................................43
2.5.1.3.5.2 Weight-regulatory hormones ...........................................................43
2.5.1.3.5.3 Melatonin ........................................................................................43
2.5.2 The laboratory rat (IV-V) .................................................................................44
2.5.2.1 Origin of the brown rat...............................................................................44
2.5.2.2 Ecology and reproductive physiology of the brown rat ...............................44
2.5.2.3 The laboratory rat ......................................................................................45
2.5.2.4 Endocrinology of the laboratory rat ............................................................46
2.5.2.4.1 Melatonin ..............................................................................................46
2.5.2.4.2 Leptin, ghrelin and GH ..........................................................................47
2.5.3 The burbot (VI-VII) .........................................................................................48
2.5.3.1 Origin of the burbot....................................................................................48
2.5.3.2 Ecology of the burbot.................................................................................48
2.5.3.3 Physiological adaptations of the burbot.......................................................49
2.5.3.3.1 Thermoregulation and energy metabolism..............................................49
2.5.3.3.2 Seasonal reproduction ...........................................................................50
3 AIMS OF THE STUDY................................................................................................50
4 MATERIALS AND METHODS...................................................................................51
4.1 Housing and caring of the experimental animals.......................................................51
4.2 Study protocols.......................................................................................................52
4.3 Measurement of growth parameters ........................................................................52
4.4 Sampling and sacrification.......................................................................................52
4.5 Biochemical determinations.....................................................................................53
4.6 Statistical analyses...................................................................................................56
5 RESULTS.....................................................................................................................56
5.1 Seasonal weight-regulation of the raccoon dog........................................................56
5.2 Influence of seasonal fasting on the energy metabolism of the raccoon dog..............58
5.3 Influence of exogenous melatonin and continuous light on energy metabolism.........60
5.3.1 Effects of melatonin treatment on melatonin levels, BM, adiposity and appetite.60
5.3.2 Effects of melatonin treatment on weight-regulatory hormones .........................61
5.3.3 Effects of melatonin treatment on intermediary metabolism...............................61
5.3.4 Effects of continuous light on intermediary metabolism.....................................62
5.4 Seasonal reproduction.............................................................................................62
5.4.1 Effects of spawning on the energy metabolism of the burbot .............................62
5.4.2 Effects of reproduction on weight-regulatory hormones....................................63
5.4.3 Effects of gender on weight-regulatory hormones .............................................64
5.5 Endocrinological and metabolic interactions ............................................................64
6 DISCUSSION...............................................................................................................66
6.1 General remarks......................................................................................................66
6.2 Seasonal weight-regulation of the raccoon dog........................................................67
6.2.1 Autumnal fat accumulation ...............................................................................67
6.2.2 Metabolic transition ..........................................................................................68
6.2.3 Wintertime energy preservation ........................................................................68
6.2.4 Vernal mating season and summer ....................................................................69
6.3 Fasting-induced winter sleep in the raccoon dog......................................................70
6.3.1 Characteristics of winter sleep in medium-sized and large carnivores.................70
6.3.2 Effects of fasting on BM, adiposity and plasma lipids ........................................71
6.3.3 Effects of fasting on protein metabolism............................................................73
6.3.4 Effects of fasting on glucose and weight-regulatory hormones ..........................75
6.3.4.1 Glucose, insulin and glucagon concentrations .............................................75
6.3.4.2 Cortisol and thyroid hormone concentrations..............................................76
6.3.4.3 Leptin, ghrelin and GH concentrations........................................................76
6.3.5 Endocrine response to fasting in canids vs. ursids..............................................77
6.3.6 Influence of re-feeding on energy metabolism ...................................................78
6.4 Melatonin in body weight-regulation .......................................................................79
6.4.1 Effects of melatonin on BM, BMI and food intake ............................................79
6.4.2 Effects of melatonin on weight-regulatory hormones.........................................80
6.4.3 Effects of melatonin and LL on intermediary metabolism ..................................82
6.4.3.1 Liver carbohydrate metabolism...................................................................82
6.4.3.2 Liver lipid metabolism ................................................................................83
6.4.3.3 Kidney energy metabolism..........................................................................84
6.4.4 Effects of melatonin on glucose, insulin, thyroid hormone, lipid and aa levels ....84
6.5 Seasonal reproduction and weight-regulatory hormones ..........................................86
6.5.1 Leptin and reproduction of the raccoon dog......................................................86
6.5.2 Connection of leptin-LI to energy metabolism of the spawning burbot ..............86
6.5.3 Roles of ghrelin and GH in reproduction...........................................................88
6.5.4 Gender differences in the levels of weight-regulatory hormones ........................88
6.6 Interactions between weight-regulatory hormones...................................................90
6.7 Liver as a possible site for leptin synthesis ...............................................................90
7 GENERAL IMPLICATIONS........................................................................................91
8 CONCLUSIONS...........................................................................................................91
ACKNOWLEDGEMENTS
REFERENCES
LIST OF ORIGINAL PUBLICATIONS
This thesis is based on the following publications, which are referred to by their Roman
numerals:
I
Nieminen P, Mustonen A-M, Asikainen J & Hyvärinen H 2002. Seasonal weight
regulation of the raccoon dog (Nyctereutes procyonoides): Interactions between
melatonin, leptin, ghrelin, and growth hormone. J Biol Rhythms 17, 155-163.
II
Mustonen A-M, Nieminen P, Asikainen J, Saarela S, Kukkonen JVK & Hyvärinen H
200x. Continuous melatonin treatment and fasting in the raccoon dog (Nyctereutes
procyonoides) – Vernal body weight regulation and reproduction. Submitted
manuscript.
III
Mustonen A-M, Nieminen P, Puukka M, Asikainen J, Saarela S, Karonen S-L,
Kukkonen JVK & Hyvärinen H 2003. Physiological adaptations of the raccoon dog
(Nyctereutes procyonoides) to seasonal fasting – Fat and nitrogen metabolism and
influence of continuous melatonin treatment. J Comp Physiol B, in press.
IV
Mustonen A-M, Nieminen P & Hyvärinen H 2001. Preliminary evidence that
pharmacologic melatonin treatment decreases rat ghrelin levels. Endocrine 16, 43-46.
V
Mustonen A-M, Nieminen P & Hyvärinen H 2002. Effects of continuous light and
melatonin treatment on energy metabolism of the rat. J Endocrinol Invest 25, 716-723.
VI
Mustonen A-M, Nieminen P & Hyvärinen H 2002. Leptin, ghrelin, and energy
metabolism of the spawning burbot (Lota lota, L.). J Exp Zool 293, 119-126.
VII
Mustonen A-M, Nieminen P & Hyvärinen H 2002. Liver and plasma lipids of spawning
burbot. J Fish Biol 61, 1318-1322.
Some unpublished results are also presented.
ABBREVIATIONS
aa
α-AB
AC
AGRP
Ala
ANOVA
ARC
Arg
AS
B
B0
BAT
BM
BMI
BS
C
cAMP
(c)DNA
CE
Chol
Cit
CNS
CREM
Cys
db/db mouse
DD
DG
DS
EDTA
fa/fa rat
FFA
FSH
GH
GH-R
GHRH
GHS
GHS-R
Gln
Glu
Gly
GnRH
G-6-Pase
HDL
HIOMT
His
I
ICER
IGF-I or -II
JAK
kb
LD
12L:12D
LDL
LH
-(L)I
LL
LPC
Lys
M
ME
amino acid
α-aminobutyrate
adenylate cyclase
agouti-related peptide
alanine
analysis of variance
arcuate nucleus
arginine
after spawning
standard or sample binding
maximum binding
brown adipose tissue
body mass
body mass index
before spawning
canines
cyclic adenosine monophosphate
(complementary) deoxyribonucleic acid
cholesteryl esters
cholesterol
citrulline
central nervous system
cAMP responsive element modulator
cysteine
a mutant lacking the OB-Rb
constant darkness
diacylglycerols
during spawning
ethylenediaminetetraacetic acid
a mutant lacking the OB-Rb
free fatty acid
follicle-stimulating hormone
growth hormone
GH receptor
GH-releasing hormone
GH secretagogue
GHS receptor
glutamine
glutamate
glycine
gonadotropin-releasing hormone
glucose-6-phosphatase
high density lipoprotein
hydroxyindole-O-methyltransferase
histidine
incisors
inducible cAMP early repressor
insulin-like growth factor I or II
Janus kinase
kilobase
long daylength
12 hr light:12 hr darkness
low density lipoprotein
luteinizing hormone
-(like) immunoreactivity
constant light
lysophosphatidylcholine
lysine
molars
median eminence
MEL
Met
3-MH
MR
(m)RNA
Myr
NA
NAT
NH3
NPY
ob/ob mouse
OB-R(b)
Orn
PC
PE
Phe
PKC
PL
PLC
Pm
PRL
Pro
PS
PVN
Px
REMS
RHP
RIA
rs
sc
SCG
SCN
SD
SE
Ser
SHAM
SM
SRIF
STAT
SWS
T3
T4
Taa
Tau
Tb
TG
Thr
TL
TP
Trp
TSH
Tw
U/C
VMN
WAT
melatonin-treated
methionine
3-methylhistidine
metabolic rate
(messenger) ribonucleic acid
millions of years
noradrenaline
serotonin-N-acetyltransferase
ammonia
neuropeptide Y
a mutant lacking functional leptin
leptin receptor (long isoform)
ornithine
phosphatidylcholine
phosphatidylethanolamine
phenylalanine
protein kinase C
phospholipids
phospholipase C
premolars
prolactin
proline
phosphatidylserine
paraventricular nucleus
pinealectomy
rapid eye movement sleep
retinohypothalamic projection
radioimmunoassay
Spearman correlation coefficient
subcutaneous
superior cervical ganglia
suprachiasmatic nuclei
short daylength
standard error
serine
sham-operated
sphingomyelin
somatostatin
signal transducers and activators
of transcription
slow-wave sleep
triiodothyronine
thyroxine
total amino acids
taurine
body temperature
triacylglycerols
threonine
total lipids
total protein
tryptophan
thyroid-stimulating hormone
water temperature
urea-creatinine
ventromedial nucleus
white adipose tissue
1
concentrations and their association with body
weight, fat storage, appetite and reproduction
of vertebrates. Also the effects of food
deprivation and winter rest on leptin and
ghrelin levels of wild animals remain unknown.
Furthermore, an excess or a lack of melatonin
could
influence
leptin
and
ghrelin
concentrations as well as intermediary
metabolism of seasonal and nonseasonal
animals.
INTRODUCTION
Arctic and boreal vertebrates have adapted to
seasonal changes of the high latitudes with a
wide variety of strategies. Their reproductive
processes are synchronized to the most
favorable time of the year with relatively high
ambient temperature and food availability. The
climatic and nutritional challenges of the winter
pose the greatest threats to the survival of wild
animals. During the course of evolution they
have generated several physiological means
such as moulting, storage of fat and winter rest
to help them cope with the cold season.
By predicting the forthcoming seasons
animals can time their physiological processes
correctly. Photoperiod is the most reliable
ambient factor staying identical between the
years. Numerous species have chosen daylength
and the darkness hormone melatonin as
proximate cues for the timing of their seasonal
rhythms. Melatonin has been shown to time
several
seasonal
processes
such
as
reproduction, pelage change and antler growth
of mammals. A possible role for melatonin in
weight-regulation has been lately re-enforced
and become an active area of investigation.
Leptin (Zhang et al., 1994) and ghrelin
(Kojima et al., 1999) are novel weightregulatory peptides. Their functions in energy
homeostasis have been mainly investigated in
laboratory rodents and humans. As these signal
peptides must have evolved to improve the
adaptation of animals to their changing
environments, it would be important to
investigate their functions also in wild
vertebrates. Species exhibiting seasonal
cyclicity of lipid metabolism with profound but
nonpathological accumulation of fat into
adipose tissue or liver would be exceptionally
fascinating models for obesity research.
Interesting starting points for the study
would be the seasonality of leptin and ghrelin
2
2.1
REVIEW OF THE LITERATURE
Melatonin
2.1.1 Background
The pineal gland (epiphysis cerebri) was
recognized more than 2000 years ago, but the
first anatomist who discovered this organ
remained unknown (Zrenner, 1985). The most
ancient written description of the pineal is that
of Galen (c. 130-200). During the classical
antiquity and the Renaissance, the pineal was
considered a mechanical system of valves
regulating the flow of pneuma or a lymphatic
gland-like structure supporting the venous
network within the brain. The first
representation of the human pineal by Vesalius
(1514-1564) dates back to the 16th century.
Descartes (1596-1650) considered the pineal as
the point at which the soul pre-eminently
controls the body and hypothesized that the
pineal controls the flow of animal spirits into
motor nerves affecting the movements of the
body. He also predicted correctly that the
stimulus for pineal function came as visual
input to the retina (Arendt, 1995).
Ancient ideas about the pineal gland
persisted for several centuries (Zrenner, 1985).
In the late 1800s it was suggested that the
pineal might function as a light-sensing organ
comparable to the lateral eyes. It was also
13
1975). Moreover, nocturnal species seem to
have smaller pineals than diurnal ones (Ralph,
1975).
The mammalian pineal gland functions as a
secretory organ, whereas in cyclostomes, fish,
amphibians and some reptiles the pineal is
directly photoreceptive (Young, 1935; Gern,
1981; Arendt, 1995). The pineal of birds and
some reptile species has a mixed photoreceptor
and secretory function. In some fish, amphibian
and reptile species, the pineal forms two
components: the intracranial pineal organ and
the extracranial parietal organ (Arendt, 1995).
The latter may lie below the skin on the dorsal
surface of the cranium as the third eye. Parietaleyeless lizards tend to be restricted to low
latitudes, whereas species with parietal eyes
inhabit also high latitudes (Gundy et al., 1975).
noticed by several scientists that the pineal
organ could represent the vestige of an
unpaired third eye of the ancestors of living
vertebrates. The first evidence that the pineal
gland affects reproductive endocrinology came
from studies investigating pineal tumors at the
end of the 1890s. Kitay & Altschule reviewed a
large body of literature about the pineal gland
in 1954 and were convinced that it was not a
functionless, vestigial organ but had an active
metabolism. Finally, Lerner and his coworkers
isolated melatonin (1958) and identified its
structure (1959), while they were searching for
the amphibian skin-lightening factor in bovine
pineal glands.
2.1.2 The pineal gland
The pineal body is a small, unpaired structure
closely associated with the third ventricle
(Arendt, 1995). It originates as an invagination
of the diencephalon and in most species retains
a stalk connection to the habenular
commissure. There is a marked variation in the
size and location of the pineal. The human
pineal gland is of the size of a pea weighing
100-150 mg and its shape resembles a
pinecone.
Representative species from all vertebrate
classes except for Pteraspidomorphi (hagfishes)
have been demonstrated to possess a pineal
gland (Oksche, 1965; Ralph, 1975). Some seal,
sea lion, walrus (Pinnipedia) and penguin
species (Sphenisciformes) inhabiting polar
regions have exceptionally large pineals,
whereas the gland is relatively small or totally
absent in elephants (Proboscidea), edentates
(Xenarthra), rhinoceroses (Rhinocerotidae),
dugongs (Sirenia), hyraxes (Hyracoidea),
pangolins (Pholidota), bats (Chiroptera),
insectivores (Insectivora), whales (Cetacea)
and crocodiles (Crocodylus, Alligator; Oksche,
1965; Cuello & Tramezzani, 1969; Ralph,
2.1.3 Melatonin synthesis and catabolism
Melatonin is mainly synthesized in and released
from the pineal gland (Axelrod, 1974). The
pineal was considered the only site of melatonin
synthesis until it was demonstrated that
pinealectomy (Px) only suppressed but did not
totally abolish blood melatonin levels (Ozaki &
Lynch, 1976). In addition to the retina (Gern &
Ralph, 1979), Harderian glands (MenendezPelaez et al., 1987) and gastrointestinal tract
(Raikhlin et al., 1975), also extra-orbital
lacrimal glands (Mhatre et al., 1988), testes
(Tijmes
et
al.,
1996),
erythrocytes
(Rosengarten et al., 1972) and platelets
(Launay et al., 1982) may have minor roles of
local importance in melatonin production.
Melatonin synthesis initiates with the uptake
of tryptophan (Trp) from the circulation to the
gland (Arendt, 1995). Trp is transformed to 5hydroxytryptophan
by
tryptophan-5hydroxylase (Lovenberg et al., 1967), the
activity of which is higher during the night
compared to daytime values (Sitaram & Lees,
14
1978).
5-Hydroxytryptophan
is
then
decarboxylated
to
serotonin
by
5hydroxytryptophandecarboxylase (Shein et al.,
1967). This enzyme exhibits little daily
variation in the pineal (Snyder & Axelrod,
1965). Serotonin is N-acetylated to Nacetylserotonin
by
serotonin-N-acetyltransferase (NAT; Weissbach et al., 1960), the
activity of which undergoes over a 15-fold
increase at night in the rat pineal gland (Klein &
Weller, 1970). Thereafter N-acetylserotonin is
O-methylated to melatonin (N-acetyl-5methoxytryptamine)
by hydroxyindole-Omethyltransferase (HIOMT; Axelrod &
Weissbach, 1960). HIOMT activity shows no
significant diurnal rhythm (Steinlechner et al.,
1984).
It remains unresolved whether melatonin is
released from the pineal to the cerebrospinal
fluid (Anton-Tay & Wurtman, 1969), to the
general circulation (Pang & Ralph, 1975) or
both (Pang et al., 1993). The half-life of
melatonin varies between 13 and 44 min
depending on the species. Primarily the liver
and secondarily the kidneys are the major sites
for melatonin metabolism. It undergoes 6hydroxylation followed by sulfate and
glucuronide conjugation in microsomal phase I
and II reactions (Kveder & McIsaac, 1961;
Arendt, 1995). The metabolites, of which 6sulfatoxymelatonin is the most important, are
excreted in the urine.
(Mel1a, 1b and 1c) coupled to G-protein have
been identified (Reppert et al., 1994, 1995a,b).
In rodents melatonin binding sites have been
discovered from the 15th day of gestation
onwards (Williams et al., 1991). Over a wide
range of species they are most consistently
located on pars tuberalis of the hypophysis
(Weaver & Reppert, 1990; Boissin-Agasse et
al., 1992; Duncan & Mead, 1992). Other
locations are the paired suprachiasmatic nuclei
(SCN; Vaněček, 1988), pars distalis (Weaver
& Reppert, 1990), paraventricular nucleus
(PVN; Williams et al., 1991), preoptic area,
cerebellum (Ekström & Vaněček, 1992),
hypothalamus, thalamus (Weaver et al., 1989),
retina, choroid plexus (Vaněček, 1988), arcuate
nucleus (ARC), median eminence (ME), pineal
gland itself (Weaver et al., 1988), spinal cord,
spinal and cranial nerves and cochlea of the ear
(Williams et al., 1997). There are melatonin
binding sites also in the periphery, e.g. in the
liver (Acuña-Castroviejo et al., 1994), kidneys
(Song et al., 1992), gastrointestinal tract
(Bubenik et al., 1993), ovaries and testes (Ayre
et al., 1992), prostate (Laudon et al., 1996),
brown adipose tissue (BAT; Le Gouic et al.,
1997), melanophores (Ebisawa et al., 1994),
Harderian glands (Lopez-Gonzalez et al.,
1991), spleen (Yu et al., 1991), arteries
(Viswanathan et al., 1990), pancreas, thyroid
gland and lungs (Williams et al., 1997).
2.1.5 Innervation of the pineal gland
2.1.4 Melatonin binding sites
The primary function of the pineal is to
transduce information about the light-dark
cycles to body physiology (Arendt, 1995). The
information
is
conveyed
via
the
retinohypothalamic projection (RHP) from the
retina to SCN and from SCN via PVN,
hindbrain, spinal cord and superior cervical
ganglia (SCG) to the pineal gland. The
melatonin rhythm is generated by SCN
Melatonin binding sites have been discovered
from teleost fish to metatherian and eutherian
mammals (Stankov et al., 1993). Fewer brain
areas seem to possess them in higher than in
lower vertebrates (Rivkees et al., 1989;
Stankov et al., 1993). At least three different
subtypes of plasma membrane-bound receptors
15
entrained to 24 hr by the light entering the
retina. The SCN receive inhibitory nerve
impulses from the retina during photophase
(Bentley, 1998), but in darkness sympathetic
activity releases the major neurotransmitter
noradrenaline (NA) from the postganglionic
sympathetic fibers of SCG terminating within
the pineal gland (Arendt, 1995).
NA interacts with β1-adrenergic receptors
on the pinealocyte, which leads to the
activation of adenylate cyclase (AC) and the
formation of cAMP (Bentley, 1998). cAMP
activates protein kinase A, which induces
transcription by phosphorylating the cAMPresponse element on the NAT gene leading to
increased melatonin synthesis. After a lag
period of several hours of darkness the
response declines, although the levels of cAMP
remain high. This effect is mediated by the
CREM gene (cAMP responsive element
modulator), which expresses a repressor of
nuclear transcription processes mediated by
cAMP. ICER (inducible cAMP early repressor)
decreases the activity of the NAT regulator
gene, when the light period approaches (Stehle
et al., 1993; Takahashi, 1993). Binding of NA
to α1-adrenergic receptors potentiates βadrenergic induction (Bentley, 1998). It leads
to the activation of phospholipase C (PLC) and
the phosphatidylinositol metabolism pathway,
resulting in synergistic activation of AC.
levels of e.g. the golden hamster (Mesocricetus
auratus) peak late in the dark period, whereas
in the laboratory rat (Rattus norvegicus)
melatonin peaks in the middle of scotophase. In
some species such as the white-footed mouse
(Peromyscus leucopus), plateau peak levels are
observed throughout the dark period.
The melatonin secretion rhythm is
endogenous persisting in continuous darkness
(DD; Klein & Weller, 1970; Ralph et al.,
1971). Constant light (LL), on the other hand,
obliberates the diurnal melatonin secretion
rhythm (Ralph et al., 1971) by suppressing the
activities of NAT (Klein & Weller, 1970) and
HIOMT (Wurtman et al., 1963b). Even a brief
exposure to light at night can suppress
melatonin production in experimental rodents
(Illnerová & Vaněček, 1979). Consequently
species such as the emperor penguin
(Aptenodytes forsteri) and the Weddell seal
(Leptonychotes weddellii) inhabiting polar
regions lack diurnal fluctuations in blood
melatonin during the maximal daylight (Barrell
& Montgomery, 1989; Miché et al., 1991).
The melatonin production rhythm is
generated within the pineal in birds and lower
vertebrates (Arendt, 1995). Due to the direct
photoreceptive capacity of the organ (Falcón &
Collin, 1989) its melatonin production rhythm
can be maintained in a culture for several days
(Deguchi, 1979; Menaker & Wisner, 1983;
Falcón et al., 1989). It is also possible to reestablish a locomotor activity rhythm of a bird
rendered arrhythmic by Px by transplanting a
pineal from a donor bird into the anterior
chamber of the eye of a recipient bird
(Zimmerman & Menaker, 1975).
On the contrary, the pineal of mammals is
not a self-sustaining rhythm generator, but the
hypothalamic SCN are their main circadian
pacemakers (Arendt, 1995). The rhythmic
activity of SCN is entrained to the 24 hr day
primarily by light-dark cycles conveyed via the
2.1.6 Control of the diurnal rhythm of
melatonin synthesis
In almost all species studied to date, whether
diurnal, crepuscular or nocturnal, melatonin is
synthesized and secreted during the dark phase
of the daily dark-light cycle (Reiter, 1986). The
duration of the melatonin secretion peak is
proportional to the length of the night.
Nocturnal melatonin patters of the pineal can
be divided into three categories. The melatonin
16
proposes that there is a circadian rhythm of
sensitivity in target organs, which can coincide
with scotophase and produce appropriate
physiological responses only in SD. The
internal coincidence hypothesis suggests that
there are two rhythms: the melatonin cycle and
the rhythm of organ sensitivity, which are
important for determining the response of a
given system to melatonin. The message is read
only if the melatonin peak coincides with the
window of sensitivity. The increased duration
of melatonin peak increases the chance that the
elevated melatonin will overlap the window of
sensitivity. When these two do not coincide,
melatonin, even though elevated for a long
period, is not capable of modifying body
physiology. The amplitude hypothesis claims
that the actions of melatonin depend on the
magnitude of its nocturnal peak (Reiter, 1991).
There are, however, more data supporting the
duration and coincidence theories, the duration
hypothesis having the most experimental
support (Tamarkin et al., 1976; English et al.,
1988; Wayne et al., 1988).
RHP. The circadian rhythm in electrical activity
of SCN is self-sustained and can be maintained
in a culture (Green & Gillette, 1982).
Transplantation of SCN to the third ventricle of
rodents restores the rhythmic locomotor
activity in animals rendered arrhythmic by SCN
ablation (Ralph et al., 1990).
2.1.7 Seasonality of melatonin secretion
The length of the nocturnal melatonin peak
reflects the duration of the dark period and thus
the length of the day and the season of the year.
The melatonin peak is longer during short days
(SD) than during long days (LD) in several
species such as the laboratory rat (Illnerová &
Vaněček, 1980), the golden hamster (Brainard
et al., 1982) and the sheep (Ovis aries; Bittman
et al., 1983), to name only a few. Some lower
vertebrates, on the other hand, can have
discrepancy between their nocturnal melatonin
peak and the length of the night (Underwood &
Hyde, 1989). Moreover, the seasonal changes
in melatonin excretion of the mink (Mustela
vison; Valtonen et al., 1992, 1995), the
raccoon dog (Nyctereutes procyonoides; Xiao,
1996) and the reindeer (Rangifer tarandus
tarandus; Eloranta et al., 1992) are more
closely associated with moulting and
reproductive processes than with the daylength.
In humans Birau et al. (1981) have measured
the highest serum melatonin levels in Jan and
June, whereas the nadir was observed in April.
Arendt et al. (1977) have detected peak human
melatonin levels in Jan and July and the lowest
concentrations in April and Oct.
There are several hypotheses explaining
possible ways of photoperiodic time
measurement by animals (Reiter, 1987).
According to the duration hypothesis, the
length of the nocturnal melatonin peak
determines its ability to modify endocrine
physiology. The external coincidence model
2.1.8 Role
reproduction
of
melatonin
in
seasonal
As reviewed by Kitay & Altschule in 1954, the
earliest studies on the role of melatonin in
reproduction focused on human pineal tumors,
which were associated with precocious
puberty. In experimental animals Px led to
gonadal hypertrophy and accelerated vaginal
opening, prolonged estrus and shortened
diestrus. Administration of pineal extracts, on
the other hand, caused gonadal atrophy,
retarded vaginal opening and decreased
sensitivity
to
gonadotropins.
These
observations led to the conclusion that the
possible physiological function of the pineal
was most likely concerned with reproduction.
So far the essential role of melatonin in
17
whereas the interpretation of the signal depends
on the species concerned.
A primary action of melatonin in the control
of reproduction seems to be the modification of
the
hypothalamic
gonadotropin-releasing
hormone (GnRH) secretion (NToumi et al.,
1994; Messager et al., 1999). Melatonin also
changes the sensitivity of the pituitary to GnRH
stimulation (Martin & Klein, 1976; MondainMonval et al., 1988). As a consequence, blood
luteinizing hormone (LH) and folliclestimulating hormone (FSH) concentrations
respond to melatonin exposure (Reiter &
Sorrentino, 1971; Mondain-Monval et al.,
1988), and melatonin also influences blood
concentrations of testosterone (Kokolis et al.,
2000), 17β-estradiol, progesterone and
prolactin (PRL; Martinet et al., 1983;
Chemineau et al., 1986; Rose et al., 1996).
The mediobasal hypothalamus is the most
probable target for melatonin action on
seasonal reproduction (Domański et al., 1975;
Malpaux et al., 1993a; Maywood & Hastings,
1995). In vitro experiments have, however,
demonstrated that the melatonin-induced
effects on reproduction can be also targeted
directly on the pituitary (Martin & Klein, 1976;
Martin et al., 1982), ovaries (Fiske et al.,
1984) and testes (Ellis, 1972).
It remains unresolved whether pineal gland
plays a role in human reproduction. Silman et
al. (1979) have shown that blood melatonin
levels of schoolboys drop before the physical
and endocrinological signs of puberty appear.
In women melatonin concentrations are high
during the menstrual bleeding, decrease to a
nadir during ovulation and rise during the luteal
phase (Wetterberg et al., 1976).
reproduction has been demonstrated beyond
question only in mammals. Although the pineal
gland has been shown to influence reproductive
processes of birds (Gupta et al., 1987;
Ramachandran et al., 1996), reptiles (Haldar &
Thapliyal, 1981), amphibians (Kupwade &
Saidapur, 1986), teleost fish (de Vlaming et al.,
1974; de Vlaming & Vodicnik, 1978) and
cyclostomes (Joss, 1973), the function of
melatonin in their reproduction is far from
being understood.
The first experiments conducted on
laboratory rodents and some seasonal mammals
indicated an antigonadotropic role of
melatonin. Exogenous melatonin treatment was
shown to induce gonadal regression in some
lagomorphs (Küderling et al., 1984; Boyd,
1985) and rodents (Turek et al., 1976; Glass &
Lynch, 1982; Carter & Goldman, 1983).
Subsequent studies, however, revealed that
melatonin could stimulate reproduction of some
mammalian species. Estrus of ewes (Arendt et
al., 1983) and domestic ferrets (M. putorius
furo, Nixon et al., 1995), ovulation of goats
(Capra hircus, Chemineau et al., 1986) and
testicular activity of rams (Lincoln & Ebling,
1985) and some mustelid (DiGregorio &
Murphy, 1987) and canid species (Forsberg et
al., 1990; Xiao, 1996) advance due to
exogenous melatonin introduced during LD.
On the other hand, melatonin implantation
during SD can delay testicular regression
(Mondain-Monval et al., 1988; Xiao, 1996).
Furthermore, melatonin regulates the duration
of the preimplantation period of fertilized
embryos in some mustelids (Martinet et al.,
1983; Berria et al., 1988). It is no longer
believed that melatonin is either anti- or
progonadotropic, but that it serves to transmit
the changes in daylength to body physiology
(Arendt, 1995; Bentley, 1998). Long duration
melatonin treatment is equivalent to SD and
short duration melatonin administration to LD,
2.1.9 Role of melatonin in thermoregulation
The regulatory effect of photoperiod on the fur
growth of mammals has been known since the
18
Berger, 1992) and mammals (Pavel et al.,
1981; Vollrath et al., 1981).
Melatonin treatment also increases BAT
mass, uncoupling protein content, cytochrome
oxidase activity and thermogenic capacity as
well as cold resistance and heat conservation of
rodents (Heldmaier & Hoffmann, 1974;
Heldmaier et al., 1981; Glass & Lynch, 1982;
Bartness & Wade, 1984; Andrews & Belknap,
1985). In birds, melatonin increases roosting
(Barchas et al., 1967), cold resistance, thermal
insulation and maximal heat production
(Saarela & Heldmaier, 1987). Moreover,
melatonin is involved in thermoregulation of
reptiles and amphibians. In a thermal gradient,
melatonin changes temperature selection of the
collared lizard Crotaphytus collaris (Cothran &
Hutchison, 1979) and the mudpuppy Necturus
maculosus (Hutchison et al., 1979). It also
lowers the panting threshold of the Jacky lizard
Amphibolurus muricatus (Firth & Heatwole,
1976).
1930s (Bissonnette & Wilson, 1939). Rust &
Meyer (1969) demonstrated the influence of
exogenous melatonin on the fur growth in the
short-tailed weasel (M. erminea). They
observed that melatonin induced moulting and
growth of white winter pelage to weasels kept
in LD. Subsequent studies reported that
melatonin treatment in LD accelerates the
growth of winter fur in hares and rabbits
(Küderling et al., 1984; Boyd, 1985), mustelids
(Allain & Rougeot, 1980; Nixon et al., 1995)
and canids (Forsberg & Madej, 1990; Xiao,
1996). If melatonin is introduced in late winter,
the vernal pelage change can be delayed
(Martinet et al., 1983; Mondain-Monval et al.,
1988). Insertion of melatonin implants to goats
in early spring increases fiber growth and yields
of cashmere (Malpaux et al., 1993b), whereas
the moult of the European badger (Meles
meles) is delayed or inhibited by vernal
melatonin (Maurel et al., 1989).
Body temperature (Tb) of diurnal animals is
known to follow a circadian rhythm opposite to
that of melatonin with a nadir at night.
Melatonin secretion seems to be a major
regulator of this rhythm (Cagnacci et al.,
1992). Melatonin treatment lowers Tb of
mammals (Arutyunyan et al., 1964) and birds
(Murakami et al., 2001), Px increases Tb, and
melatonin can reverse the hyperthermic effects
of Px (John et al., 1978). Exogenous melatonin
treatment increases spontaneous nesting
behaviour and incidence of daily torpor in the
white-footed mouse (Lynch et al., 1978) and
the incidence and duration of hibernation in the
golden-mantled ground squirrel (Citellus
lateralis; Palmer & Riedesel, 1976). It also
depresses the duration of electrical activity in
isolated hearts from hibernating and nonhibernating ground squirrels. A sleep-inducing
influence of melatonin has been observed in
birds (Bermudez et al., 1983; Phillips &
2.1.10 Melatonin
hormone
as
a
weight-regulatory
Long before the discovery of melatonin Px was
found to cause hyperglycemia and to decrease
urine nitrogen excretion in experimental
animals (reviewed by Kitay & Altschule, 1954).
Administration of pineal extracts led to
hypoglycemia, decrease of hyperketonemia and
increased blood concentrations of proteins and
amino acids (aa). Numerous earlier reports
focusing on the role of melatonin in the control
of
intermediary
metabolism
yielded
contradictory results. Perhaps for this reason,
the influence of melatonin on weight-regulation
was not a focus of intensive research until
recently.
Studies conducted on seasonal mammals
revealed that exogenous melatonin mimicking
the effects of SD can affect body mass (BM),
fat content and food intake of animals.
19
elevated total Chol and low density lipoprotein
(LDL) Chol concentrations (Hoyos et al.,
2000). On the contrary, levels of high density
lipoprotein (HDL) Chol increase due to
melatonin. Melatonin has also several
antiglucocorticoid effects; it e.g. decreases
glucosuria, hyperglycemia and blood FFA and
total Chol concentrations elevated by
glucocorticoid treatments (Aoyama et al.,
1986). In the mink melatonin decreases liver
lipase esterase activities, hepatic triacylglycerol
(TG) and Chol concentrations as well as plasma
polar lipid levels (Nieminen et al., 2001b).
Some experimental data show that
exogenous melatonin stimulates carbohydrate
metabolism in preference to utilization of lipids
(John et al., 1990), but contradictory results
also exist (Mazepa et al., 2000). Melatonin may
also affect energy metabolism via other
hormones such as insulin (Rasmussen et al.,
1999), glucocorticoids (Acuña et al., 1984),
thyroid hormones (Vriend, 1983) and growth
hormone (GH; Smythe & Lazarus, 1974)
participating in the regulation of carbohydrate
and fat metabolism. Melatonin treatment
stimulates avian lipogenesis (synthesis of fatty
acids or lipids; Osei et al., 1989), and
melatonin-induced effects on lipid metabolism
have also been observed in teleost fish (de
Vlaming et al., 1974).
Melatonin treatment during LD stimulates
appetite, weight gain and fat storage in some
mammals exhibiting pronounced autumnal
fattening followed by a seasonal rest (Wade &
Bartness, 1984; Le Gouic et al., 1996). BM
and body fatness can be decreased by melatonin
in species with decreasing appetite, BM and fat
stores in response to shortening daylength
leading to reduced energy requirements during
wintering (Dark et al., 1983; Bartness &
Goldman, 1988). Melatonin-induced changes in
weight gain and appetite have also been
demonstrated in birds (Bermudez et al., 1983;
Gupta et al., 1987; Osei et al., 1989).
Exogenous melatonin treatment has several
effects on intermediary metabolism. It can
induce hyperglycemia (Fabiś et al., 2002) and
hypoinsulinemia (Rasmussen et al., 1999) in
rodents. Liver and muscle glycogen contents of
rats increase due to melatonin treatment
(Mazepa et al., 2000). Lactate concentration of
their livers increases, whereas plasma lactate
levels decrease. In the tundra vole (Microtus
oeconomus), melatonin treatment stimulates
gluconeogenesis (i.e. de novo synthesis of
glucose), increases glycogen stores and inhibits
fat utilization in kidneys (Mustonen et al.,
2002b).
Furthermore,
liver
glucose-6phosphatase (G-6-Pase) activities of minks
increase due to exogenous melatonin
(Nieminen et al., 2001b). Contradictory data
exist concerning the influence of melatonin on
the glucose metabolism of birds (Bermudez et
al., 1983; John et al., 1990) and teleost fish
(Delahunty & Tomlinson, 1984; Soengas et al.,
1998).
Exogenous melatonin decreases blood free
fatty acid (FFA; Mazepa et al., 2000) and free
cholesterol (Chol) levels but increases
phospholipid (PL) concentrations of rats
(Esquifino et al., 1997). Also Chol
esterification index increases with melatonin. In
hypercholesterolemic rats, melatonin decreases
2.1.11 Evolution of the pineal gland and the
functions of melatonin
According to the fossil record the pineal organ
has been a typical feature throughout the
evolution of vertebrates (Gern & Karn, 1983).
The pineal and its major cellular component the
pinealocyte have, however, experienced
significant evolutionary changes (Bentley,
1998). The pineals of several cyclostome and
fish species have an adjacent part called the
parapineal (Oksche, 1965). In some teleost fish
20
rubrum, one of the oldest living organisms
(Manchester et al., 1995). This indicates that
the molecule is evolutionarily highly conserved.
Besides mammals, the diurnal secretion rhythm
of melatonin with peak values in circulation
during the night has been observed in teleost
fish (Gern et al., 1978), amphibians (Gern &
Norris, 1979), reptiles (Owens et al., 1980) and
birds (Gwinner et al., 1993). Even the
bacterium R. rubrum (Manchester et al., 1995)
and the unicellular dinoflagellate Gonyaulax
polyedra show a melatonin synthesis rhythm
with higher melatonin levels during scotophase
(Pöggeler et al., 1991). This has led Arendt
(1995) to the suggestion that melatonin might
have functioned as a transmitter of daily lightdark information since the origin of the very
first life forms.
there is a transparent or translucent window
above the pineal (Breder & Rasquin, 1950),
whereas the pineal organ of anuran amphibians
has a nerve connection to the extracranial
frontal organ with photosensor cells lying
below the skin (Oksche, 1965; Ralph, 1975).
The homologous parietal eye of some lizard
species and the tuatara (Sphenodon punctatus)
has a cornea, a lens and a retina-like structure
(Eakin, 1973).
In cyclostomes, fish, amphibians and
reptiles, the pineal organ exhibits a
photosensory-like structure resembling retinal
receptors (Oksche, 1965). The pineal of birds
consists of modified photoreceptive cells with
rhodopsin-like
photosensitivity
(Deguchi,
1981), whereas there are only remnants of the
photoreceptive structures left in mammals
(Gern & Karn, 1983). It is possible that the
pineal organ ceased to function as a
photoreceptor and started to convey light-dark
information via sympathetic innervation during
the reptile-mammal transition in the Triassic.
The timing of seasonal reproduction is one
of the most widespread functions of melatonin
being demonstrated from teleost fish to
mammals. The effects on body coloration and
thermoregulation are also common among
vertebrates. Furthermore melatonin has been
shown to control daily activity rhythms in
reptiles, birds and mammals, and to influence
energy metabolism of teleost fish, birds and
mammals. In amphibians, melatonin has a
strong regulatory effect on metamorphosis and
orientation, and its influence on sleep has been
demonstrated in birds and mammals. It may
also have ancient functions as an antioxidant
(Manchester et al., 1995).
Melatonin has been identified from edible
plant species (Dubbels et al., 1995) to humans
(Gern & Karn, 1983), and melatonin-like
immunoreactivity (-LI) has been detected in the
photosynthetic
bacterium Rhodospirillum
2.2
Leptin
2.2.1 Background
Mammals can maintain a constant BM and fat
stores despite of daily fluctuations in food
availability. According to the adipostat model
of Kennedy (1953) circulating satiety signals
related to fat stores inform the brain about
changes in energy homeostasis and lead to
adjustments in food intake and energy
expenditure to normalize the BM. In 1959
Hervey demonstrated in a parabiosis study the
presence of a satiety factor that regulated
appetite and BM through an interaction with
the hypothalamus.
Cross-circulation experiments of Coleman
(1973) verified the presence of the circulating
satiety factor. Coleman studied morbidly obese
ob/ob and db/db mice (Mus musculus) with
recessive mutations in the obese (ob) and
diabetes (db) genes. By exchanging circulation
with a normal mouse an ob/ob mouse ate less
and gained weight less rapidly. Parabiosis of an
21
ob/ob and a db/db mouse led to starvation of
the ob/ob mouse, whereas the diabetic partner
remained healthy. These studies suggested that
the ob/ob mouse has a functional satiety center
but lacks a circulating satiety factor regulating
energy balance, whereas the db/db mouse
produces the satiety factor but is insensitive to
it.
Positional cloning of the obese gene by
Zhang et al. (1994) confirmed the hormonal
link between the adipose tissue and the brain.
The hormone encoded by the ob gene was
named leptin derived from the Greek word
λεπτός [leptós] meaning thin (Halaas et al.,
1995). The C57BL/6J ob/ob mouse has a
nonsense mutation in the obese gene at codon
105 (Zhang et al., 1994). An arginine (Arg) has
been replaced by a premature stop codon
resulting in the production of an inactive form
of leptin. In the SM/Ckc-+DACob2J/ob2J mouse,
a retroviral-like transposon inserted into the
first intron of the ob gene prevents the
synthesis of mature ob mRNA (Moon &
Friedman, 1997). Apart from being morbidly
obese,
ob/ob
mice are hyperphagic,
hyperglycemic, hyperinsulinemic and infertile.
The first experiments with exogenous leptin
administrations
in
1995
demonstrated
significant leptin-induced decreases in BM,
body adiposity and food intake of ob/ob mice
(Campfield et al., 1995; Halaas et al., 1995;
Pelleymounter et al., 1995).
transcript (Zhang et al., 1994). Mouse leptin is
a 167 aa protein, but it circulates as a 146 aa
peptide after cleavage of the N-terminal signal
sequence. The leptin molecule consists of four
α-helices connected by two long crossover
links and one short loop arranged in a left-hand
twisted helical bundle (Zhang et al., 1997). The
C-terminus of the protein and the beginning of
one of the long crossover links are held
together by a disulfide bond formed by cysteine
(Cys)-96 and Cys-146.
Initially leptin protein was demonstrated to
be expressed exclusively in the white adipose
tissue (WAT; Zhang et al., 1994). Subsequent
reports revealed expression also in various
central and peripheral tissues e.g. in the brain
(Morash et al., 1999; Wiesner et al., 1999),
pituitary (Morash et al., 1999; Jin et al., 2000),
BAT (Moinat et al., 1995; Tsuruo et al.,
1996), stomach (Bado et al., 1998), skeletal
muscle (Wang et al., 1998), osteoblasts
(Reseland et al., 2000), placenta and in fetal
bone and cartilage (Hoggard et al., 1997a).
Half-life of leptin in circulation is about 1.6 hr
and it is degraded primarily by the kidneys
(Cumin et al., 1996; Meyer et al., 1997).
There are at least six isoforms of leptin
receptor (OB-Ra-f; Lee et al., 1996; Wang et
al., 1996), single membrane-spanning proteins
homologous to class I cytokine receptor family
(Tartaglia et al., 1995). OB-Rb is the long form
responsible for cell signalling containing a
cytoplasmic domain of about 300 aa (Chen et
al., 1996; Lee et al., 1996; Tartaglia, 1997).
Binding of leptin to OB-Rb activates the JAK
(Janus kinase)-STAT (signal transducers and
activators of transcription) signal transduction
pathway (Baumann et al., 1996; Vaisse et al.,
1996; Ghilardi & Skoda, 1997). The other OBR isoforms considered to lack signalling
capabilities contain a shorter cytoplasmic
domain of 30-40 aa (Tartaglia et al., 1995;
Tartaglia,
1997).
OB-Re
lacks
the
2.2.2 Structure and expression of the leptin
protein and leptin receptor
The human ob gene is located on chromosome
7q31.3 (Isse et al., 1995; Clement et al., 1996).
It consists of three exons separated by two
introns and spans 18-20 kilobase (kb) pairs,
encoding a 3.5 kb cDNA (Isse et al., 1995;
Gong et al., 1996). The murine obese gene is
located on chromosome 6 encoding a 4.5 kb
22
transmembrane and intracellular domains and
circulates as a soluble receptor (Lee et al.,
1996).
OB-Rb
predominates
in
the
hypothalamus, while the short form is detected
in most tissues. Also the long form is expressed
in peripheral tissues but in much lower levels.
OB-R are found in several organs and
tissues such as hypothalamic nuclei (Tartaglia
et al., 1995; Couce et al., 1997; Fei et al.,
1997),
leptomeninges,
choroid
plexus
(Tartaglia et al., 1995; Hoggard et al., 1997a),
cerebellum, brain microvessels (Bjørbæk et al.,
1998), pituitary (Jin et al., 2000), brain, kidney,
lung (Tartaglia et al., 1995; Hoggard et al.,
1997b), WAT, BAT (Kutoh et al., 1998),
stomach (Wang et al., 1996), intestine (Lostao
et al., 1998), pancreas (Kieffer et al., 1996),
liver, spleen, adrenals (Cohen et al., 1996;
Hoggard et al., 1997b), ovary (Karlsson et al.,
1997), testis (Hoggard et al., 1997b; Caprio et
al., 1999), osteoblasts (Reseland et al., 2000),
uterus, heart, muscle (Lin et al., 2000),
placenta and fetal lung, hair follicles, cartilage
and bone (Hoggard et al., 1997a).
follow a diurnal rhythm (Sinha et al., 1996;
Schoeller et al., 1997). Concentrations rise to a
peak during the night and fall to a nadir
between morning and early afternoon. When
meal times are shifted by 6.5 hr, the plasma
leptin rhythm is also transferred by 5-7 hr
(Schoeller et al., 1997).
Insulin (Cusin et al., 1995; Saladin et al.,
1995), glucocorticoids (Slieker et al., 1996;
Wabitsch et al., 1996a) and PRL (Gualillo et
al., 1999) stimulate leptin synthesis. In
contrast, thyroid hormones (Escobar-Morreale
et al., 1997), GH (Florkowski et al., 1996),
somatostatin (SRIF; Donahoo et al., 1997) and
FFA (Rentsch & Chiesi, 1996) have an
inhibitory action on leptin. Moreover, cold
exposure decreases leptin gene expression in
WAT (Trayhurn et al., 1995b) and BAT
(Moinat et al., 1995) and also catecholamines
inhibit leptin release (Scriba et al., 2000).
2.2.3 Regulation of leptin secretion
Exogenous leptin administrations decrease food
intake, BM and adiposity without inducing a
loss of fat-free dry mass in ob/ob mice
(Campfield et al., 1995; Halaas et al., 1995;
Pelleymounter et al., 1995; Mistry et al., 1997;
Kaibara et al., 1998). Also their metabolic rate
(MR), blood glucose and insulin levels and Tb
are normalized by leptin supply. On the
contrary, leptin has no clear effects on the
energy metabolism of db/db mice, and its
effects are not as pronounced in genetically
normal lean mice as in ob/ob mice. Central
administrations seem to induce the strongest
responses (Halaas et al., 1997; Ramsey et al.,
1998). Also MR of genetically normal
mammals can be increased by leptin, but only in
situations with already lowered MR e.g. in
2.2.4 Metabolic effects of leptin
2.2.4.1 Anorectic influence
Circulating leptin levels of humans and
laboratory rodents correlate positively with
their body fat stores and body mass indexes
(BMIs) reflecting body adiposity (Frederich et
al., 1995; Maffei et al., 1995; Ma et al., 1996).
Blood leptin levels of humans start to decline
after 12 hr of fasting with the minimum values
after 36 hr (Boden et al., 1996; Kolaczynski et
al., 1996). This decrease is not directly
proportional to the loss of fat stores. Leptin
levels return to basal values in 24 hr after refeeding. A similar fasting-induced suppression
and a re-feeding-mediated rebound occur in
WAT leptin mRNA levels of rodents
(MacDougald et al., 1995; Trayhurn et al.,
1995a). Circulating leptin levels of humans
23
ATP-sensitive potassium channels resulting in
β-cell hyperpolarization and decreased cell
excitability (Harvey et al., 1997). Leptin
specifically targets a protein kinase C (PKC)regulated component of the PLC/PKC
signalling system to prevent insulin secretion
(Chen et al., 1997). Leptin impairs in vitro
several metabolic actions of insulin such as
stimulation of glucose transport, glycogen and
protein synthesis, lipogenesis and inhibition of
lipolysis (hydrolysis of TG to FFA and glycerol;
Müller et al., 1997).
Furthermore, exogenous leptin induces redistribution of intrahepatic glucose fluxes by
stimulating liver gluconeogenesis and inhibiting
glycogenolysis (degradation of glycogen;
Rossetti et al., 1997; Liu et al., 1998; Burcelin
et al., 1999; Nemecz et al., 1999). The
inhibition of glycogen degradation leads to
preservation of liver carbohydrate stores
(Nemecz et al., 1999; O´Doherty et al., 1999;
Mustonen et al., 2002a).
Leptin also increases glucose uptake and
oxidation, lactate formation and glycogen
synthesis in skeletal muscle (Kamohara et al.,
1997; Ceddia et al., 1999). Burcelin et al.
(1999) have reported increased whole-body
glucose turnover and uptake to BAT, brain and
heart after leptin administrations. Glucose
utilization is increased in skeletal muscle and
BAT but suppressed in WAT by leptin
treatment (Siegrist-Kaiser et al., 1997; Wang et
al., 1999). Leptin is also able to inhibit gastric
emptying of glucose (Smedh et al., 1998) and
small intestine sugar absorption (Lostao et al.,
1998).
fasted or hypothermic animals (Stehling et al.,
1996; Döring et al., 1998; Geiser et al., 1998).
Neuronal targets for the anorectic actions of
leptin are the ARC, ventromedial nucleus
(VMN), PVN, dorsomedial nucleus and lateral
hypothalamus (Ahima & Osei, 2001). Several
neuropeptides involved in the control of
appetite are colocalized with OB-Rb in the
hypothalamus. Leptin administrations decrease
gene expression of the orexigenic neuropeptide
Y (NPY; Stephens et al., 1995; Ahima et al.,
1996), agouti-related peptide (AGRP), galanin
and melanin-concentrating hormone (Sahu,
1998; Mizuno & Mobbs, 1999). On the
contrary, gene expression of anorectic peptides
such as pro-opiomelanocortin (Schwartz et al.,
1997), cocaine- and amphetamine-regulated
transcript and neurotensin are stimulated by
leptin (Kristensen et al., 1998; Sahu, 1998).
Leptin also functions synergistically with
cholecystokinin to suppress food intake
(Barrachina et al., 1997).
2.2.4.2 Effects of leptin on carbohydrate
metabolism
Leptin affects glucose metabolism via the
central nervous system (CNS; Kamohara et al.,
1997). It may also have direct effects on
peripheral tissues, as OB-Rb are expressed in
the pancreas (Pallett et al., 1997), liver (Chen
et al., 1996), skeletal muscle (Liu et al., 1997)
and intestine (Morton et al., 1998) i.e. tissues
participating in carbohydrate metabolism.
Exogenous leptin supply has elevated (Kulkarni
et al., 1997), suppressed (Schwartz et al.,
1996; Wang et al., 1999) or had no effect on
circulating glucose concentrations of rodents
(Kamohara et al., 1997; Mustonen et al.,
2002a).
Leptin inhibits insulin secretion from the βcells of the islets of Langerhans (Kulkarni et
al., 1997; Pallett et al., 1997) by activating
2.2.4.3 Effects of leptin on lipid metabolism
The influence of leptin on mammalian fat
metabolism is exerted indirectly through the
CNS or directly on the peripheral tissues
(Reidy & Weber, 2000). Both of these
24
excess of fat are associated with infertility
(Frisch, 1994). As leptin levels of humans and
laboratory rodents are directly proportional to
body fat stores, a large number of studies have
focused on the possible function of leptin in the
control of reproductive processes. Evidence is
accumulating that leptin plays a role in puberty,
menstruation, pregnancy and lactation.
Women have higher leptin levels than men at
any per cent body fat (Ma et al., 1996; Saad et
al., 1997), and similar sexual dimorphism is
also observed in leptin concentrations of
rodents (Frederich et al., 1995). Women seem
to have higher total, free and protein-bound
leptin levels (McConway et al., 2000). The
gender difference has been observed from the
third postnatal day onwards (Hytinantti et al.,
1999), and it may result from several factors.
Leptin secretion rates are 2-3 times higher from
subcutaneous (sc) fat compared to visceral fat
(van Harmelen et al., 1998), and the ratio of sc
vs. visceral fat is usually higher in women. In
addition, the pulse amplitude of leptin release is
more than twice as high in women (Licinio et
al., 1998a), and the feminine adipose tissue is
more sensitive to substances such as estradiol
and glucocorticoids stimulating leptin release
(Casabiell et al., 1998).
The hypothalamus (Chen et al., 1996),
pituitary (Jin et al., 2000), uterus (Kitawaki et
al., 2000), placenta (Hoggard et al., 1997a),
ovary (Karlsson et al., 1997) and Leydig cells
of testis (Caprio et al., 1999) express OB-Rb.
Leptin stimulates hypothalamic GnRH release
by increasing frequency and amplitude of
GnRH pulses (Magni et al., 1999; Lebrethon et
al., 2000; Parent et al., 2000). In the pituitary,
leptin stimulates release of LH, FSH and PRL
(Yu et al., 1997). Leptin has also been shown
to inhibit testosterone secretion (Tena-Sempere
et al., 1999) and estradiol (Karlsson et al.,
1997) and progesterone production (Barkan et
al., 1999).
pathways decrease TG synthesis and increase
lipolysis and lipid oxidation, leading to an
elevation in circulating FFA levels (Hwa et al.,
1997) and to a suppression in blood TG
concentrations (Wang et al., 1999). In ob/ob
mice, exogenous leptin decreases the
respiratory quotient, indicating a switch from
carbohydrate to lipid oxidation (Hwa et al.,
1997).
Lipolysis in adipocytes from lean and ob/ob
mice increases with leptin treatment (Frühbeck
et al., 1997, 1998). A similar effect has also
been observed in adipocytes of lean rats
(Siegrist-Kaiser et al., 1997). Conversely, no
effects are observed, when adipocytes of db/db
mice or fa/fa rats lacking the functional OB-R
isoform are treated with leptin. Also TG
contents of liver, muscle and pancreas decrease
due to leptin (Shimabukuro et al., 1997).
Leptin mediates fatty acid metabolism by
influencing concentrations and mRNA levels of
its key enzymes. In preadipocytes, leptin
inhibits gene expression of acetyl-CoA
carboxylase, the rate-limiting enzyme of long
chain fatty acid synthesis (Bai et al., 1996). In
pancreatic islets, leptin elevates mRNA levels
of enzymes promoting FFA oxidation and
decreases levels of enzymes participating in
their esterification (Zhou et al., 1997).
Moreover, leptin reduces adipose tissue mass
by inducing adipocyte apoptosis (Qian et al.,
1998). It also modifies fat metabolism indirectly
by reducing the antioxidative and lipogenic
actions of insulin (Muoio et al., 1997, 1999). In
addition, the functions of leptin may be
mediated via other lipolytic agents such as
thyroid hormones (Cusin et al., 2000) and GH
(Barb et al., 1998).
2.2.5 Role of leptin in reproduction
It has been long recognized that body adiposity
affects reproduction as both a lack and an
25
may also participate in the determination of the
animal pole of the oocyte and in the
establishment of the inner cell mass and
trophoblast in the preimplantation stage embryo
(Antczak & Van Blerkom, 1997).
Leptin concentrations increase during
pregnancy, especially during the second
trimester (Hardie et al., 1997). After childbirth,
leptin levels decline (Butte et al., 1997; Hardie
et al., 1997). Lactation decreases leptin
concentrations (Pickavance et al., 1998;
Woodside et al., 2000) or has no effect on
them (Butte et al., 1997). Furthermore, leptin
levels decline in postmenopausal women,
possibly due to suppressed sex steroid
production (Rosenbaum et al., 1996).
Leptin treatment restores fertility in ob/ob
mice (Barash et al., 1996; Chehab et al., 1996),
and the onset of puberty of normal female mice
can be accelerated by leptin treatment (Ahima
et al., 1997; Chehab et al., 1997). A similar
effect could not be demonstrated in ad libitumfed normal female rats (Cheung et al., 1997).
Leptin was, however, able to advance puberty
in food-restricted individuals. Leptin levels
exhibit sexually dimorphic changes during the
pubertal
phases
of
humans.
Leptin
concentrations of boys peak at Tanner stage 2
and decrease thereafter (Blum et al., 1997).
Leptin levels of girls, on the other hand,
increase gradually throughout the puberty.
These developmental differences in the
leptin levels may result from the suppressive
effects of testosterone (Luukkaa et al., 1998)
and the stimulatory influence of estradiol
(Shimizu et al., 1997; Casabiell et al., 1998) on
leptin secretion. Farooqi et al. (1999) have
normalized feeding behaviour and BM, and
triggered the onset of puberty with exogenous
leptin in a girl suffering from congenital leptin
deficiency. These results suggest that leptin is a
necessary but not necessarily a sufficient factor
for the onset of mammalian puberty.
OB-Rb mRNA levels in the endometrium
change throughout the menstrual cycle
(Kitawaki et al., 2000). The early proliferative
phase is associated with low OB-Rb
expression, which increases gradually and
peaks during the early secretory phase. The
highest circulating leptin levels are encountered
during the late follicular phase and during the
luteal phase (Hardie et al., 1997; Riad-Gabriel
et al., 1998; Ludwig et al., 2000). Licinio et al.
(1998b) have documented that the ultradian
LH, estradiol and leptin rhythms are
synchronized from mid- to late follicular phase.
Moreover, leptin inhibits early follicular
development (Kikuchi et al., 2001) and
ovulation (Duggal et al., 2000) in rodents. It
2.2.6 Physiological significance of leptin
2.2.6.1 Seasonal starvation and obesity
Leptin has been hypothesized to be involved in
energy conservation during periods of energy
shortage and to prevent obesity during periods
of energy excess (Caro et al., 1996). The
possible role of leptin in the neuroendocrine
response to fasting was first demonstrated by
Ahima et al. (1996). They observed that fasting
decreases murine leptin levels together with
thyroid and reproductive hormones, whereas
stress
hormone
concentrations
and
hypothalamic NPY mRNA levels increase.
Starvation is also characterized with suppressed
GH secretion (Aubert et al., 1997) and immune
function (Lord et al., 1998). Exogenous leptin
treatment can reverse the above-mentioned
fasting-induced adaptations (Ahima et al.,
1996; Aubert et al., 1997; Finn et al., 1998;
Lord et al., 1998). The decrease in leptin levels
caused by starvation could be a critical signal
initiating the neuroendocrine response to
fasting including the stimulation of stress
response and the suppression of reproductive
26
the rhesus monkey (Macaca mulatta; Hotta et
al., 1996). Schneider et al. (2000) have
observed that the plasma leptin levels of golden
hamsters do not always reflect their body fat
content. Also several studies on Fissipedia and
Pinnipedia show dissociation of leptin levels
from fat stores.
Plasma leptin concentrations of the raccoon
dog and the blue fox (Alopex lagopus) were
low in summer and increased in Oct with
increasing body adiposity (Nieminen et al.,
2001a). However, in Nov their leptin
concentrations declined, although their BMs
remained stable. Arnould et al. (2002) reported
a negative trend between plasma leptin levels
and body condition in lactating Antarctic fur
seals (Arctocephalus gazella), whereas there
was no correlation between fat mass and serum
leptin levels in the northern elephant seal pups
(Mirounga angustirostris; Ortiz et al., 2001a).
Nieminen et al. (2000) have demonstrated a
negative relationship between plasma leptin
levels and BMIs of female minks, but Tauson &
Forsberg (2002) found a positive correlation
between mink leptin concentrations and BMs.
Furthermore, Mustonen et al. (2000)
documented a positive correlation between
leptin levels and BMIs of minks but only in a
certain time of the autumn. Rhesus monkeys of
Mann et al. (2000) showed a positive
correlation between their leptin levels and per
cent body fat at a particular point of the study,
but there was no relation between these
variables later. In the common shrew (Sorex
araneus),
the
highest
BAT
leptin
concentrations were measured in mid-winter,
when BMs of the shrews were low (Nieminen
& Hyvärinen, 2000). Also plasma leptin levels
of the little brown bat (Myotis lucifugus) were
dissociated from their body adiposity during the
prehibernatory period (Kronfeld-Schor et al.,
2000).
processes, growth, MR, thermogenesis and
immune defense.
From the evolutionary perspective the ability
of humans and other animals to adapt to
prolonged starvation is of fundamental
importance for survival in nature (Flier, 1998).
On the contrary, the ability to avoid obesity has
little survival value in natural conditions, as
wild animals seldom experience pathological
overweight causing e.g. heart and vascular
diseases, diabetes or arthrosis. For this reason it
seems logical that leptin enters the brain across
the blood-brain barrier by a saturable transport
system (Banks et al., 1996; Karonen et al.,
1998) which even physiologically low hormone
levels are able to saturate (Banks et al., 2000;
Burguera et al., 2000). During the periods of
food abundance, animals with leptin resistance
do not receive a satiety signal and thus can
consume and store large amounts of energy as
fat to be used during periods of food shortage
(Flier, 1998). The fasting-induced suppression
of leptin levels, on the other hand, induces
metabolic, endocrine and behavioural responses
that conserve energy and stimulate food intake,
thus enhancing the chances of an individual to
survive through a period of food deprivation.
2.2.6.2 Leptin in seasonal mammals
The data obtained from studies using seasonal
mammals as experimental animals are
somewhat different from human and laboratory
rodent experiments. It has been observed in
several seasonal species that the primary
physiological role of leptin is not necessarily to
function as a signal of their body adiposity. A
positive correlation between leptin levels and
BM or body fatness has been found in some
seasonal mammals such as the Djungarian
hamster (Phodopus sungorus; Klingenspor et
al., 2000), the cattle (Bos taurus; Ehrhardt et
al., 2000), the sheep (Lincoln et al., 2001) and
27
(reflects fat stores) of the fat-tailed dunnart
(Sminthopsis crassicaudata) fed on a standard
laboratory diet (Hope et al., 1999).
Concannon et al. (2001) have studied the
seasonality of leptin levels in reference to the
annual BM and food intake cycles of
woodchucks (Marmota monax). Their blood
leptin levels increase during the gain in BM in
late spring, reach peak values in summer 2-7
wk after the peak BMs and decline together
with BMs to minimum values in late winter.
The autumnal decline in food intake is fastest,
when leptin levels are close to maximum values
and food intake increases in late winter, when
leptin concentrations are low.
Leptin levels of the reindeer decrease in midwinter when also BM gain of the animals is
suppressed (Soppela, 2000). Moreover,
reindeer fed with lichens have lower leptin
concentrations than animals fed with reindeer
feed. The rhesus monkey, on the other hand,
experiences peak leptin levels in late winter and
the nadir in late summer (Mann et al., 2000).
Autumnal leptin levels of the European brown
bear (Ursus arctos arctos) increase with
increasing body adiposity with the highest
values just prior to the winter sleep (Hissa et
al., 1998a). Also the autumnal leptin
concentrations of the mink rise in association
with increasing BMs (Mustonen et al., 2000).
Photoperiod and melatonin are known to
regulate leptin secretion of some seasonal
mammals. Djungarian hamsters kept in SD have
lower BMs and blood leptin levels than animals
kept in LD, while photoperiod has no effect on
BMs or leptin levels of the golden hamster
(Horton et al., 2000; Klingenspor et al., 2000).
Also leptin and OB-Rb mRNA levels in adipose
tissue and hypothalamus are downregulated by
SD in the Djungarian hamster (Mercer et al.,
2000). Furthermore, Klingenspor et al. (1996)
have reported reduced leptin gene expression in
WAT of Djungarian hamsters during the
As mentioned earlier, fasting has a negative
influence on leptin levels of humans and
rodents (Trayhurn et al., 1995a; Kolaczynski et
al., 1996). The same phenomenon has also
been observed in the golden hamster (Schneider
et al., 2000), Djungarian hamster (Mercer et
al., 1997), sheep (Marie et al., 2001) and in
pups of the Antarctic fur seal (Arnould et al.,
2002). Leptin pulse frequency of domestic pigs
(Sus scrofa) also decreases by fasting and
increases after re-feeding (Barb et al., 2001).
On the other hand, lactating Antarctic fur seals
experience increasing leptin levels during the
first 24 hr of fasting and decreasing
concentrations during the following 48 hr
without food (Arnould et al., 2002). A total
three-wk wintertime fast has no effects on the
blood leptin levels of raccoon dogs, while there
is a trend towards lower leptin levels in fasting
blue foxes (Nieminen et al., 2001a).
The anorectic effect of exogenous leptin has
been described in the Arctic ground squirrel
(Spermophilus parryii plesius; Boyer et al.,
1996; Ormseth et al., 1996; Boyer et al.,
1997). Exogenous leptin decreases their food
intake
and
prevents
the preand
posthibernational fattening. Also the food
intake of pigs (Barb et al., 1998) and sheep
(Clarke et al., 2000) decrease with leptin
supply. Centrally administered leptin inhibits
vernal food intake in sheep, but provides no
satiety signal when administered in autumn.
This suggests that the responsiveness to leptin
depends on the season. A similar phenomenon
has also been observed in the Djungarian
hamster. Exogenous leptin causes a larger
reduction to body fat mass of SD kept hamsters
compared to animals maintained in LD,
whereas food intake is inhibited similarly in
both daylengths (Klingenspor et al., 2000). The
anorectic influence of leptin has also been
observed in marsupials as exogenous leptin
decreases energy intake, BM and tail width
28
which decline postdelivery (Henson et al.,
1999). Leptin levels of the big brown bat
(Eptesicus fuscus) also increase during
pregnancy and decrease after delivery (Kunz et
al., 1999). An increase in leptin synthesis has
been observed during sheep pregnancy
(Ehrhardt et al., 2001) whereas lactation
decreases their leptin levels (Sorensen et al.,
2002).
winter. Seasonal variations in WAT leptin
expression closely follow the changes in BM
and daylength being highest in June and lowest
in Dec. In addition, artificial SD decreases
leptin expression in WAT and BAT.
Exogenous melatonin treatment increases
autumnal leptin levels in mink plasma
(Mustonen et al., 2000) and leptin gene
expression in WAT and BAT of the garden
dormouse (Eliomys quercinus; Ambid et al.,
1998). On the contrary, melatonin treatment
suppresses leptin levels in the marginally
photoperiodic rat (Rasmussen et al., 1999;
Wolden-Hanson et al., 2000) suggesting that
the effects of melatonin on leptin
concentrations are species-specific.
Data concerning the effects of leptin on
reproductive variables of seasonal mammals are
scarce. The sexual dimorphism observed in
leptin levels of humans and rodents (Frederich
et al., 1995; Saad et al., 1997) is also present in
sheep, as ewe lambs have higher leptin
concentrations than ram lambs (Ehrhardt et al.,
2000). Gender difference is, however, absent in
several wild mammals such as the pups of the
Antarctic fur seal (Arnould et al., 2002), the
mink (Mustonen et al., 2000), the raccoon dog
and the blue fox (Nieminen et al., 2001a).
There are gender differences also in leptin
sensitivity as the vernal responsiveness to leptin
is more pronounced in ewes compared to rams
(Clarke et al., 2000). Diurnal leptin levels of
male rhesus monkeys decrease throughout the
juvenile period until the onset of puberty
without any association with the pubertal rises
in LH or testosterone concentrations (Mann et
al., 2000).
Leptin is able to facilitate lordosis in fed but
not food-deprived golden hamsters (Wade et
al., 1997). Furthermore, LH secretion of fasted
sheep is stimulated by leptin (Nagatani et al.,
2000). Baboon (Papio sp.) gestation is
associated with increased leptin concentrations
2.2.7 Molecular evolution of leptin
Leptin protein is highly conserved among
mammals. The rhesus monkey (91 %, Hotta et
al., 1996), the cattle (87 %, Ji et al., 1998), the
cat (Felis catus, 86 %, Sasaki et al., 2001) and
the mouse (84 %, Zhang et al., 1994) share the
highest aa identities with human leptin.
Moreover, in the rat (Ogawa et al., 1995), the
pig (Ramsay et al., 1998), the Djungarian
hamster (Mercer et al., 1997) and the dog
(Canis familiaris, Iwase et al., 2000) leptin
sequences are also highly homologous to
human peptide (80-83 %).
When mammalian leptin gene sequences are
analyzed phylogenetically, the large hairy
armadillo (Chaetophractus villosus) and the
fat-tailed dunnart are at the base of the tree
(Doyon et al., 2001). The primates group
together, and humans are closer to other
members of the family Hominidae (orangutan
Pongo pygmaeus, gorilla Gorilla gorilla and
chimpanzee Pan troglodytes) than the Old
World monkeys (rhesus monkey). The
carnivores (raccoon Procyon lotor, striped
skunk Mephitis mephitis, dog and cat) group
together, the beluga whale (Delphinapterus
leucas) is grouped with artiodactyl species (pig,
sheep and cattle) and rodents (mouse and rat)
group together with the little brown bat. When
avian (chicken Gallus gallus and turkey
Meleagris gallopavo) sequences are included in
the analysis, they group with mouse and rat
29
2.2.8.2 Reptilia and Amphibia
leptins. Doyon et al. (2001) consider that
convergent or parallel evolution could be the
most plausible hypotheses for explaining the
unexpected similarity between bird and rodent
leptins.
Doyon et al. (2001) have failed to clone leptin
from adipose tissue and liver of the snapping
turtle (Chelydra serpentina) and the leopard
frog (Rana pipiens). However, polyclonal
antibodies against mouse leptin recognize a
protein of a similar molecular weight in brain of
the fence lizard (Sceloporus undulatus;
Niewiarowski et al., 2000) and in plasma of the
Italian wall lizard (Podarcis sicula; Paolucci et
al., 2001). Moreover, leptin-LI has been
observed in stomach of the African clawed frog
(Xenopus laevis), viperine (water) snake
(Natrix maura) and Iberian wall lizard (P.
hispanica; Muruzábal et al., 2002).
Daily injections of recombinant murine leptin
are able to produce similar phenotypic effects
to fence lizards as observed in mammals
(Niewiarowski et al., 2000). Leptin increases
their Tb and resting MR as well as suppresses
food intake and activity rates. Paolucci et al.
(2001) have found leptin-LI in plasma, liver and
fat body of P. sicula. Their leptin-LI
concentrations
fluctuate
during
the
reproductive cycle, plasma and fat body leptinLI levels being highest during the quiescent
period simultaneously with large corpora
adiposa fat reserves.
2.2.8 Leptin of nonmammalian vertebrates
2.2.8.1 Aves
The functions of avian leptin have been mainly
investigated in the domestic fowl. There is
some controversy about the cloning of the
leptin gene of the chicken. Taouis et al. (1998)
have cloned and sequenced a candidate for the
chicken leptin gene 83, 96 and 97 % identical
to human, rat and mouse sequences, and this
sequence was independently confirmed with a
single nucleotide difference by Ashwell et al.
(1999a). On the contrary, Friedman-Einat et al.
(1999) could not confirm the presence of a
chicken leptin gene sequence with close
sequence similarity to mouse leptin.
Leptin is mainly secreted by the liver and
adipose tissue of chickens (Taouis et al., 1998;
Ashwell et al., 1999a). Leptin expression has
also been located in the embryonic liver and
yolk sac. The hepatic expression has been
hypothesized to result from the major role of
the liver in avian lipogenesis. As in mammals,
fasting suppresses leptin concentrations of
chickens (Dridi et al., 2000), and injections of
chicken leptin decrease their food intake (Raver
et al., 1998). On the contrary, murine leptin
administrations have no influence on appetite of
chickens (Bungo et al., 1999).
Leptin expression in chicken liver is
increased by insulin, dexamethasone (a
synthetic glucocorticoid) and GH and
decreased by glucagon and estrogen (Ashwell
et al., 1999a,b). In addition, estrogen decreases
leptin expression in the adipose tissue of
chickens.
2.2.8.3 Osteichthyes & Cephalaspidomorphi
The original report on the identification of the
leptin gene (Zhang et al., 1994) indicated the
presence of a homologous DNA fragment in
the eel (Anguilla sp.). This finding suggests
that the leptin gene is highly conserved among
vertebrates. Doyon et al. (2001), however,
failed to clone leptin gene from adipose tissue
and liver of the rainbow trout (Oncorhynchus
mykiss), the American eel (A. rostrata) and the
goldfish (Carassius auratus). Although the fish
leptin gene has not yet been cloned,
30
2.3
immunoreactive material of similar size to
mammalian leptin has been described in various
tissues of several fish species using anti-mouse
leptin antibodies (Johnson et al., 2000).
Leptin-immunoreactivity (-I) is expressed in
blood, brain, heart and liver of the green sunfish
(Lepomis cyanellus), bluegill sunfish (L.
macrochirus), largemouth bass (Micropterus
salmoides), white crappie (Pomoxis annularis),
channel catfish (Ictalurus punctatus) and
rainbow trout. Leptin-LI is present also in the
stomach of the rainbow trout (Muruzábal et al.,
2002). Blood leptin concentrations of the green
sunfish decrease during a two-wk fast, and
brain leptin concentrations correlate positively
with per cent body fat in the white crappie and
the bluegill sunfish (Johnson et al., 2000). On
the other hand, there is no relationship between
blood leptin levels and body adiposity in fish.
Moreover, Yaghoubian et al. (2001) have
identified four proteins that are immunoreactive
with antibody against a human leptin fragment
in serum and fat-containing tissues of the sea
lamprey (Petromyzon marinus).
Baker et al. (2000) have not found any
effects of human recombinant leptin on growth,
energy stores, gonad weight, pituitary content
of FSH or on plasma levels of insulin-like
growth factor-I (IGF-I), insulin, GH or
thyroxine (T4) of the immature Coho salmon
(O. kisutch). In the goldfish, central and
peripheral administrations of murine leptin
decrease food intake (Volkoff et al., 2003).
Peyon et al. (2001) have reported that murine
recombinant leptin also affects LH secretion
from the pituitary cells of the European sea
bass (Dicentrarchus labrax) suggesting that
leptin may regulate reproductive functions of
fish. Furthermore, Londraville & Duvall (2002)
have recently stimulated fat metabolism of
green sunfish with recombinant mouse leptin
without any leptin-induced effects on their BM,
fat content or appetite.
Ghrelin
2.3.1 Background
It was long assumed that the secretion of GH
from the pituitary was primarily regulated by
antagonistic
interactions
between
two
hypothalamic peptides: the stimulatory GHreleasing hormone (GHRH) and the inhibitory
SRIF (Bentley, 1998). A new independent
pathway controlling GH release was recently
discovered from GH secretagogue (GHS)
experiments. GHS are small synthetic
molecules developed to release GH in vitro
through a specific GHS receptor (GHS-R;
Casanueva & Dieguez, 1999). The first highly
potent
GH-releasing
hexapeptide
1
6
[His ,Lys ]GHRP was developed in the 1980s
(Bowers et al., 1984).
The cloning of the G-protein-coupled GHSR in the hypothalamus and pituitary
demonstrated that GHS function through
different receptors from those of GHRH
(Howard et al., 1996). GHS increase the
intracellular Ca2+ levels via inositol 1,4,5trisphosphate signal transduction, whereas
GHRH increases intracellular cAMP serving as
a second messenger (Kojima et al., 2001). The
endogenous ligand for GHS-R remained
unknown until 1999 when a Japanese research
group purified and identified it from rat
stomach extract (Kojima et al., 1999). This
endogenous GHS was named ghrelin: "ghre” is
the Proto-Indo-European root for the word
“growth” and the suffix “relin” signifies
“releasing substances”. In the same paper the
ghrelin-induced stimulation of rat GH secretion
was demonstrated in vitro as well as in vivo.
2.3.2 Structure of ghrelin
Rat and human ghrelins consist of 28 aa
(Kojima et al., 1999). Serine (Ser)-3 residue is
31
post-translationally n-octanoylated, which is
essential for the biological activity of the
molecule (Kojima et al., 1999; Bedranek et al.,
2000). In addition to ghrelin-28, a 27-residue
ghrelin called des-Gln14-ghrelin has been found
from the rat stomach (Hosoda et al., 2000a).
Its sequence is identical to ghrelin-28 except
for one deleted glutamine (Gln). Des-Gln14ghrelin is produced through an alternative
splicing of the ghrelin gene, and the n-octanoyl
modification at Ser-3 is indispensable for its
activity, as well. This second endogenous
ligand for GHS-R has also been experimentally
shown to stimulate rat GH secretion.
Moreover, Hosoda et al. (2000b) have
found a new form of ghrelin in rat plasma and
gastrointestinal tissues. This des-n-octanoyl
form of ghrelin, named des-acyl ghrelin, is not
capable of activating GHS-R. It, however,
constitutes the major fraction of ghrelin-I found
in the rat stomach. The chicken ghrelin consists
of 26 aa, and its Ser-3 is n-octanoylated or ndecanoylated (Kaiya et al., 2002). The bullfrog
(R. catesbeiana) has three forms of ghrelin,
each consisting of 27-28 aa (Kaiya et al.,
2001). At position 3 the bullfrog ghrelin has a
threonine (Thr) which is acylated by either noctanoic or n-decanoic acid. The goldfish
ghrelin consists of 19 aa and has two putative
cleavage sites and amidation signals after 12
and 19 aa (Unniappan et al., 2002). The second
aa has been substituted from Ser to Thr.
Human ghrelin is homologous to rat ghrelin
except for two aa (Kojima et al., 1999).
Bullfrog ghrelin shares a 29 % sequence
identity with mammalian ghrelins (Kaiya et al.,
2001), whereas chicken ghrelin has 54 and 19
% homologies to human and bullfrog peptides
(Kaiya et al., 2002). The goldfish ghrelin shares
47, 36 and 31 % identities to human, rat and
bullfrog ghrelins (Unniappan et al., 2002). The
N-terminal aa 1 and 4-7 are highly conserved in
all the above-mentioned species.
2.3.3 Ghrelin and GHS-R expression
Mammalian ghrelin is primarily expressed in the
gastrointestinal tract. The levels are highest in
the gastric fundus and decrease distally towards
the colon (Kojima et al., 1999; Date et al.,
2000a; Ariyasu et al., 2001). Gastrectomy
leads to a 65 % reduction in circulating ghrelinLI levels (Ariyasu et al., 2001). Cells with
ghrelin-I are abundant from the neck to the
base of the oxyntic gland but rare in the pyloric
gland (Kojima et al., 1999; Date et al., 2000a;
Hayashida et al., 2001). In addition to the
gastrointestinal tract, ghrelin is also synthesized
e.g. in the hypothalamic ARC (Kojima et al.,
1999; Lu et al., 2002), pituitary (Korbonits et
al., 2001), kidney (Mori et al., 2000),
pancreatic α-cells (Date et al., 2002), lungs
(Ariyasu et al., 2001), heart (Kaiya et al.,
2001), immune cells (Hattori et al., 2001),
placenta (Gualillo et al., 2001a) and testes
(Tena-Sempere et al., 2002).
In contrast to mammals, ghrelin-I is not
detectable in the proventriculus, stomach, ileum
or colon of the domestic fowl (Ahmed &
Harvey, 2002). It is, however, present in
hypothalamic neurons, although not in the
ARC. In contrast, Kaiya et al. (2002) have
located ghrelin synthesis to the proventriculus,
duodenum, ileum, cecum, rectum, spleen,
lungs, corpus striatum, cerebellum, optic lobe
and brain stem of chickens. Bullfrog ghrelin is
mainly synthesized by the stomach, like in the
rat (Kaiya et al., 2001). Low levels of gene
expression are also observed in the heart, lungs,
gall bladder, pancreas, small intestine and
testes, but not in the brain. In contrast, ghrelinLI has been observed in the stomach mucosa
and in several locations of the brain in the
European green frog (R. esculenta; Galas et al.,
2002). Moreover, goldfish show ghrelin mRNA
expression in the brain, pituitary, intestine,
liver, spleen and gills (Unniappan et al., 2002).
32
Ghrelin does not seem to require GH for its
orexigenic effect, as it is capable of increasing
food intake and BM gain of GH-deficient
rodents (Tschöp et al., 2000; Shintani et al.,
2001). Neither are circulating ghrelin and GH
concentrations always correlated with each
other (Tolle et al., 2002).
The ghrelin-induced stimulation of appetite
has been observed e.g. in rats (Tschöp et al.,
2000; Wren et al., 2000), mice (Asakawa et al.,
2001) and humans (Arvat et al., 2000; Broglio
et al., 2001; Wren et al., 2001). The orexigenic
activity of ghrelin may be exerted by its
stimulatory action on the genes encoding
hypothalamic NPY and AGRP, potent feeding
stimulators. Ghrelin administrations augment
NPY (Asakawa et al., 2001; Nakazato et al.,
2001; Shintani et al., 2001) and AGRP gene
expression (Kamegai et al., 2000) in rodent
hypothalamus. Antibodies and antagonists of
NPY and AGRP abolish the ghrelin-induced
feeding (Nakazato et al., 2001; Shintani et al.,
2001). Furthermore, ghrelin is able to reverse
the inhibition of appetite induced by exogenous
leptin, whereas the ghrelin-induced feeding is
suppressed by leptin administration. These data
indicate that ghrelin may antagonize leptin
action in the hypothalamus.
Ghrelin administration stimulates gastric
mobility, emptying and gastric acid secretion in
rodents (Masuda et al., 2000; Asakawa et al.,
2001; Date et al., 2001). It also increases
gastrin excretion (Lee et al., 2002) and inhibits
exocrine secretion of the pancreas (Zhang et
al., 2001). Ghrelin administrations increase
blood
glucose,
adrenaline,
cortisol,
adrenocorticotropic hormone and PRL
concentrations (Broglio et al., 2001; Nagaya et
al., 2001). On the other hand, ghrelin inhibits
thyroid-stimulating hormone (TSH) and
pancreatic SRIF secretion of rats (Wren et al.,
2000; Egido et al., 2002). The ghrelin-induced
effects on insulin secretion have been either
There are at least two different GHS-R
subtypes named 1a and 1b (Howard et al.,
1996). Ghrelin receptors have been found e.g.
in hypothalamic nuclei, hippocampus, pituitary
(Howard et al., 1996; Guan et al., 1997),
stomach, intestine (Date et al., 2000a), kidneys
(Mori et al., 2000), pancreas (Date et al.,
2002), myocardium, liver, adrenals, lungs,
thyroid, adipose tissue, blood vessels, skeletal
muscle, skin, lymphnodes, uterus, gonads
(Papotti et al., 2000) and immune cells (Hattori
et al., 2001).
2.3.4 Functions of ghrelin
After the discovery of ghrelin (Kojima et al.,
1999), its major action was supposed to be the
stimulation of GH secretion. Subsequent
studies, however, revealed that ghrelin
participated also in the control of feeding
behaviour and energy balance. The stimulatory
effect of ghrelin on GH release has been
documented in vivo e.g. in rats (Kojima et al.,
1999; Date et al., 2000b) and humans (Arvat et
al., 2000). A similar effect has also been
observed in the goat with rat ghrelin
administrations (Hayashida et al., 2001) and in
the domestic fowl treated with human (Ahmed
& Harvey, 2002) or chicken ghrelin (Kaiya et
al., 2002). Bullfrog and rat ghrelins have
stimulated in vitro GH secretion of the bullfrog
(Kaiya et al., 2001) and the Mozambique
tilapia (Oreochromis mossambicus; Riley et al.,
2002).
In mammals exogenous ghrelin stimulates
appetite leading to weight gain (Tschöp et al.,
2000). It also decreases fat utilization (Tschöp
et al., 2000) and oxygen consumption
(Asakawa et al., 2001). Conversely, central
injections of rat ghrelin inhibit food intake of
neonatal chickens (Furuse et al., 2001). Fish
ghrelin, on the other hand, stimulates food
intake of the goldfish (Unniappan et al., 2002).
33
Secretion decreases with high-fat food but
increases with a low-protein diet (Lee et al.,
2002). Low ghrelin levels of obese individuals
increase due to weight loss (Hansen et al.,
2002). Ghrelin levels are elevated by fasting
and exogenous insulin but reduced by refeeding and glucose administration (Tschöp et
al., 2000; Toshinai et al., 2001; Shiiya et al.,
2002). They also rise due to vagotomy and
gastrin administration (Lee et al., 2002).
Human ghrelin levels follow a diurnal
pattern that is in phase with the leptin rhythm
(Cummings et al., 2001). The levels increase
throughout the day to a peak at 0100 hr and fall
thereafter to a nadir at 0900 hr. Ghrelin may
play a role as a meal initiator as its levels rise 12 hr before meals and fall within one hr after
eating (Cummings et al., 2001; Shiiya et al.,
2002; Tolle et al., 2002). The ghrelin
concentrations of cattle also decrease one hr
after feeding and recover 4 hr after food intake
(Hayashida et al., 2001).
Ghrelin expression in rat pituitary increases
by GHRH treatment (Kamegai et al., 2001).
GH administration, on the other hand,
decreases ghrelin expression in rat stomach
(Lee et al., 2002). There are contradictory data
from rodent studies showing both positive
(Toshinai et al., 2001) and negative (Asakawa
et al., 2001) regulation of ghrelin synthesis by
leptin. In some cases, leptin has not affected
ghrelin secretion to either direction (Lee et al.,
2002).
stimulatory (Date et al., 2002; Lee et al., 2002)
or inhibitory (Broglio et al., 2001; Egido et al.,
2002). Ghrelin seems to have a strong
stimulatory influence on chicken corticosterone
secretion (Kaiya et al., 2002), and also in vitro
PRL secretion of the bullfrog (Kaiya et al.,
2001) and the Mozambique tilapia (Riley et al.,
2002) is stimulated by ghrelin.
Ghrelin may also be involved in the
regulation of reproductive processes. GHS-R
are localized in testes of men and rats and in
ovaries and uterus of women (Papotti et al.,
2000; Tena-Sempere et al., 2002). Ghrelin
mRNA is expressed in bullfrog and rat testes
(Kaiya et al., 2001; Tena-Sempere et al.,
2002). Recent studies show that ghrelin induces
in vitro a dose-dependent inhibition of
testosterone secretion in male rats (TenaSempere et al., 2002) and suppresses pulse
frequency of LH secretion in ovariectomized
females (Furuta et al., 2001). However, ghrelin
infusion does not affect circulating LH or FSH
concentrations of humans (Nagaya et al.,
2001). A similar lack of effects has been
observed in rat pituitary cells in vitro (Kojima
et al., 1999). Human ghrelin levels do not
express any changes with pubertal status
(Bellone et al., 2002), but ghrelin expression in
placenta fluctuates according to the phase of
pregnancy (Gualillo et al., 2001a).
2.3.5 Regulation of ghrelin secretion
Ghrelin is secreted in a pulsatile fashion and its
half-life in plasma is about 30 min (Tolle et al.,
2002). Blood ghrelin concentrations correlate
inversely with body adiposity and BMI in
humans (Ariyasu et al., 2001; Ravussin et al.,
2001; Shiiya et al., 2002). Ghrelin levels are
markedly elevated in anorexia and bulimia
nervosa and suppressed in overweight (Ariyasu
et al., 2001; Tschöp et al., 2001; Bellone et al.,
2002; Shiiya et al., 2002; Tanaka et al., 2002).
2.4
Growth hormone
2.4.1 Regulation of GH secretion
GH or somatotropin is a 22 kilodalton protein
of about 191 aa (Scanes & Campbell, 1995a)
synthesized in the somatotrophs of the anterior
pituitary gland (Harvey, 1995a) and secreted in
a pulsatile manner (Martin et al., 1974). Other
34
exposure (Olsen & Trenkle, 1973) have a
positive influence on GH. On the contrary, GH
release is suppressed by insulin (Melmed &
Slanina, 1985), FFA (Imaki et al., 1985) and
obesity (Veldhuis et al., 1995).
sites of GH synthesis are reproductive
(Frankenne et al., 1987; Selman et al., 1994),
immune (Weigent et al., 1988) and nerve
tissues (Hojvat et al., 1982). Vertebrate GH
molecules form a single chain polypeptide with
two intrachain disulfide bonds between Cys-57
and Cys-165 and between Cys-182 and Cys189 (Scanes & Campbell, 1995a).
GH secretion is regulated by two peptides of
hypothalamic origin: the stimulatory GHRH
(Krulich & McCann, 1969; Guillemin et al.,
1982; Rivier et al., 1982) and the inhibitory
SRIF (Krulich & McCann, 1969; Brazeau et
al., 1973). GHRH is synthesized by neurons
located in the ARC and VMN (Frohman et al.,
1968; Bloch et al., 1983) and SRIF by neurons
in the periventricular nucleus (Hökfelt et al.,
1975). Both of these peptides are released from
the nerve endings of the ME via the portal
vessels to the adenohypophysis (Hökfelt et al.,
1975; Bloch et al., 1983; Werner et al., 1986).
Three variants of human GHRH with 37, 40
and 44 aa have been identified (Guillemin et al.,
1982). SRIF-14 is a tetradecapeptide with a
disulfide bridge linking two Cys residues
(Brazeau et al., 1973), whereas SRIF-28
contains the sequence of SRIF-14 at its
COOH-terminal end (Bentley, 1998). In
addition of blocking GH secretion, SRIF also
inhibits the secretion of insulin, glucagon
(Koerker et al., 1974), gastrin (Bloom et al.,
1974) and secretin (Boden et al., 1975). GH
displays a negative feedback to its own
secretion by stimulating SRIF production
(Zeitler et al., 1990) and by inhibiting GHRH
synthesis (Chomczynski et al., 1988).
Fasting and hypoglycemia (Roth et al.,
1963), infusion of certain essential aa (Knopf et
al., 1965), anorexia nervosa (de Rosa et al.,
1983), exercise (Hunter et al., 1965), stress
(Abplanalp et al., 1977), glucocorticoids
(Samuels et al., 1977), ghrelin (Kojima et al.,
1999), sleep (Takahashi et al., 1968) and cold
2.4.2 GH receptor and IGF-I
GH receptor (GH-R) is a single-chain
polypeptide of approximately 620 aa containing
an extracellular, a transmembrane and a
cytoplasmic domain (Leung et al., 1987). The
extracellular domain of GH-R is identical to the
circulating GH-binding protein. GH-R are
located in most tissues and organ systems e.g.
in the hypothalamic ARC (Chan et al., 1996),
chondrocytes, osteocytes (Barnard et al., 1988;
Werther et al., 1990), adipocytes (Fagin et al.,
1980; Vikman et al., 1991), liver, kidney, heart,
muscle, skin, adrenals, intestine, lungs,
pancreas (Mathews et al., 1989; Carlsson et al.,
1990), thymocytes (Arrenbrecht, 1974) and
reproductive system (Lobie et al., 1990).
Binding of GH to GH-R results in receptor
dimerization (Cunningham et al., 1991; de Vos
et al., 1992). GH may act via a tyrosine kinase,
probably JAK2, associated with the GH-R
(Scanes, 1995). JAK2 might phosphorylate
other kinases such as the extracellular signalregulated kinase/mitogen-activated protein
kinase. GH is mainly degraded in the liver and
kidneys (Harvey, 1995b).
All the actions of GH are not mediated
directly via GH-R, but indirectly through the
secretion of IGF-I synthesized mainly in the
liver, but also in several other tissues (Lowe et
al., 1987). It is a single chain polypeptide
consisting of 70 aa cross-linked by three
disulfide bridges (Rinderknecht & Humbel,
1978). IGF-I promotes growth by stimulating
incorporation of sulfate by cartilage (Salmon &
Daughaday, 1957) especially during the
embryonic and postnatal growth (Baker et al.,
35
GH is essential for the normal postnatal
growth of nearly all animal species studied
(Scanes & Daughaday, 1995). The guinea pig
(Cavia porcellus) seems to be an exception, as
GH administrations do not affect its somatic
growth (Mitchell et al., 1954). GH does not
have a specific target organ but affects the
growth and development of most tissues
(Simpson et al., 1950). It stimulates the
absorption and retention of Ca and P (Carter et
al., 1999), reduces blood urea-N levels (Andres
et al., 1991) and promotes N retention (Snyder
et al., 1988; Tomas et al., 1992). Aa uptake
and muscle protein synthesis are stimulated
(Kostyo, 1968; Nutting, 1976) and efficiency of
aa utilization for protein deposition improved
(Eisemann et al., 1986; Tomas et al., 1992)
while hepatic capacity for aa catabolism
decreases (Blemings et al., 1996). These
responses lead to increased growth of muscle,
cartilage and bone (Simpson et al., 1950;
Isaksson et al., 1982; Solomon et al., 1994).
Also the mechanical strength and collagen
content of skin are improved (Jørgensen et al.,
1989) and wound healing stimulated (Steenfos
& Jansson, 1992) due to GH. The growthpromoting effect of somatotropin has been
observed in all vertebrate groups studied as
well as in Mollusca and Arthropoda, indicating
that it could have been the most ancient
function of GH having been emerged for over
500 millions of years (Myr) ago (Scanes &
Campbell, 1995b).
1993). IGF-I forms a negative feedback loop by
inhibiting GHRH synthesis and GH release as
well as by stimulating SRIF synthesis
(Berelowitz et al., 1981; Sato & Frohman,
1993). IGF-II is not as potent as IGF-I having
only a modest influence on the growth of
cartilage (Schoenle et al., 1985).
2.4.3 Metabolic effects of GH
2.4.3.1 Effects of GH on appetite
The effect of GH on appetite of animals seems
to be species-specific. GH decreases food
intake of pigs (Andres et al., 1991) and
chickens (Wang et al., 2000), whereas the
appetite of humans (Blissett et al., 2000), rats
(Azain et al., 1995) and Nile crocodiles (C.
niloticus; Kimwele et al., 1992) is stimulated
by it. The GH-induced changes in the appetite
may be mediated by hypothalamic NPY
production (Chan et al., 1996; Wang et al.,
2000).
2.4.3.2 GH and protein metabolism
In the early 1920s Evans and Long (1921,
1922a,b) suggested that the adenohypophysis
contained a certain principle which maintained
and stimulated the growth of animals. Li and
coworkers demonstrated in 1945 that this
unknown growth-promoting factor was
somatotropin. GH administrations have been
successfully utilized as a replacement therapy
for GH-deficient humans since the 1950s
(Raben, 1958). They have also been introduced
to meat-producing animals to promote their
growth rate, feed-to-gain efficiency, carcass
quality and milk production (Etherton &
Bauman, 1998). With the help of modern DNA
technologies recombinant bovine and porcine
GHs have been produced for research purposes
since the beginning of the 1980s.
2.4.3.3 GH and carbohydrate metabolism
GH regulates glucose metabolism via acute
insulin-like effects, which occur within an hour
after exposure (Goodman, 1968a; Davidson,
1987). GH treatment increases glucose uptake
(Altszuler et al., 1968; Goodman, 1968a) and
utilization (Goodman, 1968a; Honeyman &
Goodman, 1980) causing a decrease in blood
36
Davidson, 1987). FFA turnover and oxidation
increase due to somatotropin (Altszuler et al.,
1968). GH-induced stimulation of lipolysis is
indicated by elevated hormone-sensitive lipase
activities and increased glycerol and FFA
release (Dietz & Schwartz, 1991; Richelsen et
al., 1994). The lipolytic sensitivity of adipose
tissue for catecholamines is also increased by
GH (Marcus et al., 1994). Somatotropin seems
to require the presence of glucocorticoids for
the exertion of its lipolytic action (Fain et al.,
1965; Goodman, 1968b).
GH prevents the accumulation of TG to
adipose tissue by inhibiting lipoprotein lipase
activity (Richelsen et al., 1994; Ottosson et al.,
1995). Activities of lipogenic enzymes are also
suppressed by GH (Schaffer, 1985; Lee et al.,
2000).
Somatotropin
decreases
insulin
sensitivity (Bolinder et al., 1986) e.g. by
suppressing expression of insulin-responsive
glucose transporters (Zhao et al., 1996). In
addition to mammals, GH has been shown to
affect fat metabolism of e.g. birds (Hall et al.,
1987), teleost fish (Sheridan, 1986) and insects
(Bhakthan & Gilbert, 1968).
glucose concentrations (Altszuler et al., 1968).
It also promotes the conversion of glucose to
fatty acids (Goodman, 1968a), lipids and
glycogen (Honeyman & Goodman, 1980).
Antiinsulin-like effects of GH occur after an
interval of 2-4 hr (Goodman, 1968a; Davidson,
1987). GH promotes glucosuria (Cotes et al.,
1949; Altszuler et al., 1968) and increases
circulating glucose and insulin concentrations
(Altszuler et al., 1968) by decreasing glucose
uptake, utilization (Bratusch-Marrain et al.,
1982) and incorporation into fatty acids and
lipids (Goldman, 1973) as well as by elevating
hepatic gluconeogenesis and glucose release
(Altszuler et al., 1968; Knapp et al., 1992;
Bentley,
1998).
GH
also
causes
unresponsiveness of tissues to the effects of
insulin (Altszuler et al., 1968). Besides
mammals, GH has been demonstrated to
influence carbohydrate metabolism of e.g. birds
(Hall et al., 1987).
2.4.3.4 GH and lipid metabolism
It has been known for decades that GH
administrations decrease the amount of carcass
lipids in mammals (Lee & Schaffer, 1934).
Evidence has accumulated that this decrease in
fat mass results from increased lipolysis and
fatty acid oxidation (Eisemann et al., 1986;
Dietz & Schwartz, 1991; Harant et al., 1994),
decreased lipogenesis (Bornstein et al., 1983;
Rosenbaum et al., 1989; Lee et al., 2000) and
inhibited differentiation of adipocyte precursor
cells (Wabitsch et al., 1996b). GH treatment
also decreases circulating Chol, apolipoprotein
B and A1 (Blackett et al., 1982) and HDLChol concentrations as well as lipoprotein
lipase and hepatic TG lipase activities
(Asayama et al., 1984).
The influence of GH on fat mobilization can
be an acute insulin-like effect (storage of fat) or
a delayed antiinsulin-like effect (lipolysis;
2.4.4 GH in the regulation of sleep
The close association between GH release and
sleep was noticed in humans in the 1960s
(Quabbe et al., 1966). The major GH secretion
pulse occurs during slow-wave sleep (SWS) at
the beginning of the sleeping phase (Takahashi
et al., 1968). The stimulatory influence of GH
administrations on rapid eye movement sleep
(REMS) of rats was demonstrated in the 1970s
(Drucker-Colín et al., 1975). Subsequent
studies reported that also the hypothalamic
regulatory hormones affect sleep: GHRH was
found to increase the amount of non-REMS
and REMS (Obal et al., 1988), whereas SRIF
stimulated REMS (Danguir, 1986). In addition,
recent papers have demonstrated that ghrelin
37
(Barb et al., 1998), and it also increases
hypothalamic GHRH mRNA levels while
suppressing SRIF mRNA levels in rats
(Quintela et al., 1997; Carro et al., 1999;
Cocchi et al., 1999). Exogenous leptin can
reverse the fasting-induced suppression of GH
secretion (Aubert et al., 1997; Carro et al.,
1997) probably by preventing the inhibitory
action of NPY on GH release (Vuagnat et al.,
1998). GH (Florkowski et al., 1996) and SRIF
treatments (Donahoo et al., 1997), on the other
hand, reduce circulating leptin levels of
humans. Melatonin treatment inhibits GH
secretion of rat pituitary in vitro (Griffiths et
al., 1987), but increases blood GH levels of
humans (Smythe & Lazarus, 1974).
can promote SWS and decrease REMS
duration (Tolle et al., 2002; Weikel et al.,
2003).
2.4.5 Seasonality of GH secretion
Seasonal variations in GH levels of wild
mammals have been investigated only in a few
studies. Bubenik et al. (1975) have found a
gradual increase in GH levels of the whitetailed deer (Odocoileus virginianus) from Dec
to April with low levels during the rest of the
year. GH levels of the Spitzbergen reindeer
(Rangifer tarandus platyrynchus) are higher in
winter compared to summer values (Ringberg,
1979), and GH levels of the red deer (Cervus
elaphus) are low and stable between mid-May
and mid-Sep (Curlewis et al., 1992). GH
concentrations of the blue fox increase in Nov
and GH levels of the raccoon dog in Dec from
lower autumnal values (Mustonen et al., 2001).
In calves, the GH peak length is higher in Jan
than in July or Oct (Kazmer et al., 1992). In
contrast to studies conducted on mammals, GH
levels of the red grouse (Lagopus lagopus
scoticus) display peak levels in spring and early
summer and the nadir during the autumn and
winter (Harvey et al., 1982).
2.4.7 Evolution of GH
GH has evolved from a common ancestral
molecule with PRL, placental lactogen and
somatolactin (Bentley, 1998). Genes encoding
these hormones exhibit a common pattern of
five exons and four introns. It is possible that
GH and PRL derive from a single ancestral
proto-GH-PRL, which has undergone a gene
duplication (Scanes & Campbell, 1995b). This
duplication might have occurred prior to
separation of Protostomata (e.g. Annelida,
Arthropoda, Mollusca) and Deuterostomata
(e.g. Chordata, Echinodermata) in the
Precambrian era over 590 Myr ago.
GH has been isolated from representative
species from elasmobranchs to mammals.
Furthermore, a GH-like peptide has been
located in distinct pituitary cells of Agnatha
(Wright, 1984). Particular aa of GH have been
preserved during evolution, as thirty-five of
them are invariant among vertebrates (Bentley,
1998). Nonprimate mammals share 135 aa
positions, but the rate of evolutionary change
has been more rapid in primates. Cattle share a
67 % sequence homology with humans,
2.4.6 Interactions of GH with leptin and
melatonin
As both obesity and undernutrition are
associated with abnormal GH (de Rosa et al.,
1983; Veldhuis et al., 1995) and leptin
concentrations (Maffei et
al., 1995;
Kolaczynski et al., 1996), numerous studies
have focused on the possible GH-leptin
interactions. Spontaneous GH secretion of rats
has been shown to be inhibited by
administration of leptin antiserum (Carro et al.,
1997). Leptin supply stimulates GH secretion
of rats (Tannenbaum et al., 1998) and pigs
38
whereas the per cents for the domestic fowl and
the bullfrog are 59 and 46. Also Teleostei,
Holostei, Chondrostei and Elasmobranchii have
relatively conserved GH peptides (34-54 %
homologous to humans). A GH-like peptide
has also been found in insects (Swinnen et al.,
1990; Vanden Broeck et al., 1990) and
molluscs (Toullec et al., 1992), suggesting that
the proto-GH-PRL has existed before the
divergence between arthropods/insects and
vertebrates in the Precambrian era (Scanes &
Campbell, 1995b).
GHRH
has
been
identified
from
chondrostean fish to mammals (Bentley, 1998).
The homology between the human and fish
GHRH sequences is about 40 %. SRIF-I
peptide has been detected from mammals to
cyclostomes (King & Millar, 1979). It is also
present in molluscs (Grimm-Jørgensen, 1983),
insects (Romeuf & Rémy, 1984), flowering
plants (LeRoith et al., 1985b) and in unicellular
protozoans (Berelowitz et al., 1982) and
prokaryotes (LeRoith et al., 1985a). The
structure of SRIF-14 has been conserved
extremely well: the molecules of Amphibia and
Teleostei differ by only two aa and those of
Chondrostei, Holocephali and Cyclostomata by
one residue from the mammalian SRIF
(Bentley, 1998).
2.5
Fig. 1. The raccoon dog Nyctereutes procyonoides
(Gray, 1834).
muzzle, a moderately long body and short legs
and tail (Siivonen, 1972). Its back is grayish
brown, whereas the belly is yellowish and the
chest and legs are dark brown or black. Dark
coloration of cheeks circles the eyes creating a
mask. The dental formula is 2 (I 3/3, C 1/1, Pm
4/4, M 2/3) = 42 (Heptner et al., 1974).
The genus Nyctereutes of late Pliocene
epoch is known through N. donnezani (Fig. 2),
which was about one fourth bigger than the
modern raccoon dog (Nowak, 1993). During
Villafranchian there were several species of the
genus Nyctereutes extending from Europe to
China. The great raccoon dog N.
megamastoides lived in southern Europe
between late Pliocene and early Pleistocene. It
was of a large body size and fed on fruits, green
plant parts and meat. N. sinensis, discovered in
China, Mongolia, Transbaikalia and Poland,
lived between Villafranchian and midPleistocene. In China its body size gradually
reduced and later Pleistocene Chinese forms
carried through a complete transition to the
modern species (Kurtén, 1968).
N. procyonoides originates from eastern
Asia: Middle-Amur, Ussuria, Manchuria,
China, Korea and Japan (Siivonen, 1972). It
can be divided into three subspecies: the
The experimental species of the thesis
2.5.1 The raccoon dog (I-III)
2.5.1.1 Origin of the raccoon dog
The raccoon dog (Nyctereutes procyonoides
Gray, 1834) is a middle-sized (4-10 kg)
carnivore of the family Canidae (Fig. 1).
“Nyctereutes” refers to the nocturnal life-style
of the species and “procyonoides” to its similar
habitus with the raccoon. The raccoon dog has
thick fur, a small and short head, a pointed
39
estimated size of the Finnish raccoon dog
population in the mid-1990s was about 60-70
000 individuals in early spring and 165-190 000
individuals in autumn (Valste, 2001). The
density is highest in southern Finland and the
northern distribution limit of the species lies
between 65 °N and the Arctic circle (Kauhala,
1992). Summer is too short for the cubs to
grow and store enough adipose tissue for the
winter in Lapland preventing the species from
colonizing the northernmost areas of Finland.
Finnish raccoon dog farming began in 197172 with animals originating from the wild
population (Mäkelä, 1973). At present, Finland
is the leading country in raccoon dog farming
with an annual production of about 80 000
skins (Lindh, 2001).
Fig. 2. A fossilized skull of Nyctereutes donnezani
Depéret photographed at the National Museum of
Natural Sciences in Madrid, Spain.
southern N. p. procyonoides and the northern
N. p. ussuriensis in the continental Asia and N.
p. viverrinus in Japan (Heptner et al., 1974).
However, depending on interpretation the
number of subspecies can be higher. The
Japanese raccoon dog called tanuki was
isolated from the raccoon dogs of the EastAsian mainland by the Sea of Japan 12 000
years ago (Kauhala, 2000). Most of the
chromosomes of the tanuki (2n=38) are
metacentric, whereas the Finnish raccoon dog
(2n=54) has mainly acrocentric chromosomes
(Mäkinen et al., 1986).
Between 1927-57, raccoon dogs of the
subspecies N. p. ussuriensis were introduced as
a fur-bearing game animal to the European part
of the former Soviet Union (Siivonen, 1972).
Due to its great colonizing ability, the raccoon
dog successfully expanded its geographical
distribution to Sweden, Lithuania, Poland,
Germany, the Netherlands, Czech Republic,
Austria, Romania and Bulgaria (Viro, 1984).
The first raccoon dogs were noted in Finland in
the 1930s (Kauhala, 1992). They had probably
dispersed from the Leningrad and Novgorod
regions. The southern and central parts of
Finland were colonized in a couple of decades.
Nowadays the raccoon dog has a vacant
ecological niche in Finnish nature, over 1000
km north from its original habitats. The
2.5.1.2 Ecology of the raccoon dog
The habitat of the raccoon dog is usually moist
deciduous or mixed forest with abundant
undergrowth (Nowak, 1993). It also inhabits
coniferous forest, marshy and moist meadows
with bushes as well as lake- and riversides. The
maximum home range of adults is 9.5 km2 and
its core area 3.4 km2 (Kauhala, 1992). Home
ranges of a paired male and female raccoon dog
overlap almost totally, whereas the core areas
of adjacent adult individuals or pairs do not
usually overlap in the cub-rearing season.
The sex ratio of the raccoon dog population
is close to 1:1, possibly due to its monogamous
breeding system. The maximum life span is 7-8
years but the life expectancy at birth only 0.7
years, the generation time being 2.1 years. The
annual mortality rate of the population is 81 %,
being 54 % for the adults and 88 % for the
juveniles.
The raccoon dog is an opportunistic
omnivore being independent of any specific
food items. Food composition and diet diversity
are affected by the availability of different food
40
autumn. Winter underfur hairs start to develop
in mid-Aug and the moult is completed by the
end of Nov. The dense winter pelage is
maintained until the spring moult.
Despite of its thick and warm coat, the
raccoon dog is rather poorly adapted to the
cold Finnish climate (Korhonen & Harri, 1989).
The lower critical temperature of the species is
relatively high, about +11 °C in winter and +15
°C in summer (Korhonen & Harri, 1984).
Below these temperatures, raccoon dogs have
to increase energy metabolism to keep their Tb
constant. The resting MR of the farmed
raccoon dog is 11-12 ml of oxygen * kg-0.75
min-1 (Korhonen, 1987).
Heat loss is greatest from the chest, head,
abdomen and feet (Korhonen & Harri, 1986).
Raccoon dog cubs are capable of BAT
nonshivering thermogenesis, but adults remain
homeothermic
by
muscular
shivering,
locomotion and sunbasking (Korhonen &
Harri, 1989). In contrast to the Finnish raccoon
dog, the Japanese tanuki is adapted to live in a
temperate marine climate. For this reason, it
has a thin fur coat of lower insulative value and
smaller seasonal changes of its body energy
stores (Korhonen et al., 1991).
items fluctuating between seasons and years.
The relatively weak canines and carnassials and
the long intestine are associated with the ability
to use a versatile diet (Heptner et al., 1974).
Plant material (berries, cereals, vegetables and
fruits), small mammals (voles Microtus spp.,
Clethrionomys spp., shrews Sorex spp.),
carcasses, waste and birds (passerines,
Passeriformes) occur in the diet throughout the
year (Kauhala, 1992). Insects (wasps,
Vespidae) and large beetles (Carabidae,
Scarabaeidae) are frequently encountered in the
summer and autumn diets. Frogs (B. bufo,
Rana spp.) and common lizards (Lacerta
vivipara) are captured in late spring and early
summer and fish are used in early spring and
autumn. In addition, hares (Lepus spp.),
gallinaceous birds (pheasant Phasianus
colchicus, black grouse Tetrao tetrix, hazel hen
Bonasa bonasia), waterfowl (mallard Anas
platyrhynchos, teal A. crecca, great merganser
Mergus merganser) and eggs can occur in the
diet.
Natural enemies of the raccoon dog are few.
In addition to wild dogs, potential predators are
the gray wolf (C. lupus), the lynx (L. lynx), the
red fox (V. vulpes) and large birds of prey (B.
bubo, Haliaeetus albicilla, Aquila chrysaetos,
Accipiter gentilis; Heptner et al., 1974; Viro,
1984). Moreover, large numbers of raccoon
dogs are killed each year by traffic and hunters
(Kauhala, 1997).
2.5.1.3.2 Seasonal BM cycle
Profound seasonal fattening is a natural
characteristic of the life history of the raccoon
dog (Korhonen, 1987). BMs of wild and
farmed raccoon dogs fluctuate 30-40 %
throughout the year, being highest in Oct-Dec
and lowest in spring and summer (Korhonen,
1987; Kauhala, 1992). The seasonal variations
in BM are mainly due to changes in sc fat
stores, which are the main energy source and
provide thermal insulation during the coldest
part of the winter (Korhonen, 1987, 1988).
Wild raccoon dogs prepare themselves for the
winter rest by autumnal hyperphagia, during
2.5.1.3 Seasonal physiology of the raccoon
dog
2.5.1.3.1 Thermoregulatory adaptations
The raccoon dog has one complete annual
pelage change (Xiao, 1995). Moulting of the
underfur is characterized by a heavy loss of old
winter hairs in spring. The new guard hairs
develop in April-May and mature in late
41
state can be induced to the animals by
withdrawing the food and providing them with
nestboxes (Asikainen et al., 2002). The rate of
weight loss is about 1.5 kg month-1 during a
total fast. A slight but significant decrease in
the rectal Tb (0.5-1.5 °C) has been measured
during the fasting-induced winter sleep. A total
fast of two months is without any detrimental
effects on the health of the animals. Neither is
the reproductive success of the raccoon dog
impaired by a wintertime fast.
which they consume mainly berries (Kauhala,
1997). The weight gain and fat storing of
farmed raccoon dogs are the most pronounced
in late autumn (Korhonen, 1987). During the
coldest part of the winter farmed raccoon dogs
decrease their food intake and locomotor
activity (Korhonen, 1987, 1988). Wintertime
hypophagia leads to weight loss that is also
observed in wild raccoon dogs during their
seasonal rest (Kauhala, 1992). After arousal
from the winter sleep, the raccoon dog starts to
restore its depleted fat stores.
2.5.1.3.4 Reproductive processes
2.5.1.3.3 Winter sleep
The extremely high reproductive capacity of
the wild raccoon dog is due to its monogamous
breeding system, omnivory, winter sleep and
high mortality rate (Kauhala, 1992).
Reproductive success of the species is mainly
affected by climate, weather and food
availability. A proportion of 78 % of the wild
female raccoon dogs reproduces annually, and
the reproductive value is highest among twoyear old females. Mean productivity (mean
birth litter size * proportion of reproducing
females) for the raccoon dog is 6.9.
Wild raccoon dogs mate in Finland between
Feb and April with the peak in March. In farm
conditions the mating season is in Feb-March
(Valtonen et al., 1977). The mean duration of
pro-estrus of the females is about 8 days. The
estrus lasts for 4 days and the gestation 59-64
days. The female gives birth in April-May.
The litter size of the raccoon dog varies
geographically and annually (Nowak, 1993). In
Finnish fur farms, the mean litter size per mated
female was 6.07 cubs in 2002 (Finnish Fur
Breeders’ Association, pers. commun., 2003).
The average litter size at birth is about 8.8 cubs
in the wild population and it correlates
positively with the body adiposity of adult
females in March (Kauhala, 1992). In young
Wild raccoon dogs spend the coldest part of
the winter in a superficial winter sleep in
northern and eastern Europe, Poland and Far
East, i.e. areas of cold and harsh winters
(Nowak, 1993). The seasonal rest makes the
raccoon dog independent of food during the
most unproductive time of the year. Finnish
raccoon dogs winter in a burrow or a den for
up to 4-5 months from Nov to March-April
(Siivonen, 1972). The metabolism of raccoon
dogs can decrease by 25 % during the winter
sleep (Heptner et al., 1974).
Compared to e.g. bears (Ursus spp.), the
facultative winter sleep of the raccoon dog is
shallow with periodic arousal and foraging
during the warmer periods (Siivonen, 1972).
The raccoon dogs are, however, poor hunters
and cannot find much to eat in mid-winter. For
this reason they usually lose their fat stores by
wandering
around
(Kauhala,
1992).
Furthermore, the ability of the raccoon dog to
move on snow is limited due to the small
footpads, which are also sensitive to frostbites
(Korhonen & Harri, 1989).
The raccoon dogs do not usually exhibit
winter sleep in fur farms probably due to the
lack of nests (Korhonen, 1987). In
experimental conditions a winter sleep-like
42
2.5.1.3.5.2 Weight-regulatory hormones
females the body fat reserves in late autumn
affect the age at first reproduction.
The male raccoon dog helps to rear the
offspring and stays at the den with the cubs,
while the female searches for food due to her
high energy requirements. The juveniles reach
sexual maturity at the age of ten months during
the next winter (Kauhala, 1997).
The plasma leptin levels of the raccoon dog
remain low from July to Sep and increase in
Oct (Nieminen et al., 2001a). The
concentrations decrease rapidly in early Nov,
although the BMs of the animals remain
constant. Thereafter the leptin levels stay low
and stable until Dec. A 3-wk fast does not
affect the leptin levels and neither is there any
correlation between the leptin concentrations
and BMIs. These findings suggest that the
circulating leptin concentrations of raccoon
dogs are not solely determined by the amount
of fat in their bodies.
The circulating GH concentrations of the
raccoon dog remain low and stable between
July and Nov (Mustonen et al., 2001). There
are no marked differences in the GH levels
between
adults
and
juveniles.
The
concentrations increase significantly in early
Dec, but a three-wk fast does not affect them.
The plasma triiodothyronine (T3) and T4
concentrations of the raccoon dog are higher in
the summer than during the winter (Korhonen,
1987; Nieminen et al., 2001a). A three-wk fast
does not affect the T4 levels, which, however,
increase after re-feeding (Nieminen et al.,
2001a). The T3 and T4 levels are elevated in
overfed animals (Korhonen, 1987).
2.5.1.3.5 Seasonal endocrinology
2.5.1.3.5.1 Sex steroids
The testicular width and score count of
spermatogenesis of male raccoon dogs increase
in early Nov (Xiao, 1996). Testosterone
secretion starts to increase in Jan-Feb and
peaks during the mating season. After the
mating period the testes regress and their
testosterone production returns to low values.
The testes remain quiescent until recrudescence
in late autumn.
The 17β-estradiol concentrations of the
females reach the highest levels during proestrus and at the beginning of estrus (Valtonen
et al., 1978). The levels decrease postcoitally
and remain low during the early gestation.
Between days 13 and 26 of gestation, they
increase slightly and decrease thereafter
towards parturition.
The progesterone concentrations of the
females are low during pro-estrus. During
estrus, they increase rapidly reaching the
maximum values during the first half of
gestation. From the middle of gestation, the
concentrations fall to reach very low
postpartum levels. The ovaries remain
quiescent outside the reproductive season
(Asikainen et al., 2003).
2.5.1.3.5.3 Melatonin
The urinary melatonin secretion of male
raccoon dogs increases during the autumn with
a peak in early Nov followed by a rapid
decrease in Dec (Xiao, 1996). The rise from
July to Aug and the stable concentrations until
Oct are associated with the growth of winter
fur. The further increase in Nov, on the other
hand, may be connected to the increased
testicular activity.
43
only on their anterior surface. The diploid
number of chromosomes is 42 (Becker, 1978).
The brown rat originates from the temperate
regions of Asia, specifically the area between
the Caspian sea and Tobolsk, possibly
extending as far east as Lake Baykal (Lindsey,
1979). Brown rats spread via an unknown
route from Asia to Europe during the Middle
Ages (Becker, 1978). The first reports about
their dispersal to Denmark, England, Germany
and France derive from the 18th century. Rats
have inhabited the present European region
also during the Plio- and Pleistocene epochs, as
indicated by fossil records (Sharp & La Regina,
1998).
With the help of man-made vehicles, the
brown rat rapidly expanded its geographical
distribution to Palearctic, Nearctic, Oriental,
Ethiopian, Neotropical and Australian regions
as well as to oceanic islands (Becker, 1978). To
North America it arrived around 1775. Even
the northernmost latitudes of Europe have been
occupied by the species. On the contrary, only
moist areas are inhabited in the Mediterranean
region and the distribution in the tropics and
subtropics is restricted to seaports and
metropolises. The brown rat arrived to Finland
during the late 1700s and early 1800s
(Myllymäki, 1972).
Continuous-release melatonin implants
introduced into the interscapular sc fat elevate
serum and urine melatonin concentrations of
raccoon dogs for several months. Increased
circulating melatonin concentrations induce
physiological changes that are normally
connected to SD. The melatonin treatment
introduced in July suppresses PRL secretion of
the raccoon dogs. It also advances testicular
recrudescence, growth of winter underfur and
maturation of both the underfur and guard
hairs. In addition, melatonin decreases BM of
the raccoon dogs at the end of the year. It
seems likely that the increasing endogenous
melatonin secretion after the summer solstice
triggers the autumn moult of the species, as
previously suggested for the mink (Valtonen et
al., 1992, 1995). Melatonin treatment in March
slows down testicular regression and inhibits
PRL secretion (Xiao, 1996). It also inhibits the
initiation of the growth of guard hairs and
stimulates the growth of underfur.
2.5.2 The laboratory rat (IV-V)
2.5.2.1 Origin of the brown rat
The laboratory rat bred for experimental
purposes derives from the domesticated brown
rat (Rattus norvegicus, Berkenhout 1769,
Muridae, Rodentia; Gill et al., 1989).
According to Robinson (1979), brown rats can
be divided into five different subspecies: R. n.
norvegicus, R. n. caraco, R. n. praestans, R. n.
primarius and R. n. socer. The snout of the
brown rat is blunt and ears are small and round
(Becker, 1978). The color of the fur varies
from reddish and grayish brown to black on the
back, whereas the belly is white or gray. The
brown rat has five digits, but the pollex is
rudimentary (Vaughan et al., 2000). Its dental
formula is 2 (I 1/1, C 0/0, Pm 0/0, M 3/3) = 16.
The incisors are ever-growing having enamel
2.5.2.2 Ecology and reproductive physiology
of the brown rat
The enormous dispersal success of the brown
rat is based on its efficient reproduction,
neophobia, omnivory, aggressiveness and on
the flexibility of its behaviour (Myllymäki,
1972). In the wild, the brown rat inhabits dense
vegetation near waterways (Becker, 1978).
However, the biotype of the species is primarily
commensal, as the brown rat has colonized
man-made habitats of almost every kind. It is
an omnivore feeding on cereals, fresh plant
44
three wk (Myllymäki, 1972). In captivity, only
a small number of females reproduce at the age
of 3-4 months, but the majority is pregnant by
the age of seven months (Becker, 1978). The
mean number of pups is 4-8. Until the
menopause at the age of 15-18 months, a
normal female has delivered 6-8 litters with a
total of 41 puppies. Also in natural conditions,
the puberty is reached at the earliest at the age
of 3-4 months.
parts, fruits, vegetables, meat and fish. In
addition, rats are able to kill chickens, young
rabbits and birds as well as small rodents,
mollusks and frogs. The species is considered
to be the worst mammalian pest of all times. It
causes damage by spreading infectious diseases
such as bubonic plague, typhus, trichina,
cholera and jaundice and by destroying crops,
foodstuffs and buildings (Myllymäki, 1972).
The brown rat is active year around. It is a
crepuscular species with activity peaks shortly
after the sunset and just before the sunrise,
whereas the animal rests during midnight and
photophase (Becker, 1978). The brown rat
moves mainly on the ground but it is also a
good jumper, swimmer and climber (Hirsimäki,
1996; Myllymäki, 1997). The radius of its home
range seldom exceeds 50 m (Myllymäki, 1972).
Rats live in a family community, the ranking
order of which is re-newed by fighting (Becker,
1978). The territory belonging to one male and
his harem lies in the vicinity of the nest, and the
male defends this area against other males. An
increase in population density makes the
identification of individuals more difficult,
leading to weakening of the social hierarchy.
In captivity, the maximum life span of the
brown rat seldom exceeds three years.
Mortality increases after one year, and 80 % of
males and 77 % of females reach the age of 20
months. In the wild, the annual mortality rate is
90-95 %. Besides humans, also cats, dogs, least
weasels (M. nivalis), ermines, European
polecats (M. putorius) and some owl species
(B. bubo, Strix aluco, Tyto alba) are important
enemies for the brown rat.
The brown rat is an r-selected species and it
can reproduce year around. The largest amount
of pregnant females is encountered in spring
and autumn and the lowest number in summer
and winter. These seasonal changes are absent
in the constant conditions of the sewer system.
The gestation lasts about 24 days and lactation
2.5.2.3 The laboratory rat
Albino mutants of the brown rat with red eyes
and pink ears, legs and tail (Fig. 3), which
appeared in the wild brown rat population,
were captured and tamed (Lindsey, 1979). In
the 1800s rats were commonly used in ratbaiting, in which the time a trained terrier
needed for killing 100-200 rats in a fighting pit
was recorded. The domestication of the species
probably derived as a by-product of this sport,
as white rats were removed from rat pounds for
shows and breeding. These show rats
eventually became the first mammalian species
domesticated for scientific purposes.
The first scientific work using albino rats as
experimental animals investigated the effects of
adrenalectomy in the middle of the 19th century.
The first breeding experiments with different
color variants were performed at the end of the
Fig. 3. The laboratory rat
(Berkenhout, 1769).
45
Rattus norvegicus
sexual maturity earlier and has a better
fecundity than its wild counterpart.
The laboratory rat reaches sexual maturity at
the age of 50 ± 10 days. It is polyestrous and
its estrus cycle lasts 4-5 days consisting of proestrus (12 hr), estrus (12 hr), metestrus (21 hr)
and diestrus (57 hr). The length of the gestation
is 21-23 days, and the number of pups 3-18.
The menopause begins at the age of 450 days,
and the average life span is 1000 days for a
female and 1300 days for a male (Hirsimäki,
1996).
1800s, and albino rats have been used in
neuroanatomical research since the 1890s. The
Wistar Institute in Philadelphia, USA created
the foundation for the standardization of the
laboratory rat in scientific research.
Nowadays the laboratory rat is the most
commonly used species with the laboratory
mouse, especially in genetics, immunogenetics,
neurosciences, pharmacology, physiology,
toxicology, transplantation, and in behavioural,
cancer and cardiovascular research (Gill et al.,
1989; Hirsimäki, 1996). The laboratory rat is
easy to handle and care and small and
inexpensive to maintain. It is popular also due
to its high fecundity and short life span and
generation time. A large body of literature
exists about the anatomy, endocrinology,
histology, metabolism, nutrition and physiology
of the laboratory rat (Robinson, 1979), and
several hundreds of well-defined strains of rats
have been developed for experimental purposes
(Hirsimäki, 1996). The laboratory rat is one of
the most important animal species in reference
to its contribution to the advancement of
science (Lindsey, 1979).
The length of the laboratory rat from the
nose to the tip of the tail is 40-46 cm and the
maximum weight about 400 g for a female and
800 g for a male (Hirsimäki, 1996). The animal
is most active during the night and early
morning (Sharp & La Regina, 1998) and shows
maximum energy intake during scotophase
(Rietveld et al., 1980). Compared to wild
brown rats, the laboratory rat has 8.3 % less
brain substance as a result of domestication
(Kruska, 1975a). The reduction is especially
pronounced in the corpus striatum, neocortex,
forebrain, cerebellum and limbic structures,
whereas the olfactory regions have degenerated
less (Kruska, 1975a,b). The laboratory rat has
also smaller adrenals and preputial glands than
wild rats (Sharp & La Regina, 1998).
Moreover, it is a nonseasonal breeder reaching
2.5.2.4 Endocrinology of the laboratory rat
2.5.2.4.1 Melatonin
The laboratory rat is considered to be a
marginally photoperiodic rodent, as exposure
to melatonin or different photoperiods induces
only small effects on its reproduction (Turek et
al., 1976; Wallen & Turek, 1981). There are,
however, data demonstrating that the
laboratory rat has remains of a photoperiodic
machinery left, and in some experimental
conditions its physiology responds to
daylength. The melatonin secretion rhythm of
rats follows the length of the night in
experimental conditions as well as in the natural
photoperiod (Illnerová & Vaněček, 1980), and
their blood melatonin concentrations reduce
with aging (Pang et al., 1984).
LL abolishes the diurnal melatonin secretion
rhythm of rats, and it can regress ovaries
(Reiter & Klein, 1971) and inhibit ovulation in
female rats (Campbell & Schwartz, 1980).
Sperm production of males, on the other hand,
can be increased by LL exposure during
neonatal testis development (Rocha et al.,
1999). Exogenous melatonin delays sexual
maturation (Lang et al., 1983) and decreases
the weight of the anterior pituitary, testes and
accessory sex organs of male rats (Debeljuk,
46
of rats correlate positively with their body
fatness being elevated in obesity (Maffei et al.,
1995). Their leptin levels also increase with
aging (Iossa et al., 1999). Blood leptin
concentrations and WAT leptin mRNA levels
of rats are suppressed by fasting and they
recover to normal levels by re-feeding
(MacDougald et al., 1995; Hardie et al., 1996).
Moreover, leptin treatment inhibits ovarian
steroidogenesis (Barkan et al., 1999), testicular
testosterone secretion (Tena-Sempere et al.,
1999) and ovulation (Duggal et al., 2000) of
rats. On the contrary, pituitary LH and FSH
secretion are stimulated by leptin (Yu et al.,
1997). Rat leptin concentrations increase due
to Px but decrease with exogenous melatonin
(Rasmussen et al., 1999; Canpolat et al.,
2001).
Rat ghrelin is identical to human peptide
except for two aa (Kojima et al., 1999).
Ghrelin increases GH release of rats in vivo as
well as in vitro. Also their insulin secretion is
stimulated by ghrelin (Lee et al., 2002).
Moreover, a ghrelin-induced suppression of LH
release has been documented in rats (Furuta et
al., 2001). Exogenous ghrelin administration
increases their food intake, BM gain and fat
accumulation (Tschöp et al., 2000), and ghrelin
antagonizes leptin action in the rat
hypothalamus and vice versa (Nakazato et al.,
2001).
The GH molecule of the rat consists of 190
aa and differs by 34 % from the human GH
(Seeburg et al., 1977). In male rats
somatotropin is secreted in pulses at 3-4 hr
intervals with almost undetectable levels
between bursts, whereas the secretion pattern
of females is more continuous with a lower
amplitude (Jansson et al., 1985). GH stimulates
growth, BM gain, food intake, feed conversion
efficiency, carcass protein content and protein
accretion, but decreases carcass fat content and
rate of lipid accretion in rats (Azain et al.,
1969; Gilad et al., 1998). Melatonin also
suppresses their spermatogenesis (Mandal et
al., 1990), steroidogenesis (Valenti et al.,
1995) and blood testosterone, LH and FSH
levels (Lang et al., 1983). In female rats,
melatonin stimulates progesterone and estrogen
secretion of granulosa cells (Fiske et al., 1984).
Incidence of estrus and ovarian weight decrease
(Wurtman et al., 1963a) and ovulation (Reiter
& Sorrentino, 1971) and pituitary LH release
(Symons et al., 1985) are inhibited by
exogenous melatonin. Also the sensitivity of the
rat pituitary to GnRH is reduced by melatonin
treatment (Martin & Klein, 1976; Martin et al.,
1982).
The circadian activity rhythms of rats are
abolished by LL, whereas daily melatonin
injections can re-synchronize them (Chesworth
et al., 1987). Melatonin, LL or DD do not
influence BMs or food intake of rats (Dark et
al., 1980). However, melatonin treatment has
particular effects on their intermediary
metabolism. Visceral fat mass as well as blood
insulin and free Chol levels decrease with
exogenous melatonin, whereas muscle and liver
glycogen contents and blood PL concentrations
increase (Esquifino et al., 1997; Rasmussen et
al., 1999; Mazepa et al., 2000). Rat thyroid
activity is inhibited by melatonin administration
(Vriend, 1983). Furthermore, melatonin has
induced a suppression in blood total and LDLChol levels and an elevation in HDL-Chol
concentrations in hypercholesterolemic rats
(Hoyos et al., 2000).
2.5.2.4.2 Leptin, ghrelin and GH
As the rat has been a popular experimental
animal in endocrinological research, there is a
large body of literature concerning rat leptin,
ghrelin and GH. The rat leptin aa sequence is
83 % homologous to human leptin (Ogawa et
al., 1995), and circulating leptin concentrations
47
1995). The increase in food intake may be
caused by stimulation of the hypothalamic NPY
expression by GH (Chan et al., 1996). Rat
testosterone and estradiol secretion can be
stimulated by GH treatment (Maran et al.,
2000). Melatonin, on the other hand, inhibits
GH synthesis and secretion of rat pituitary in
vitro (Griffiths et al., 1987), but leptin
stimulates GH secretion in fasted rats (Carro et
al., 1997) by preventing the inhibitory action of
NPY on GH release (Vuagnat et al., 1998).
Conversely, GH inhibits leptin synthesis in rat
adipose tissue (Isozaki et al., 1999).
Fig. 4. The burbot Lota lota (L. 1758).
divided into three subspecies: L. l. lota in
Europe and western Siberia, L. l. leptura in
Alaska and north-eastern Siberia and L. l.
maculosa (lacustris) in south-eastern North
America (Hubbs & Schultz, 1941). The
enormous fecundity and profundal life-style of
the burbot are considered to be the major
reasons for its wide geographical distribution
(Pääkkönen, 2000). Its present distribution
probably results from dispersal through sea
(Lehtonen, 1998). The burbot still has got
several characteristics typical to marine species:
i.e. spawning season in winter, a high number
of eggs with a large oil globule and pelagic
larvae. It also shares parasites with marine fish
species (Sterud, 1998).
2.5.3 The burbot (VI-VII)
2.5.3.1 Origin of the burbot
The burbot (Lota lota L. 1758, Gadidae,
Gadiformes, Fig. 4) is the only freshwater
species belonging to the codfish family. It has
an elongated and cylindrical body shape, the
length of which is 30-70 cm (Koli, 1998). The
head is flattened and has a pointed snout, a chin
barbel and tubular extensions at nostrils. The
burbot has two dorsal fins, the second of which
is long, and a single anal fin. Both jaws have
numerous small teeth in bands. The scales are
small, soft and cycloid. The dorsal color of
adults varies from yellow and green to light
brown and black, whereas the belly is light. The
diploid chromosome number is 48 (Rab, 1986).
Cumbaa et al. (1981) have discovered late
Pleistocene fossils of L. lota of a large body
size in North America. The taxon may have
existed prior to and survived the Wisconsinan
glaciation in the Beringian refugium. The
burbot probably migrated to Finland from the
Baltic Sea over 10 000 years ago (Raitaniemi,
1998). The species has a holarctic distribution
from Europe to Asia and North America (Koli,
1998). It mainly inhabits lakes, brackish waters
and rivers (Lehtonen, 1998). Burbots can be
2.5.3.2 Ecology of the burbot
The burbot exhibits a benthic life-style, and it
has been videotaped to create biogenic shelter
structures in bottom conditions (Boyer et al.,
1989). Its activity periods are longest (10-11
hr) in Sep-Oct and in Feb (Müller, 1973). The
burbot becomes nocturnal after spawning, and
in autumn it switches back to diurnal activity
(Wikgren, 1955; Müller, 1973). The activity
rhythm is desynchronized in midsummer, as the
activity time shifts from midnight to afternoon
or over the full 24 hr (Müller, 1973).
The burbot has a well-developed sense of
smell, and for this reason it is able to catch prey
successfully in lake-bottom conditions with
zero illumination (Koli, 1998; Lehtonen, 1998).
Its predation is especially efficient in winter,
48
2.5.3.3 Physiological
burbot
when several prey species are inactive and easy
to catch by this slow swimmer (Lehtonen,
1998). The diet of the burbot depends on the
season and the stage of its development. Young
burbots feed on invertebrates: rotifers
(Rotifera), copepods (Cyclopoida, Calanoida),
water fleas (Cladocera), stoneflies (Plecoptera),
dayflies (Ephemeroptera) and dipterous insects
(Diptera; Chen, 1969; Ghan & Sprules, 1993).
Adult burbots are ambush predators feeding
mainly on fish and crayfish (Hewson, 1955;
Chen, 1969; Pulliainen & Korhonen, 1990).
The most important prey species in Finnish
lakes are the vendace (Coregonus albula), the
ruffe (Gymnocephalus cernuus), the perch
(Perca fluviatilis), the roach (R. rutilus), the
smelt (Osmerus eperlanus) and the bullhead
(Cottus gobio; Pulliainen & Korhonen, 1990;
Koli, 1998; Lehtonen, 1998). In the Baltic sea,
the diet includes e.g. the Baltic herring (Clupea
harengus membras), the eelpout (Zoarces
viviparus) and gobids (Pomatoschistus spp.).
Fish are mainly consumed during the winter and
early summer, whereas invertebrates (Mysis
relicta, Pallasea quadrispinosa, Chironomidae) are common in the summer and autumn
diets (Pääkkönen, 2000).
The stomachs of the burbots contain
numerous different food items before
reproduction (McCrimmon & Devitt, 1954).
Food intake is suppressed during spawning,
after which the fish consume large quantities of
any food items available. The weight of the full
stomach and intestine of the burbot can be 25
% of its BM, whereas the empty
gastrointestinal tract accounts for only 4 % of
BM (Chen, 1969). Pääkkönen (2000) has
recorded that the stomach content can weigh as
much as 37 % of the burbot´s own mass.
Gastric evacuation rate of the species is low,
indicating that the stomach can function as a
food reserve during periods of irregular
feeding.
adaptations
2.5.3.3.1 Thermoregulation
metabolism
and
of
the
energy
The burbot is a stenothermic fish species and it
prefers cold waters (Koli, 1998). For this
reason, adult burbots are rarely encountered at
water temperatures (Tw) over +12 °C
(Lehtonen, 1973). The MR of the species is
relatively low, and the Q10 value is 2.97,
indicating that the MR is tripled for a 10 °C
increase in Tw (Pääkkönen, 2000). The
optimum Tw for food intake has been +13.6 °C
in experimental conditions.
The liver is the main fat storing organ of the
burbot and its weight undergoes seasonal
variation (Chen, 1969; Lehtonen, 1973;
Karhapää, 1978). The liver weight has been
measured to be highest in Aug (7.6 % BM,
Chen, 1969) or in Nov (5.6 % of BM,
Karhapää, 1978). Its lipid content has been
greatest in May (32 % of liver fresh weight,
Karhapää, 1978). Fat is transported from the
liver to the gonads during the autumn and
winter leading to a reduction of liver weight,
while the opposite occurs in the gonads (Chen,
1969; Karhapää, 1978). The liver lipase
esterase activity, indicating mobilization of
hepatic fat, is highest before spawning, when
the hepatic fat content is lowest (17 % of liver
fresh weight, Karhapää, 1978). The liver
weight is at its lowest (3 % BM) after
spawning, when the burbot starts to restore its
hepatic fat stores (Chen, 1969; Karhapää,
1978). TG are the most abundant lipid class in
the livers (Karhapää, 1978).
49
per m2 (Sorokin, 1971). The adults do not
provide their offspring with any parental care.
The fry hatch after 70 days of incubation
simultaneously with the breaking up of the ice
(Koli, 1998). The length of the newly hatched
fry is 3-5 mm. The larvae live pelagically until
they reach the length of 15-20 mm and begin a
benthic life. After one year, the body length of
the burbot attains about 10-11 cm (Chen, 1969;
Koli, 1998), and it increases rapidly during the
first 4-6 years of life (McCrimmon & Devitt,
1954; Chen, 1969). Sexual maturity is reached
at the age of 3-7 years (Chen, 1969; Koli,
1998), and it is associated more with the size
than with the age of the individual (Hewson,
1955). Female burbots grow faster and live
longer than the males (Chen, 1969). Most
burbots, however, die before the age of 15
years. Adult burbots do not spawn each year,
which is independent of their nutritional status
(Pulliainen & Korhonen, 1990). Infertility has
been observed among burbots inhabiting waters
contaminated with pulp mill effluents
(Korhonen, 2000).
2.5.3.3.2 Seasonal reproduction
Spawning of the burbot occurs in mid-winter
(Jan-March), later in the north than in the south
(Koli, 1998; Lehtonen, 1998). Gonadal
development starts in the previous autumn and
continues throughout the autumn and winter
(Chen, 1969; Karhapää, 1978). Simultaneously
with the increasing gonadal weight, the liver
mass decreases, as the hepatic fat is transported
to the gonads. The highest gonadal weights of
burbots have been measured in Jan-Feb (15-21
% of BM), whereas the lowest weights are
encountered in May after spawning (0.4-0.5 %
of BM). The lipid content of the ovary fresh
weight is highest in Feb (9 %) and lowest in
May (1 %), the most abundant lipid classes
being PL and Chol (Karhapää, 1978). The
ovaries contain 35 000-5 million eggs with a
diameter of about 1 mm (Muus, 1968).
The burbot displays strong homing
behaviour and spawns in the same area where it
was born (Curry-Lindahl, 1985). Burbots of
some regions are considered migratory, as they
wander from seas and lakes to rivers for
spawning (Sorokin, 1971; Müller, 1982). The
mean rate of spawning migration has been
measured to be about 0.7-2.5 km day-1
(Sorokin, 1971; Hedin, 1983). The nocturnal
migration has been associated with moonphases
with peak activities before the new moon and
decreasing activities thereafter (Hedin, 1983).
Males arrive to the spawning areas earlier than
females (Lehtonen, 1998). The spawn takes
place on silt, sand, gravel or stony bottoms. It
occurs at the depth of 1-3 m under the ice
cover during the time of the coldest Tw
(Hewson, 1955; Koli, 1998). Sophisticated
courtship behaviour is displayed before the
simultaneous release of eggs and sperm, and
the whole procedure is repeated several times
(Fabricius, 1954). Egg density in the spawning
ground varies from 200 eggs to 800 000 eggs
3
AIMS OF THE STUDY
The studies in this thesis were undertaken to
investigate the effects of particular abiotic
factors i.e. season, light and nutritional status
on the endocrinological weight-regulation of
vertebrates. Measured variables were mainly
weight-regulatory hormones and blood
metabolites, and a special emphasis was on the
concentrations of newly-discovered signal
peptides leptin and ghrelin. The raccoon dog
and the laboratory rat served as a seasonal and
a nonseasonal mammalian species for the
experiments, whereas the burbot was chosen as
a seasonal nonmammalian model with
exceptional fat metabolism. The more specific
aims of the studies were the following:
50
1. Do circulating leptin, ghrelin and GH
concentrations of wild mammals follow
seasonal rhythms (I-II)?
under roof in cages (150 cm * 107 cm * 70 cm)
in natural temperature and photoperiod (Liperi,
Eastern Finland; 62° 33´N; 29° 7´E). They were
fed with commercial fur animal diets (Suomen
Rehu Oy, Helsinki, Finland): Aug 16th-Nov 26th
2000 ad libitum, Nov 27th 2000-Jan 24th 2001
200 kcal animal-1 d-1, Jan 25th-Feb 1st 2001 400
kcal animal-1 d-1, Feb 2nd-Feb 18th 2001 410
kcal animal-1 d-1, Feb 19th-April 1st 2001 320
kcal animal-1 d-1, April 2nd-April 30th 2001 350
kcal animal-1 d-1 and May 1st-Aug 6th 2001,
individual feeding for the lactating females.
Water or ice was available ad lib.
Forty barrier-bred Wistar rats (20 males and
20 females) were obtained from the National
Laboratory Animal Centre of the University of
Kuopio (Kuopio, Finland; IV-V). The animals
were maintained conventionally in a dark room
with artificial illumination from 0600 to 1800 hr
(12L:12D) at 20 ± 1 °C at the Department of
Biology, University of Joensuu, Finland. They
were housed singly in solid-bottom plastic
cages (Makrolon; 42 cm * 22 cm * 15 cm) with
wood shavings for bedding and had free access
to tap water and a pelleted commercial diet
(Avelsfoder för råtta och mus R36; 18.5 % raw
protein, 4.0 % raw fat, 55.7 % raw
carbohydrates; 301 kcal metabolizable energy
100 g-1, Lactamin, Stockholm, Sweden). The
rats were 9 wk of age at the beginning of the
study.
Thirty-eight adult burbots (20 males and 18
females) were caught with nets from Lake
Pyhäselkä in Eastern Finland (62° 30´N; 29°
45´E) before and during the spawning season in
Jan-Feb 2001 (VI-VII). The prespawning
burbots were caught between Jan 15th-29th and
the spawning animals on Feb 8th. The 13
burbots captured before the spawn (BS group,
8 males and 5 females) and 13 of the burbots
caught during the spawning season (DS group,
7 males and 6 females) were sacrificed
immediately after they were transported to the
2. What are the effects of exogenous
melatonin treatment on leptin, ghrelin and
GH levels of wild mammals (I-II)?
3. How does long-term fasting influence
circulating leptin, ghrelin and GH
concentrations of wild mammals (I-II)?
4. What are the effects of fasting-induced
winter sleep on the fat and protein
metabolism of the raccoon dog, and which
hormones participate in the induction of
their endocrine response to food
deprivation (III)?
5. What are the effects of exogenous
melatonin and continuous illumination on
the intermediary metabolism and weightregulatory hormones of a nonseasonal
mammal (IV-V)?
6. Does the liver, which is the main fat storing
organ of the burbot, synthesize a leptin-like
peptide like WAT of mammals (VII)?
7. Are there seasonal changes in plasma or
liver leptin-LI concentrations in a teleost
fish in reference to spawning (VI-VII)?
4
MATERIALS AND METHODS
4.1 Housing and caring of the experimental
animals
Thirty-two raccoon dogs (16 males and 16
females) born in May 2000 were purchased
from a commercial fur farm in Northern Karelia
in late summer 2000 (I-III). The animals were
housed in male-female pairs in a shadow house
51
The BS and DS burbots were sacrificed
immediately after transportation to the
university in Jan-early Feb 2001 (VI-VII). In
contrast, the AS group was fasted in laboratory
conditions in 750 l tanks at +4 °C for 2 wk until
sacrification in late Feb 2001.
University of Joensuu. The rest (AS, n = 12, 5
males and 7 females) were fasted in laboratory
conditions in 750 l tanks (1.1 m * 1.1 m * 0.62
m) with a flow rate of 1.5 l min-1 in darkness at
+4 °C for 2 wk.
4.2
Study protocols
4.3
Half of the raccoon dogs (the MEL group)
received a continuous-release melatonin
implant (12.0 mg PRIME-X® melatonin
implant, Wildlife Pharmaceuticals, Fort Collins,
CO, USA) inserted into the interscapular sc
tissue by a sterile syringe on Aug 16th 2000 and
on Feb 8th 2001 (I-III). The control group was
sham-operated (the SHAM group). Sixteen
animals (4 SHAM males and females and 4
MEL males and females) were put to a total
60-d fast from Nov 27th 2000 to Jan 25th 2001
(the fasted animals); the other group was fed
with 200 kcal animal-1 d-1 at the same time (the
fed animals). All the pairs were provided with
wooden nestboxes (75 cm * 45 cm * 40 cm)
and straw. After fasting, all the animals were
fed until the end of the study in Aug 2001.
On d 1 of the experiment with rats during
the summer 2000 (IV-V), half the animals were
maintained in 12L:12D photoperiod, the
randomly assigned other half was moved to
24L to suppress their melatonin secretion
(Reiter & Klein, 1971). Half of the rats of both
lighting conditions received a 12.0 mg sc
PRIME-X melatonin implant under sterile
conditions and the other half was shamoperated (IV-V). The study groups were as
follows: group 1: control males in 12L:12D;
group 2: control females in 12L:12D; group 3:
control males in 24L; group 4: control females
in 24L; group 5: melatonin-treated males in
12L:12D; group 6: melatonin-treated females in
12L:12D; group 7: melatonin-treated males in
24L and group 8: melatonin-treated females in
24L.
Measurement of growth parameters
BMs of the raccoon dogs were measured every
1-3 wk from Aug 2000 to Aug 2001 at blood
samplings (I-III). Their voluntary energy intake
was
recorded
throughout
the
study
approximately every third wk by providing the
animals with ad lib. feeding followed by the
weighing of the uneaten food 24 hr later. The
body lengths were measured from the tip of the
nose to the anus along the ventral midline
several times during the autumn 2000 and on
Jan 10th 2001 (I; III). Locomotor activity of the
raccoon dogs was measured during the fast
with mechanical sensors placed at the entrance
of their nestboxes (I). The sensors registered
the times, when an animal exited or entered the
nestbox.
BM gain and food intake of the rats were
recorded every fifth d throughout their 4-wk
study period (IV-V). Their body lengths were
measured in a similar way as in the raccoon
dogs on the last d of the experiment after
sacrifice. BMIs reflecting body adiposity of the
raccoon dogs and the rats were calculated by
the formula: BM (kg) [body length3 (m)]-1 (IV). BMs (- weight of gonads) and lengths of
the burbots from the tip of the nose to the tip of
the caudal fin were measured after sacrifice
(VI-VII).
4.4
Sampling and sacrification
Blood samples of the raccoon dogs were drawn
from a superficial vein of their left hind leg
during the morning hours between 0900 and
52
after sacrifice, and all the samples were
immediately frozen in liquid nitrogen and stored
at –40 °C (I-VII).
4.5
Biochemical determinations
Plasma hormone concentrations were mostly
determined with the radioimmunoassay (RIA)
method (I-VII). Diurnal plasma melatonin
concentrations were measured with the
Melatonin-RIA kit of DLD Diagnostika GmbH
(Hamburg, Germany) in order to verify
melatonin release from the implants (I-V).
Plasma leptin concentrations were determined
with the Multi-Species Leptin RIA kit of Linco
Research Inc. (St. Charles, MO, USA; I-II; IV;
VI). For the determination of hepatic leptin
(VII), the liver samples (200 mg) were
homogenized in 1 ml of a commercial 0.05 M
phosphosaline assay buffer (pH 7.4) from the
Multi-Species
Leptin
RIA
kit.
The
homogenized samples were centrifuged at 1500
g, and the water-soluble fraction was extracted
and used for the analysis with the Multi-Species
Leptin RIA kit. Plasma ghrelin levels were
determined with the Ghrelin (Human) RIA kit
of Phoenix Pharmaceuticals Inc. (Belmont, CA,
USA; I-II; IV; VI) and GH levels with the hGH
Human Growth Hormone Double Antibody kit
of DPC (Los Angeles, CA, USA; I-II; IV).
Plasma T3, T4 (III; V-VI), cortisol (III),
testosterone, estradiol (VI) and progesterone
(II) concentrations were measured with the
Spectria [125I] Coated Tube Radioimmunoassay
kits of Orion Diagnostica (Espoo, Finland).
T3T4 ratio was calculated by dividing T3
concentrations by T4 levels (III; V-VI). Plasma
TSH concentrations were determined with the
Spectria
TSH
[125I]
Coated
Tube
Immunoradiometric
Assay
kit
(Orion
Diagnostica; VI). Glucagon concentrations
were measured with the Glucagon Double
Antibody kit (III) and insulin levels with the
Fig. 5. Blood sampling from a raccoon dog (I-III).
1100 hr before the animals were fed every 1-3
wk (I-III; Fig. 5). Females, who gave birth,
were not sampled for 3 wk after parturition to
avoid disturbance (II-III). The male raccoon
dogs were sacrificed with an electric shock on
Mar 27th 2001, and their final blood samples
were obtained with cardiac punctures. The
females were sacrificed on Aug 7th 2001 with
the same procedure.
The rats were sacrificed at 1000-1400 hr by
diethyl ether at the end of their 4-wk study
period (IV-V). Their blood samples were
drawn by cardiac punctures. The BS and DS
burbots were sacrificed immediately after their
transportation to the university in Jan-Feb 2001
(VI-VII). AS burbots were sacrificed on Feb
22nd one wk after the spawning had ceased. The
fish were euthanized by a blow on the head and
their blood samples were obtained from the
ventral aorta.
Blood samples were obtained with aseptic
needles into test tubes containing EDTA and
centrifuged at 1000 g to obtain plasma (I-VII).
The liver and kidney samples of the raccoon
dogs (Nevalainen, 2002; unpubl. data) and the
rats (V) and the liver and trunk white muscle
samples of the burbots (VI-VII) were dissected
53
1966). The homogenization was carried out in
cold 0.85 % NaCl for the lipase esterase
measurement. The lipase esterase activity (VVI; Nevalainen, 2002; unpubl. data) was
measured using 2-naphthyl-laurate as substrate
without taurocholate (Seligman & Nachlas,
1962). Also the glycogen concentrations of the
liver, kidney and white muscle samples (V-VI;
unpubl. data) were measured spectrophotometrically according to Lo et al. (1970).
For lipid chromatography, total lipids (TL)
in the plasma of the raccoon dogs (III), in the
liver of the rats (V) and in the plasma and liver
of the burbots (VII) were extracted according
to the method of Folch et al. (1957). The
recovered chloroform extracts were separated
and
quantified
using
a
thin
layer
chromatography-flame ionization detection
system (IATROSCAN TLC-FID Analyser,
Iatron Laboratories Inc., Tokyo, Japan). The
chromatorods
were
developed
with
chloroform/methanol/water (50 / 20 / 2.5 by
vol.) ad 5 cm and n-hexane/diethyl ether/formic
acid (65 / 5 / 0.15) ad 10 cm. Detector
responses were calculated with authentic lipid
standards (Sigma Ltd., St. Louis, MO, USA).
Coat-A-Count Insulin kit (III; V) of DPC. For
the actual measurements the 1480 Wizard™ 3’’
Gamma Counter (Wallac Oy, Turku, Finland)
was used (I-VII). The intra- and interassay
variations of the hormone assays are presented
in Table I. The peptide assays were validated
such that serial dilutions of the raccoon dog, rat
and burbot plasma or burbot liver homogenate
showed linear changes in BB0-1 values that
were parallel with the standard curves
produced with the human peptides (II; VI; Fig.
6a-g). Glucose levels were determined with the
D-glucose UV method of R-Biopharm GmbH
(Darmstadt, Germany; III) or of Boehringer
Mannheim (Mannheim, Germany; V).
Liver and kidney samples of the raccoon
dogs (Nevalainen, 2002; unpubl. data) and the
rats (V), and liver and white muscle samples of
the burbots (VI-VII) were weighed and
homogenized. Homogenization was carried out
in cold citrate buffer for the G-6-Pase (pH 6.5)
and glycogen phosphorylase measurements (pH
6.1). The activity of G-6-Pase was measured
according to Hers & van Hoof (1966) using
glucose-6-phosphate as substrate in the
presence of EDTA after an incubation time of
30 minutes at 37.5 °C (V; unpubl. data) or at
25 °C (VI). Glycogen phosphorylase activity
(V-VI; unpubl. data) was measured in the
presence of glucose-1-phosphate, glycogen,
sodium fluoride and AMP (Hers & van Hoof,
Table I. The intra- and interassay variations for the hormone assays used in studies I-VII.
Hormone assay
intraassay variation (% CV)
interassay variation (% CV)
Leptin
2.8-3.6
6.5-8.7
Ghrelin
<5
< 14
Growth hormone
1.5-5.9
1.8-8.3
Insulin
3.1-9.3
4.9-10.0
Glucagon
3.2-6.5
6.0-11.9
Thyroxine
3.3-6.8
3.3-8.0
Triiodothyronine
3.3-6.1
4.5-7.5
Thyroid-stimulating hormone
0.9-5.8
1.4-4.9
Cortisol
2.6-5.4
6.5-7.3
Melatonin
4.3-7.4
11.7-12.1
Testosterone
3.8-7.5
4.8-7.0
Estradiol
2.9-9.7
2.3-10.2
Progesterone
2.9-5.8
4.7-5.1
54
Fig. 6a-g. Validation curves of peptide assays not presented graphically in the
substudies (III-V; VII), B=standard or sample binding, B0=maximum binding.
55
MEL and the SHAM animals. The results are
presented as the mean ± SE (I-VII).
Plasma total protein (TP) content was
determined spectrophotometrically according
to Lowry et al. (1951; III). Plasma urea,
ammonia (NH3) and aa concentrations were
analyzed at the Laboratory of Oulu University
Hospital (Oulu, Finland) with ion-exchange
chromatography (Biochrom 20 Amino Acid
Analyzer, Pharmacia Biotech Ltd., England).
The plasma creatinine concentrations were
determined spectrophotometrically with the
Creatinine Colorimetric Method of Randox
Laboratories Ltd. (Crumlin, UK) using the
Technicon RA-XTTM Analyser (Swords,
Ireland).
4.6
5
RESULTS
5.1 Seasonal
raccoon dog
weight-regulation
of
the
The BMs, BMIs and food intake of the raccoon
dogs increased in Aug-Oct 2000 (I; III). At the
same time, their plasma leptin and GH
concentrations increased and reached a peak in
late Oct (I; Fig. 7). The ghrelin levels and
ghrelin-leptin ratios were high in the autumn.
The gain in the BMs and BMIs ceased in OctNov and the food intake of the raccoon dogs
started to decline (I; III). Also the higher
autumnal FFA concentrations in plasma
reduced during Oct (III). The high leptin and
GH levels decreased to nadirs in early Nov (I;
Fig. 7). On the contrary, the ghrelin
concentrations and the ghrelin-leptin ratios
remained high.
The BMs and BMIs of the raccoon dogs
decreased gradually between Nov 2000 and
Feb 2001 (I; III). Also the voluntary food
intake of the raccoon dogs reduced further. The
low leptin and GH levels rose to higher
wintertime concentrations during the first half
of Nov and in late Dec, respectively (I; Fig. 7).
The ghrelin levels and the ghrelin-leptin ratios
decreased during the winter. The lower
autumnal TG, diacylglycerol (DG) and TL
concentrations in the plasma increased in OctNov to higher wintertime levels (III).
Furthermore, increases in the Chol and PL
concentrations were observed in early Feb and
between Oct and Jan, respectively. The most
pronounced elevation was documented in the
plasma FFA concentrations between late Nov
and early Dec.
Statistical analyses
Multiple comparisons were performed with the
one-way, two-way or repeated measures
analyses of variance (ANOVA) (I-VII).
Comparisons between two study groups were
performed with the Student´s t-test and the
Mann-Whitney U test for parametric and
nonparametric data. The normality of
distribution and the homogeneity of variances
were tested with the Kolmogorov-Smirnov test
and the Levene test. Correlations were
calculated with the Spearman correlation
coefficient (rs). P value less than 0.05 was
considered to be statistically significant.
When there was no sexual dimorphism in a
measured variable, the data for the males and
the females have been pooled together. As the
two-way ANOVA revealed no statistically
significant interactions between the melatonin
treatment and fasting, the data for the SHAM
and the MEL raccoon dogs have been pooled
across the feeding regimes, when reporting the
melatonin-induced effects on the variables (IIII). In a similar way, when the fasting-induced
changes are reported, the data for the fed and
the fasted groups are composed of both the
56
Fig. 7. The plasma leptin, ghrelin and growth hormone concentrations of the raccoon dogs (I-II)
according to melatonin treatment (a, c, e) or fasting (b, d, f) (mean ± SE). Both sexes participated in the
study until March 2001, thereafter the data consist of females only. * significant difference between the
MEL and the SHAM groups or between the fed and the fasted animals.
57
The BMs of the fed raccoon dogs decreased
gradually from Feb to March 2001, but the
fasted animals did not experience vernal weight
loss (II-III). The BMIs reached the lowest
levels in April and increased towards the
autumn 2001. Also the lower wintertime food
intake changed into an increase from Feb to
Aug. The plasma TG, DG and FFA
concentrations were relatively high during the
mating season (III). The leptin and ghrelin
concentrations of the male raccoon dogs did
not go through any vernal fluctuations before
the end of their study period on March 27th
2001 (II). Their GH levels, however, rose in
Feb and decreased at the end of March.
The leptin concentrations of the SHAM
female raccoon dogs fluctuated during the
spring (Fig. 7). The levels decreased in late
Feb, increased to a peak at the end of April,
reduced in late May, rose during June-July and
decreased again at the end of the study in Aug.
The ghrelin levels of the SHAM females
decreased from Feb to March, increased in late
April and remained relatively stable until Aug.
Their GH concentrations rose from early Feb to
early March, decreased in late March and
increased in April. Thereafter the GH levels
remained relatively high and stable until the end
of the experiment. The ghrelin-leptin ratios of
the females increased during Feb, decreased in
April, increased at the end of May, reduced in
late July and rose again in Aug.
fastest during the first 2 wk without food (III).
Also the weight loss between 4-6 wk of food
deprivation was faster than during the last 2 wk
of the fast. As % of BM, the rate of weight loss
was about 8 % in every 2 wk throughout the
fast. It was slightly slower between 2-4 wk than
during 4-6 wk. The locomotor activity of the
fasted animals decreased to 50-70 % of that of
the fed raccoon dogs (I). The differences
between the BMs and BMIs of the fed and the
fasted raccoon dogs remained significant for 6
and 2 wk after the end of the fast, respectively
(I-III). The voluntary food intake was higher in
the fasted raccoon dogs during most of the
vernal food intake measurements (II-III).
The plasma FFA concentrations of the fasted
raccoon dogs were 39 and 84 % higher than in
the fed animals after 2 and 4 wk of fasting,
respectively (III). Also their DG levels were
43-74 % higher after 2-4 wk without food. The
Chol levels of the fasted animals were 17 %
lower than in the fed group after 6 wk of
fasting, and their TG concentrations were 33 %
lower after 8 wk without food. The TG levels
decreased further 2-6 wk after the animals had
resumed eating. Also the FFA levels of the
fasted raccoon dogs were 39 % lower than in
the fed animals during the first month of refeeding. Furthermore, the fasted animals had
27-30 % lower TL concentrations after the fast
in March and May. The plasma cholesteryl
ester (CE), phosphatidylcholine (PC), -serine
(PS), -ethanolamine (PE), lysophosphatidylcholine (LPC) or sphingomyelin (SM)
concentrations were not clearly influenced by
fasting or re-feeding. The lipid profile of the
raccoon dog plasma is presented in Table II.
The total aa (Taa) concentrations of the
fasted raccoon dogs were lower after 4-8 wk of
fasting (III; Fig. 8). The levels of essential aa
were lower in the fasted animals throughout the
fast, whereas the concentrations of nonessential
aa were lower only after 4 wk of fasting. The
5.2 Influence of seasonal fasting on the
energy metabolism of the raccoon dog
A total fast of 8 wk in mid-winter resulted in a
3.1 ± 0.07 kg weight loss in the raccoon dogs
(28 ± 3.4 % of BM and BMI; I; III). Significant
differences in the BMs and BMIs between the
fed and the fasted raccoon dogs were first
observed after 4 and 2 wk of fasting,
respectively. The rate of weight loss (g) was
58
levels of urea, alanine (Ala), Arg, methionine
(Met), Thr, tyrosine and valine were lower in
the fasted raccoon dogs during the whole fast.
In contrast, the Gln levels were higher in the
fasting group throughout the food deprivation
(Fig. 8). The α-aminobutyrate (α-AB),
asparagine, carnosine, citrulline (Cit), glycine
(Gly), histidine (His), isoleucine, leucine, 3methylhistidine (3-MH), ornithine (Orn),
phenylalanine (Phe), proline (Pro), Ser and
taurine (Tau) concentrations were lower in the
fasted animals during some phases of the fast.
The levels of glutamate (Glu), phosphoserine
and Ser were higher in the fasting raccoon dogs
at one measurement, whereas aspartate, Cys,
lysine (Lys), 1-methylhistidine, NH3 and TP
were not influenced by fasting. After 2 wk of
re-feeding, the fasted animals had lower α-AB,
NH3, 3-MH and Phe concentrations than the
fed raccoon dogs, whereas their Gly levels
were higher. The plasma profile of the most
important aa of the raccoon dog is presented in
Table II. The plasma creatinine concentrations
were higher in the fasted animals after 8 wk of
fasting, and they had lower plasma ureacreatinine (U/C) ratios than the fed animals
throughout the fast (III).
Table II. Lipid and amino acid profiles of the experimental species (III; V; VII).
Plasma lipids
mg ml-1
TG
DG
FFA
Chol
CE
PS+PE
PC
LPC
SM
PL
TL
Liver lipids
TG
µg mg-1
DG
FFA
Chol
CE
PS+PE
PC
LPC
SM
TL
The most prevalent Gln
plasma amino acids Ala
Gly
µmol l-1
Ser
Pro
Taa
The raccoon dog (III)
% of TL
Mean
or Taa
concentration
16-32
1-4
3-8
0.2-1
1-11
0.1-1.5
11-19
1-2
bd-0.5
bd-0.04
bd-8
bd-0.8
40-50
3-6
bd-0.5
bd-0.05
1-2
0.1-0.3
37-53
2-7
100
8-13
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
17-31
600-1100
10-13
200-700
7-8
200-400
6-9
200-400
6-7
100-400
100
3000-4000
The rat (V)
% of TL
Mean
concentration
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
34-42
15-17
3-5
1-2
13-15
5-6
11-13
5-6
0.5-0.7
0.2-0.3
2-4
0.6-1.5
27-31
11-14
0.2-0.7
0.1-0.3
0.4-1.0
0.2-0.6
100
40-45
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
The burbot (VII)
% of TL
Mean
concentration
20-40
1-5
bd
bd
2-4
0.1-0.3
13-15
1-2
16-22
1-2
2-4
0.1-0.3
22-35
1.5-3.0
bd
bd
2
0.1-0.2
27-38
2-3
100
6-13
94-96
260-380
bd
bd
0.2-0.8
0.6-2.1
3-4
10-11
1-2
3-7
bd
bd
bd-1.3
bd-5
bd
bd
bd
bd
100
300-400
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
bd = below the detection limit, nd = not determined, TG = triacylglycerols, DG = diacylglycerols, FFA = free fatty acids, Chol = cholesterol, CE = cholesteryl
esters, PS = phosphatidylserine, PE = phosphatidylethanolamine, PC = phosphatidylcholine, LPC = lysophosphatidylcholine, SM = sphingomyelin,
PL = phospholipids, TL = total lipids, Gln = glutamine, Ala = alanine, Gly = glycine, Ser = serine, Pro = proline, Taa = total amino acids.
59
The glucose concentrations of the fasting
raccoon dogs decreased by 32 % after the first
2 wk of fasting. After 4 wk without food, they
had 18 % lower glucose levels compared to the
fed animals. The insulin concentrations of the
fasted raccoon dogs were 50-70 % lower
during 2-6 wk of fasting. Their glucagon
concentrations did not respond to food
deprivation, but the glucagon-insulin ratios
were 2-3-fold higher in the fasters after 4-6 wk
without food. They had also 50-70 % lower
cortisol concentrations than the fed animals
after 4-8 wk of fasting. The T3 and T4
concentrations were 20-40 and 15-20 % lower
in the fasters during 2-6 and 2-4 wk of the fast,
respectively. The leptin, ghrelin or GH
concentrations of the raccoon dogs were not
affected by a total 8-wk fast (I; Fig. 7).
After the fasted animals resumed feeding,
their insulin levels increased by 200 %, and they
had 23 and 15 % higher T3 concentrations and
T3T4 ratios than the fed raccoon dogs,
respectively (III). The fasted male raccoon
dogs had lower leptin concentrations than the
fed males in early March (II). The same was
observed in the fasted females in late Feb and
again in late March. Moreover, the fasted males
had lower GH concentrations compared to the
fed males at the beginning of March. The fooddeprived females had lower GH levels in April
and higher ghrelin concentrations in May
compared to the fed females.
5.3 Influence of exogenous melatonin and
continuous light on energy metabolism
5.3.1 Effects of melatonin treatment on
melatonin levels, BM, adiposity and appetite
Autumnal melatonin implantation (Aug 16th
2000) increased the diurnal plasma melatonin
concentrations of the raccoon dogs from Sep to
Oct (I; III). Vernal melatonin treatment (Feb 8th
2001) led to higher melatonin concentrations
from late Feb to the end of the experiment in
Aug 2001 (II; III). The melatonin-implanted
rats of the both lighting conditions (12L:12D
and LL) had higher diurnal plasma melatonin
Glutamine
Total amino acids
Fasting period
5000
1200
1000
4000
3500
800
3000
2500
2000
11/2000
Glutamine µmol l-1
Total amino acids µmol l-1
4500
600
12/2000
1/2001
2/2001
3/2001
Date
Fig. 8. The plasma glutamine and total amino acid concentrations (mean ± SE) of the fasted raccoon dogs (III).
60
concentrations than the control rats measured
at the end of their 4-wk study period (IV-V).
The MEL male raccoon dogs had higher
BMs than the SHAM males in Sep, but the
BMIs of the SHAM group were higher in Nov
(I; III). The MEL group consumed more
energy in Aug-Sep, whereas the SHAM group
had higher voluntary food intake in Oct–Jan.
The vernal BMs and food intake of the raccoon
dogs were not affected by melatonin (II-III). In
contrast, the BMIs were higher in the MEL
females than in the SHAM females in July-Aug
2001. The BMs, BM gain, BMIs or food intake
of the rats were not affected by melatonin (IVV).
The ghrelin concentrations of the MEL
raccoon dogs were higher compared to the
SHAM group in Oct (I; Fig. 7). Furthermore,
the MEL females had higher ghrelin
concentrations in early Nov, but the ghrelin
concentrations of the SHAM raccoon dogs
were higher in mid-Dec. Vernal ghrelin
concentrations of the male raccoon dogs were
not influenced by melatonin during their study
period, whereas the MEL females had higher
ghrelin concentrations than the SHAM females
in Feb, March and April (II). When the whole
ghrelin data of the raccoon dogs (I-II) were
analyzed together, the MEL group had slightly
higher ghrelin concentrations than the SHAM
group (2.24 ± 0.06 vs. 2.08 ± 0.06 ng ml-1, ttest, p < 0.05; unpubl. data). Furthermore, the
MEL females had higher ghrelin-leptin ratios
than the SHAM females in early May (II).
The plasma ghrelin concentrations of the
rats decreased by 35 % in the both lighting
regimes (12L:12D and LL) due to the 4-wk
melatonin treatment (IV). In contrast, their
leptin or GH concentrations were not affected
by melatonin.
5.3.2 Effects of melatonin treatment on
weight-regulatory hormones
The plasma leptin levels of the raccoon dogs
decreased 3 wk after the autumnal melatonin
implantation (I; Fig. 7). Thereafter the 2
autumnal leptin peaks were advanced by one
wk in the MEL group. Moreover, the MEL
males had lower leptin concentrations at the
end of March, whereas the leptin levels of the
MEL females were higher in early March
compared to their SHAM counterparts (II).
Autumnal melatonin treatment did not
acutely influence the GH or ghrelin
concentrations of the raccoon dogs (I; Fig. 7).
However, the first autumnal GH peak was
advanced by one wk in the MEL group and
their GH levels were higher than those of the
SHAM group in Nov. Moreover, the MEL
males had higher GH levels than the SHAM
males at the end of Dec. The vernal GH
concentrations of the male raccoon dogs were
not affected by melatonin (II). In contrast, the
vernal drop and the subsequent rise of GH
levels of the females were advanced by 3 and 2
wk by melatonin.
5.3.3 Effects of melatonin treatment on
intermediary metabolism
The MEL male raccoon dogs had lower hepatic
lipase esterase activities than the SHAM males
at the end of their study period in late March
2001, whereas the kidney lipase esterase
activities did not differ between the treatments
(Nevalainen, 2002). Neither were their liver
and kidney glycogen contents nor G-6-Pase and
glycogen phosphorylase activities affected
(unpubl. data). The liver and kidney
intermediary metabolism was not influenced by
melatonin in the female raccoon dogs at the end
of their study period in early Aug 2001.
Exogenous melatonin treatment decreased
the liver glycogen content of the female rats
61
maintained in 12L:12D (V). The liver glycogen
phosphorylase activities of the rats increased
due to melatonin, whereas the hepatic G-6-Pase
activities did not respond. The liver lipase
esterase activities increased by melatonin in the
12L:12D kept males, but decreased in the 24L
maintained males and in the female rats of the
both lighting regimes. Melatonin increased the
hepatic DG concentrations of the rats in
12L:12D, but decreased them in the animals
kept in 24L. The liver PE levels increased by
exogenous melatonin in both lighting regimes,
but the hepatic SM concentrations only in LL.
The lipid profile of the rat liver is presented in
Table II.
The kidney glycogen contents and G-6-Pase
activities of the rats decreased due to
exogenous melatonin treatment, but the renal
glycogen phosphorylase activities remained
unaffected (V). The kidney lipase esterase
activity levels increased due to melatonin in the
LL kept rats, but decreased in the 12L:12D
maintained females.
The effects of melatonin treatment on the
plasma glucose, insulin, glucagon, cortisol,
thyroid hormone and lipid concentrations as
well as on the parameters of nitrogen
metabolism were minor and mostly inconsistent
in the raccoon dogs (III). The MEL group had,
however, lower plasma FFA concentrations at
the end of Sep and higher levels in mid-Nov
compared to the SHAM raccoon dogs. The
MEL females also experienced a decline in their
FFA levels in Aug 2001, but the drop was
absent in the SHAM females. A one-month
melatonin treatment elevated the plasma T4
concentrations of the female rats kept in
12L:12D (V). The T3, glucose or insulin levels
were not affected, but the T3T4 ratio increased
due to exogenous melatonin in the male rats
kept in 12L:12D.
5.3.4 Effects of continuous
intermediary metabolism
light
on
The 4-wk exposure of rats to LL did not affect
their BMs, BM gain, BMIs or food intake (IVV). Neither had constant light any influence on
their diurnal melatonin levels. The liver
glycogen
concentrations
and
glycogen
phosphorylase activities of the control and the
melatonin-treated rats decreased due to LL
(V). It also increased the liver lipase esterase
activities in the control males but decreased
them in the melatonin-treated males. The liver
DG concentrations increased by LL in the
control rats and decreased in the melatonintreated animals. In addition, the hepatic CE
levels of the control rats and the SM
concentrations of the melatonin-treated animals
increased due to LL. The liver G-6-Pase
activities were not affected by 24L.
Constant light decreased the renal glycogen
concentrations and G-6-Pase activities of the
rats. The kidney phosphorylase activity levels
decreased with 24L in the males, and the renal
lipase esterase activities increased in the
melatonin-treated rats by LL.
LL did not influence the plasma thyroid
hormone or glucose concentrations of the rats.
Neither had it any influence on their leptin,
ghrelin or GH levels (IV). The T3T4 ratio
increased in the control males and the insulin
concentrations in the females due to LL (V).
5.4
Seasonal reproduction
5.4.1 Effects of spawning on the energy
metabolism of the burbot
The BMs of the BS and DS burbots were
higher than those of the AS fish (VI-VII). Also
the absolute and relative liver weights were
highest in the BS group followed by the DS
and AS burbots. The latter study groups had 51
62
The plasma testosterone concentrations of
the BS males were higher than the levels of the
DS and AS males. The estradiol concentrations
of the female burbots were highest in the BS
group. The plasma T4 concentrations of the AS
group were higher than those of the other
groups, whereas the T3 levels were higher in
the BS and AS groups compared to the DS
group. There were no differences in the plasma
T3T4 ratios or in the TSH concentrations in
reference to reproduction.
and 64 % less hepatic TL than the BS group
(VII). The proportions of TL in the liver fresh
weight (w/w) of the study groups were 40, 28
and 32 %. Also the plasma TL levels were
highest in the BS group.
TG accounted for 94-96 % of the liver TL
of the burbots, and the hepatic TG levels were
highest in the BS group. The proportions of
TG in the liver fresh weight were 38, 26 and 30
% in the BS, DS and AS groups. Also the
plasma TG levels were highest in the BS group.
Moreover, the proportions of TG in the plasma
TL were higher in the BS and DS groups (40
%) than in the AS burbots (20 %).
The FFA levels of the livers were lowest in
the BS group and increased during spawning.
The plasma FFA concentrations, on the other
hand, were highest before spawning and
decreased during reproduction. The hepatic
Chol concentrations of the BS and DS groups
were higher than in the AS group, and the
plasma Chol concentrations were highest in the
BS group. The levels of hepatic CE were low
in the DS group and increased after spawning,
but in the plasma the highest concentrations
were encountered before reproduction. Also
the plasma PL levels were highest in the BS
group. The hepatic lipase esterase activities of
the BS burbots were higher than the activity
levels of the other groups (VI). The lipid
profiles of the plasma and liver of the burbot
are presented in Table II.
Glycogen accounted for 2 % of the liver
fresh weight in all burbot study groups (VI).
The liver glycogen contents of the BS group
were higher than in the AS burbots, whereas
the muscle glycogen concentrations did not
differ between the study groups. The liver
glycogen phosphorylase activities of the BS
group were higher than those of the AS group.
On the other hand, the muscle phosphorylase
activities were highest in the DS group and the
liver G-6-Pase activities in the AS burbots.
5.4.2 Effects of reproduction on weightregulatory hormones
The SHAM female raccoon dogs had high
plasma leptin concentrations before the mating
season (I-II). The levels decreased before proestrus and estrus occurring during the later half
of March (II). The leptin concentrations
increased during the first half of pregnancy
from late March to late April, whereas
decreasing leptin concentrations were observed
during the later half of gestation in May. When
all the leptin data were organized according to
the reproductive status of the females
independently of the time of sampling, the
females, who had whelped but lost their litters
had lower leptin concentrations than the
pregnant or barren ones. Also the ghrelin and
GH concentrations of the SHAM females were
higher before the mating season and decreased
before estrus. The same was observed in the
GH levels of the male raccoon dogs.
The burbots had higher plasma leptin-LI
concentrations after reproduction than before
or during spawning (VI). Their liver leptin-LI
levels ranged from 5-34 ng g liver-1, the mean
being 8-12 ng g-1 (VII). The hepatic leptin-LI
concentrations were lower in the AS burbots
compared to the BS fish. Moreover, the plasma
ghrelin-LI concentrations were highest in the
AS burbots (VI).
63
5.4.3 Effects of gender on weight-regulatory
hormones
at the end of their 4-wk study period (3.8 ±
0.16 vs. 1.7 ± 0.20 ng ml-1, t-test, p < 0.0004).
The plasma leptin levels of the male raccoon
dogs were higher than those of the females at
the end of Sep (1.3 ± 0.17 vs. 0.7 ± 0.23 ng ml1
), Oct (2.9 ± 0.28 vs. 1.9 ± 0.13 ng ml-1) and
Feb (II), but lower in mid-Aug (0.7 ± 0.13 vs.
1.1 ± 0.06 ng ml-1), mid-Nov (3.0 ± 0.16 vs.
3.7 ± 0.22 ng ml-1), late Nov (2.9 ± 0.10 vs. 3.4
± 0.10 ng ml-1), mid-Dec (3.1 ± 0.19 vs. 3.9 ±
0.08 ng ml-1), late Dec (3.0 ± 0.16 vs. 4.3 ±
0.16 ng ml-1), early Jan (2.4 ± 0.11 vs. 3.5 ±
0.13 ng ml-1) and late Jan (2.1 ± 0.11 vs. 2.8 ±
0.14 ng ml-1, II; unpubl. data, t-test, p < 0.05).
There were no gender differences in the leptin
levels of the rats (IV), but the female burbots
had higher plasma leptin-LI levels than the
males (VI). The liver leptin-LI concentrations
of the burbots did not differ by gender (unpubl.
data).
The female raccoon dogs had higher ghrelin
levels than the males at the beginning of Nov
(4.6 ± 0.68 vs. 2.8 ± 0.43 ng ml-1, t-test, p <
0.041; unpubl. data). When the whole ghrelin
data from Aug 16th 2000 to March 27th 2001
(i.e. the time, when the both sexes participated
in the study) were analyzed together, the
female raccoon dogs had higher ghrelin
concentrations than the males (2.4 ± 0.08 vs.
2.2 ± 0.08 ng ml-1, t-test, p < 0.016). On the
contrary, there were no gender differences in
the plasma ghrelin or ghrelin-LI levels of the
rats (IV) or burbots (VI).
The male raccoon dogs had higher plasma
GH concentrations than the females in Oct (1.3
± 0.16 vs. 0.6 ± 0.14 ng ml-1), whereas the GH
concentrations of the females were higher in
late Dec (1.3 ± 0.07 vs. 0.9 ± 0.07 ng ml-1) and
Jan (1.4 ± 0.09 vs. 1.0 ± 0.10 ng ml-1, t-test, p
< 0.014; unpubl. data). The GH levels of the
female rats were higher than those of the males
5.5 Endocrinological
interactions
and
metabolic
When the whole raccoon dog data were
analyzed together, the plasma leptin
concentrations of the animals correlated
positively with their BMs (rs=0.426, p < 0.01)
and BMIs (rs=0.371, p < 0.01), and there was a
negative correlation between the leptin levels
and the voluntary food intake of the animals
(rs=-0.295, p < 0.01; I-II, unpubl. data). The
leptin concentrations of the raccoon dogs
correlated positively with their plasma TG
(rs=0.380, p < 0.01), DG (rs=0.116, p < 0.05),
PL (rs=0.167, p < 0.01) and TL (rs=0.282, p <
0.01) levels (unpubl. data). When the leptin
levels and nitrogen metabolism data of the
raccoon dogs were pooled from Nov 23rd 2000
to Feb 8th 2001, a positive correlation between
the leptin and urea concentrations (rs=0.300, p
< 0.05) and a negative correlation between the
leptin and NH3 (rs=-0.572, p < 0.01) and Gln
concentrations (rs=-0.400, p < 0.01) were
observed.
The leptin concentrations and the BMIs of
the 24L maintained rats correlated positively
with each other (IV). There was also a positive
correlation between the leptin concentrations
and the final BMs of the rats (rs=0.900, p <
0.05; unpubl. data). The leptin-LI levels of the
burbot plasma correlated negatively with the
BMs and relative liver weights as well as with
the
hepatic
glycogen
concentrations,
phosphorylase activities and TG (rs=-0.435, p <
0.01), Chol (rs=-0.425, p < 0.01) and TL
contents (rs=-0.435, p < 0.01; VI; unpubl.
data). The liver and plasma leptin-LI levels of
the BS and AS burbots correlated positively
with each other (VII). Moreover, the liver
64
leptin-LI concentrations correlated inversely
with the liver TL levels of the DS burbots.
The ghrelin levels of the raccoon dogs
correlated positively with their BMs (rs=0.136,
p < 0.01), BMIs (rs=0.267, p < 0.01) and
voluntary food intake (rs=0.533, p < 0.01; I;
unpubl. data). Also their ghrelin-leptin ratios
correlated positively with the voluntary food
intake (rs=0.402, p < 0.01) but negatively with
the BMs (rs=-0.168, p < 0.01) and GH
concentrations (rs=-0.122, p < 0.05). In
contrast, the plasma ghrelin and FFA
concentrations correlated negatively with each
other (rs=-0.286, p < 0.01; unpubl. data). There
was also an inverse correlation between the
ghrelin concentrations and the BMIs of the
control rats (rs=-0.543, p < 0.01). The ghrelinLI concentrations of the burbots correlated
negatively
with
their
liver
glycogen
concentrations and positively with their muscle
glycogen contents (VI).
The plasma GH concentrations of the
raccoon dogs correlated inversely with their
BMIs (rs=-0.170, p < 0.01) and voluntary food
intake (rs=-0.479, p < 0.01), but there was a
positive correlation between the GH and FFA
levels (rs=0.138, p < 0.05; I; unpubl. data). The
GH levels of the rats correlated inversely with
their final BMs (rs=-0.810, p < 0.01) and with
their cumulative food intake (rs=-0.770, p <
0.01), but there was a positive correlation
between the GH concentrations and the BMIs
in the 12L:12D kept rats (rs=0.664, p < 0.05;
unpubl. data).
The leptin and ghrelin concentrations of the
raccoon dogs correlated negatively with each
other (rs=-0.116, p < 0.05; I; unpubl. data). A
similar relationship was also observed between
the leptin and ghrelin concentrations of the
12L:12D kept control rats (IV). On the
contrary,
the
leptinand
ghrelin-LI
concentrations of the burbots correlated
positively with each other (VI).
There was a negative correlation between
the ghrelin and GH concentrations of the
raccoon dogs (rs=-0.302, p < 0.01; I, unpubl.
data). On the contrary, the ghrelin and GH
levels of the female rats correlated positively
with each other (rs=1.000, p < 0.01; unpubl.
data). There was also a positive correlation
between the leptin and GH concentrations of
the raccoon dogs (rs=0.126, p < 0.01; I; unpubl.
data), but this relation was absent in the
experiment with rats (rs=-0.167, p > 0.05;
unpubl. data).
The leptin (rs=0.315, p < 0.01) and GH
levels (rs=0.417, p < 0.01) correlated positively
with the testosterone concentrations of the
male raccoon dogs, but negatively with the
testosterone levels of the females (rs=-0.221, p
< 0.01; rs=-0.190, p < 0.05, unpubl. data). On
the other hand, the correlation between the
ghrelin and testosterone concentrations was
negative in the male raccoon dogs (rs=-0.366, p
< 0.01) but positive in the females (rs=0.255, p
<
0.01).
Furthermore,
the
ghrelin
concentrations of the female raccoon dogs
correlated positively with their estradiol levels
(rs=0.235, p < 0.01). In the SHAM females, the
plasma leptin and progesterone concentrations
correlated positively with each other between
March and May 2001 (II).
The plasma leptin-LI and testosterone levels
correlated inversely in the male burbots (VI).
There was also a negative correlation between
the GH and testosterone concentrations of the
male and female rats (rs=-0.764, p < 0.01;
unpubl. data). The ghrelin concentrations of the
female rats correlated negatively with their
testosterone levels (rs=-1.000, p < 0.01). There
was also an inverse correlation between the
Chol and estradiol levels of the female raccoon
dogs (rs=-0.160, p < 0.05) and a positive
correlation between the DG and testosterone
concentrations of the males (rs=0.300, p <
0.01).
65
6
6.1
As leptin and ghrelin molecules have been
identified in several mammalian species, it is
likely that also the homologous peptides
measured in the blood of the raccoon dog are
true leptin and ghrelin. The presence of true
leptin in the burbot plasma is less certain, as a
gene encoding fish leptin has not yet been
identified. On the contrary, fish ghrelin has
been recently identified e.g. in the goldfish
(Unniappan et al., 2002) supporting the
presence of ghrelin also in the burbot.
In contrast to the raccoon dog and the
burbot, the rat leptin, ghrelin, GH and insulin
molecules have been identified. There is an 83
% homology between the rat and human leptins
(Ogawa et al., 1995). The leptin concentrations
of the rats of the thesis (IV) were measured
with a multi-species RIA kit with human
standards, having a 61 % specificity to rat
leptin. The rat ghrelin molecule differs from its
human homologue by two aa (Kojima et al.,
1999), and the human ghrelin RIA kit that was
used has a 100 % cross-reactivity to rat ghrelin.
The rat and human GH molecules differ by 34
% (Seeburg et al., 1977), whereas the A-chains
of human and rat insulin molecules differ by
one aa and the B-chains by three aa (Bentley,
1998). The leptin, ghrelin, GH and insulin kits
that were used were validated for the rat
plasma in a similar way as described for the
raccoon dog and the burbot (Fig. 6c-f).
The rats (IV-V) were sampled only at the
end of their study period, whereas the burbots
(VI-VII) were sampled three times, but each
time the blood was drawn from different
individuals after sacrifice. Naturally these single
point measurements must be interpreted with
caution. In contrast to the rats and burbots,
serial blood samples were obtained from the
same raccoon dog individuals throughout a
year (I-III). As several hormones are known to
display diurnal variations in their release
(Bentley, 1998), repeated blood samples were
DISCUSSION
General remarks
Specificity of RIA kits can be a problem when
measuring concentrations of peptide hormones
from unconventional animal models such as the
raccoon dog and the burbot (I-III; VI-VII).
The peptide hormones measured in this thesis
have not been isolated or their genes cloned in
the above-mentioned species. For this reason it
cannot be said with certainty that the
determined molecules were true leptin, ghrelin,
GH, insulin or glucagon. Instead they might
have been molecules with high homology to
these peptides. However, the leptin, ghrelin,
GH, insulin and glucagon RIA kits that were
used were validated such that serial dilutions of
the raccoon dog and burbot plasma, and in the
case of leptin, burbot liver homogenate,
showed linear changes in the BB0-1 values that
were parallel with the standard curves
produced with the human peptides (II; VI; Fig.
6a-b,g).
As there are differences in the molecular
structures of peptide hormones depending on
the species, only the relative concentrations of
hormones can be measured without speciesspecific kits. The human and canine leptin
molecules share an 82 % homology (Iwase et
al., 2000), whereas the dog ghrelin shares a 93
% identity to humans (Tomasetto et al., 2001).
The GH of the mink (a mustelid) is 66 %
homologous to humans, whereas the fox (a
canid) GH shares a 67 % homology with us
(Shoji et al., 1990; Noso et al., 1995). It is also
known that the A-chains of the human and
canine insulin molecules are identical, and their
B-chains differ by one aa (Bentley, 1998).
Moreover, the structure of glucagon is wellconserved and displays virtually no variation
among mammals except of some rodent
species.
66
always taken approximately at the same time of
the day (I-III).
Continuous-release melatonin implants were
used in the raccoon dog studies instead of
timed injections, which would have been
laborious and practically impossible to carry
out daily throughout a year. The release of
melatonin from the implant did not follow any
diurnal rhythm unlike endogenous melatonin
secretion. Furthermore, continuous release of
melatonin could have caused down-regulation
of melatonin receptors of the raccoon dogs
(Gauer et al., 1993). Similar melatonin implants
have, however, been successfully used in other
raccoon dog experiments, and they have been
able to induce advancements to the autumn
moult and testicular activity of the species
(Xiao, 1996). The rationale behind the vernal
melatonin implantation (II-III) was to obtain
continuously higher melatonin concentrations
to the MEL group. The male raccoon dogs
were sacrificed earlier (on March 27th 2001)
than the females (on Aug 7th 2001) after the
mating season for financial reasons.
6.2 Seasonal
raccoon dog
weight-regulation
of
The voluntary food intake of the
experimental raccoon dogs increased in the
autumn 2000 leading to accumulation of fat and
BM gain (I; III). The plasma FFA
concentrations decreased in the autumn, as they
were transported to WAT to be esterified into
TG (III). Also the low circulating TG and DG
levels indicate their accumulation to sc fat. The
autumnal fat storage is a well-known
characteristic of the species (Korhonen, 1987).
During the fattening process the plasma leptin
and GH concentrations of the raccoon dogs
increased from low levels on Aug 16th to the
peaks on Oct 25th (I). The ghrelin levels and
ghrelin-leptin ratios were high in the autumn.
The autumnal leptin concentrations of the
raccoon dogs reflected their body adiposity.
Rising leptin levels during fat accumulation
have been previously observed in some other
seasonal mammals (European brown bear:
Hissa et al., 1998a; woodchuck: Concannon et
al., 2001). Exogenous leptin is known to
reduce appetite, BM and adiposity in laboratory
rodents (Campfield et al., 1995; Halaas et al.,
1995; Pelleymounter et al., 1995), and
continuous
leptin
infusion
inhibits
prehibernatory fattening in arctic ground
squirrels (Ormseth et al., 1996). In contrast,
the increasing autumnal leptin concentrations of
the raccoon dogs were not able to induce a
weight loss to the species, which may be due to
seasonal differences in the sensitivity to
anorectic effects of leptin (I). This hypothesis is
supported by the findings of Clarke et al.
(2000)
demonstrating
that
centrally
administered leptin decreases vernal food intake
of sheep but is ineffective when administered in
the autumn.
The results also indicate that the
physiological function of endogenous leptin is
not necessarily anorectic (I). This is in concert
with the results of Maffei et al. (1995), who
observed that overweight humans and rodents
the
6.2.1 Autumnal fat accumulation
Raccoon dog cubs are born in May-early June
in the wild (Siivonen, 1972). They have to
gather sufficient fat stores until the end of the
year in order to survive through the cold season
and to be able to reproduce during the next
spring. Fat storage is completed later in the
juveniles than in the adults, as the young have
to invest first in the attainment of the adult
body length (Kauhala, 1992). This takes 5-7
months, and only thereafter can they fully focus
on the accumulation of fat. The mortality rate
of juvenile raccoon dogs is 77 % during their
first autumn and winter.
67
Divergent seasonal changes in leptin
concentrations and body adiposity have been
previously reported in some wild mammals
(little brown bat: Kronfeld-Schor et al., 2000;
blue fox and raccoon dog: Nieminen et al.,
2001a). According to Nieminen et al. (2001a),
plasma leptin levels of blue foxes and raccoon
dogs decline in a wk in early Nov without
changes in their BMs. Blood leptin
concentrations of the little brown bat, on the
other hand, rise before the prehibernatory
increase in adiposity, and thereafter their leptin
levels decrease, while body adiposity increases
(Kronfeld-Schor et al., 2000).
Decreasing leptin levels have been
hypothesized to function as a trigger for the
neuroendocrine response to fasting inducing the
suppression of reproduction, thermogenesis,
growth and immune defense and the stimulation
of stress response (Ahima et al., 1996; Aubert
et al., 1997; Lord et al., 1998). The metabolic,
endocrinological and behavioural changes
induced by low leptin levels may lead to energy
preservation enhancing the survival of raccoon
dogs during the most challenging time of the
year.
have elevated leptin concentrations, which are
unable to induce a weight loss. The
transportation of leptin to the brain seems to be
saturable even at physiologically low
concentrations (Banks et al., 2000). Leptin
resistance of the raccoon dogs during the
autumnal food abundance (I) could permit the
continuation of active foraging and fat storing
for the cold season (see also Flier, 1998).
In contrast to leptin, exogenous ghrelin
increases appetite and suppresses lipolysis of
rodents leading to accumulation of fat (Tschöp
et al., 2000). The high autumnal ghrelin
concentrations and ghrelin-leptin ratios of the
raccoon dogs probably contributed to their
hyperphagia and fat storing (I). Also GH is
known to increase appetite of some mammalian
species (rat: Azain et al., 1995; human: Blissett
et al., 2000). It is, however, unlikely that the
increasing GH levels could have contributed to
the autumnal hyperphagia of the raccoon dogs,
as there was an inverse correlation between
their GH levels and voluntary food intake (I).
6.2.2 Metabolic transition
Young raccoon dogs have to be able to
transform their metabolic adaptations very
rapidly from autumnal energy storage to
wintertime energy preservation. This period of
metabolic transition was clearly observable in
the seasonal physiology of the raccoon dogs (I;
III). At the end of Oct, their autumnal BM and
BMI gains started to retard. At the same time,
their voluntary food intake reduced with
decreasing ambient temperature, confirming the
previous results of Korhonen (1987). The leptin
and GH concentrations declined rapidly
between late Oct and early Nov (I).
In contrast to the unidirectional changes in
the leptin levels and body adiposity in Sep-Oct,
the leptin concentrations and fat stores of the
raccoon dogs dissociated in late autumn.
6.2.3 Wintertime energy preservation
The raccoon dog pairs settle in their burrows
for the winter rest, when the ambient
temperature decreases below –5 - –10 °C, and
there are several cm of snow on the ground
making foraging difficult (Juha Asikainen, pers.
commun.). The winter lethargy of the raccoon
dogs on the study area begins usually in NovDec. At that time of the year, the voluntary
food intake of the experimental raccoon dogs
was at a low level and their BMs and BMIs
decreased gradually (I; III) in concordance with
the previous results of Korhonen (1987). As
the seasonal fat storage was completed and
replaced with the mobilization of lipids, the
68
concentrations of the raccoon dogs indicates
the role of GH in fat mobilization of the
species. In addition to lipolysis, GH may also
induce lethargy and sleepiness (Drucker-Colín
et al., 1975; Lachmansingh & Rollo, 1994)
leading to energy preservation during the
winter rest. Furthermore, GH can stimulate
gluconeogenesis (Knapp et al., 1992; Bentley,
1998) and prevent protein degradation (Vann et
al., 2000), both of which are important features
of successful wintering during the seasonal
food scarcity.
circulating FFA, TG, DG and TL
concentrations increased (III). Also the
European badger exhibits the highest plasma
TG, PL and Chol concentrations as well as CEtotal Chol ratios in late autumn-early winter
(Laplaud et al., 1980).
The GH concentrations of the raccoon dogs
remained low for five wk in Nov-Dec, whereas
their leptin levels increased during the first half
of Nov (I). Also the GH concentrations
increased to higher wintertime levels but not
until late Dec. On the contrary, the ghrelin
levels and ghrelin-leptin ratios of the raccoon
dogs were lower than in the autumn. The high
leptin concentrations together with the low
ghrelin levels could have worked together to
suppress the appetite of the raccoon dogs
(Nakazato et al., 2001; Shintani et al., 2001)
providing them with a satiety signal during the
seasonal rest (I). High lipolytic rates required
for sufficient WAT mobilization could have
been enabled by the high leptin and GH levels
and low ghrelin concentrations (Richelsen,
1997; Reidy & Weber, 2000; Tschöp et al.,
2000). This is supported by the observed
positive correlation between the leptin
concentrations and the TG, DG, PL and TL
levels and between the GH and FFA
concentrations. Also the observed inverse
correlation between the ghrelin and FFA levels
supports this view.
The high wintertime GH concentrations (I)
probably function as an adaptation to the
seasonal rest of the raccoon dog (Mustonen et
al., 2001). Also several cervid species have
higher blood GH levels in the cold season than
during the rest of the year (white-tailed deer:
Bubenik et al., 1975; Spitzbergen reindeer:
Ringberg, 1979; red deer: Curlewis et al.,
1992), suggesting that GH could play an
important role in winter catabolism of mammals
inhabiting higher latitudes. The observed
positive correlation between the FFA and GH
6.2.4 Vernal mating season and summer
Wild raccoon dogs awake from the winter sleep
in March-April (Siivonen, 1972), and the
overwintered pairs come into heat and mate
around the time of the arousal (J. Asikainen,
pers. commun.). The cubs are usually born in
May-early June (Siivonen, 1972). During the
food abundance of spring and summer the
depleted fat stores are replenished.
The experimental winter sleep of the
raccoon dogs was discontinued in late Jan 2001
(I; III). The BMs and BMIs of the fed raccoon
dogs decreased gradually during the spring, but
the fasted animals did not experience any vernal
changes in their BMs or BMIs (II-III). The
voluntary food intake of the raccoon dogs
increased during the spring and the fasted
animals had higher appetite during most of the
vernal food intake measurements. The plasma
FFA, TG and DG concentrations were
relatively high during the reproductive season,
probably due to the high metabolic
requirements of mating and gestation (III). The
TG and DG levels decreased in March-April,
whereas the FFA concentrations remained high
during the whole spring. According to Laplaud
et al. (1980), also the European badger
experiences decreases in plasma TG, PL and
Chol concentrations and in CE-total Chol ratios
69
in early spring. As the leptin, ghrelin and GH
concentrations of the raccoon dogs fluctuated
during the reproductive season (II), they might
have specialized functions in their reproductive
processes, as discussed later. Also the ghrelinleptin ratios of the raccoon dogs fluctuated
during the spring and were not directly
proportional to the voluntary food intake of the
animals unlike in the autumn 2000 (I).
During the summer and early autumn 2001,
the BMs and BMIs of the raccoon dogs started
to increase gradually as the fat accumulation
proceeded (II-III). Their voluntary food intake
increased during the summer reaching the
maximum values at the end of the study in Aug.
The rise was simultaneous with increasing
ghrelin-leptin ratios possibly inducing autumnal
hyperphagia. The leptin concentrations and the
fat gain of the raccoon dogs were decoupled as
the leptin levels rose in June-July but decreased
at the end of the study. Their ghrelin and GH
levels remained stable during the summer and
early autumn 2001 (II). The concentrations of
the weight-regulatory hormones, especially
those of GH, did not return to the same levels
as recorded in the autumn 2000 (I-II), which
may be associated with the aging of the
animals. The seasonal changes in the weightregulatory hormones and their connections with
the annual physiological phases of the raccoon
dog are represented in Table III.
6.3 Fasting-induced winter sleep in the
raccoon dog
6.3.1 Characteristics of winter
medium-sized and large carnivores
sleep
in
Winter sleep is among the most remarkable
physiological adaptations for conquering the
cold season. In contrast to e.g. bears, the
winter rest of the raccoon dog has not been
studied intensively, although the species would
be an ideal model for investigating seasonal
obesity. Blood samples can be obtained at
frequent intervals without anesthesia from
animals of a moderate BM and easy to handle.
Raccoon dogs are also readily accessible from
the wild and can be bred efficiently in captivity.
The winter lethargy of the species mostly
resembles the winter sleeps of true badgers
(Melinae; Long & Killingley, 1983) and bears
(Nelson et al., 1983b), but is probably the
shallowest one of these winter rests.
The winter sleep of the raccoon dog can last
for up to 4-5 months in the northernmost areas
of its geographical distribution (Siivonen,
1972). It is facultative and characterized with
occasional periods of arousal and food intake
during thaws. The animals probably eat snow,
urinate and defecate during the denning period
(J. Asikainen, pers. commun.). Young raccoon
dogs, which have not been able to gather
Table III. Endocrinology of seasonal weight-regulation of the raccoon dog (I-II).
Month Adaptational behaviour
Weight-regulatory hormones
Probable metabolic effect
II-VII
Normal activity
High GH concentrations
Intermediate leptin levels
Low ghrelin levels
Supply of nutrients for
mating and nursing the young
VIII-XI
Hyperphagia
High ghrelin concentrations
Low but increasing leptin and GH levels
Lipid accumulation
XII-I
Winter sleep
High leptin and GH levels
Low ghrelin concentrations
Lipolysis
Protein conservation
Induction of sleepiness
70
sufficient fat stores, can be active also during
the colder periods. Metabolism of raccoon dogs
has been reported to decrease by 25 % during
the seasonal rest (Heptner et al., 1974).
Moreover, an experimental winter sleep-like
state can be induced to raccoon dogs in farm
conditions by withdrawing their food and
providing them with nestboxes and straw
(Asikainen et al., 2002). The rectal Tb of
raccoon dogs decreases by 0.5-1.5 °C during
this fasting-induced winter sleep.
The American badger (Taxidea taxus) can
store up to 31 % of its BM as fat for the winter
and remain beneath the ground in burrows for
at least 85 consecutive days (Harlow, 1981a).
Between Nov and March the fat stores of
overwintering badgers decrease by 37 %.
During torpor their heart rate can decrease by
50 % and Tb by 9 °C as measured in field
enclosures. In the laboratory total metabolism
of badgers decreases by 26 % and their Tb by
1.7 °C after 30 days of fasting (Harlow,
1981b). The Tb of the European badger
decreases by 9 °C and the time it spends
outside the sett by 90 % in mid-winter when
compared to values measured in spring (Fowler
& Racey, 1988).
The winter sleep of the European brown
bear (Hissa et al., 1994) and the American
black bear (U. americanus, Nelson et al., 1973,
1983a,b) can last from 3 to 7 months, during
which their Tb decreases only by 2-5 °C and
heart rate as much as 75-84 %. Like raccoon
dogs and badgers, also bears use their fat stores
as the principal metabolic fuel during denning
leading to a 22-25 % weight loss in
approximately three months. In contrast to
raccoon dogs and badgers, who have been
observed to forage occasionally during the
winter, bears stay normally in their dens
throughout the resting period without drinking,
eating, urinating or defecating. However, bears
can arouse quickly from their winter rest and
defend themselves if required. The brown and
black bears give birth during the denning
period, whereas the raccoon dog delivers in late
spring after arousal (Siivonen, 1972). The
European badger, on the other hand, delivers
around the time of the arousal, as in Finland the
cubs are born in March-April and the winter
sleep terminates in April-May (Nyholm, 1972).
Presumably the metabolic response to
starvation in the raccoon dog, like in several
other animal species (Goodman et al., 1980;
Cherel et al., 1988a), can be divided into three
phases. During early starvation (phase I), which
lasts for only a couple of days, glycogen is the
primary substrate for metabolic energy. Fat
stores provide the major energy during
prolonged starvation (phase II), while the BM
loss and protein catabolism remain at a low
level. Fat stores are exhausted during terminal
starvation (phase III) leading to stimulated
proteolysis and BM loss prior to death.
Table IV compares the fasting-induced
physiological adaptations of the raccoon dog (I;
III) and selected animal species experiencing
long seasonal fasts. Also the domestic dog is
included, as it is a comparatively nonseasonal
relative of the raccoon dog. Since there are
very few complete studies on the effects of
fasting on the endocrinology and blood
chemistry of seasonal mammals with frequent
sampling and proper control animals, it is
difficult to compare the results of this thesis
with other data.
6.3.2 Effects of fasting on BM, adiposity and
plasma lipids
The total fast of 60 days caused a 28 %
reduction in the BMs and adiposity (BMIs) of
the raccoon dogs (I; III). The rate of weight
loss was steady throughout the fast (0.47 %
BM day-1, III) and confirms earlier results
(Asikainen et al., 2002). Compared to other
71
species with long seasonal fasts, the raccoon
dog loses weight more rapidly than the denning
black bear (0.25 % BM day-1, Nelson et al.,
1973) but at a slower rate than the fasting
American badger (0.92 % BM day-1, Harlow &
Seal, 1981) or the fasting king penguin (A.
patagonica, 0.94 % BM day-1, Cherel et al.,
1988b).
The plasma FFA and DG concentrations
were higher in the fasted raccoon dogs than in
the fed animals after two and four wk of fasting
(III). This indicates increased TG hydrolysis in
sc WAT. An increase in blood FFA levels is a
common response to food deprivation shared
by several species experiencing periodic or
long-term seasonal fasts (American badger:
Harlow & Seal, 1981; king penguin: Cherel et
al., 1988b; European brown bear: Hissa et al.,
1994;
European
hedgehog
Erinaceus
europaeus: Cherel et al., 1995; arctic fox:
Fuglei, 2000; black bear: LeBlanc et al., 2001).
The fasted raccoon dogs had also lower
Chol and TG levels than the fed animals after
six and eight wk of fasting, respectively (III).
These findings differ from the data of Nelson et
al. (1973), who found increased blood Chol
and TG concentrations in denning black bears.
Also several other studies conducted on black
bears show elevated blood Chol concentrations
during the winter sleep (Franzmann &
Schwartz, 1988; Hellgren et al., 1993; LeBlanc
et al., 2001), but also contradictory results with
Table IV. Effects of fasting on endocrinology and blood chemistry of the raccoon dog (I; III), the dog and selected
species with long seasonal fasts (B = blood, U = urine).
Parameter
Raccoon A. black E. brown Gray American Northern elephant Dog6
King
dog
bear1
bear2
wolf 3
badger4
seal5
penguin7
B glucose
↓
↓/–
↓
–
↓
↓/–
↓
↓
B insulin
↓
–
↓
↓
–
B glucagon
–
–
–
↑
B cortisol/corticosterone
↓
↑
–
↑
↑
B triiodothyronine
↓
↓/–
↓
↓
↓
↑
↓
↓
B thyroxine
↓
↓/–
↓
↑
–
↓
B leptin
–
–
↓
B growth hormone
–
–
B ammonia
–
–
–
B/U urea/urea-N/total-N
↓
↓/–
↓
↓
↓/–
↓
↑
B creatinine
↑
↑
↑
↑
–
↓
B urea-creatinine ratio
↓
↓
↓
↓
–
–
↑↓
↑8 ↓9
↑9
B amino acids
↓ ↑8
B total protein
–
↑/–
–
↓/–
B triacylglycerols
↓
↑/–
↓
↓
↑
–
↓/–
B diacylglycerols
↑
B free fatty acids
↑
↑
↑
↑
↑
↑
B cholesterol
↓
↑/–
↓
↓
↑/–
B phospholipids
–
↑
1
data based on results of Nelson et al., 1973; Azizi et al., 1979; Palumbo et al., 1983; Hellgren et al., 1990, 1993; Herminghuysen et al., 1995;
LeBlanc et al., 2001
2
Hissa et al., 1994, 1998b
3
DelGiudice et al., 1987
4
Harlow & Seal, 1981; Harlow & Nelson, 1990
5
Costa & Ortiz, 1982; Ortiz et al., 2001a,b
6
de Bruijne et al., 1981; Miller et al., 1983; Ishioka et al., 2002
7
Cherel et al., 1988b
8
glutamine
9
alanine
72
only after four wk of fasting, indicating that the
raccoon dogs were able to synthesize them
efficiently. Previously Nelson et al. (1973) have
not found any influence of winter sleep on
circulating aa concentrations of two black bears
and one Himalayan bear (U. thibetanus). This
differs from the data of Hissa et al. (1998b)
showing elevated (Ala, His, Lys, Met, 3-MH,
Orn and Phe) or suppressed levels (Arg and
Tau) of particular aa in the denning European
brown bear. Unfortunately, there is a lack of
other papers on the effects of fasting on
circulating aa concentrations of wild mammals.
The only aa in the raccoon dog plasma with
continuously elevated levels during the fast was
Gln (III). High circulating Gln concentrations
have been previously measured from the fooddeprived dog (Miller et al., 1983), a relative of
the raccoon dog. The fasted raccoon dogs had
20 % higher Gln concentrations than the fed
animals throughout the fast (III), indicating that
Gln may be crucial for successful wintering of
the species. Gln is an important carrier of
carbon, nitrogen and energy between organs
(Curthoys & Watford, 1995). It also
participates in liver urea synthesis, kidney
ammoniagenesis and hepatic and renal
gluconeogenesis. Furthermore, many cells use
Gln as a respiratory fuel. It is also an important
donor of α-amino group to other aa and
participates in the syntheses of purine and
pyrimidine nucleotides, aminosugars and
glycoproteins (Mathews & van Holde, 1996).
Muscle tissue synthesizes Gln via αketoglutarate and Glu from branched-chain aa
that enter the tricarboxylic acid cycle after
protein breakdown, and releases Gln into
circulation for the use of other tissues
(Goldberg & Chang, 1978). The high Gln
concentrations of the raccoon dogs may not
necessarily derive from the muscle proteolysis
(III), as it has been observed in rats (Espat et
al., 1993) and dogs (Miller et al., 1983;
stable or reduced Chol levels exist (black bear:
Hellgren et al., 1990; European brown bear:
Hissa et al., 1994). The lowered Chol
concentrations of the fasting raccoon dogs (III)
may have derived from suppressed Chol
absorption from the gut or from decreased
hepatic Chol synthesis.
The blood Chol and TG levels of the gray
wolf follow the pattern of the raccoon dog and
reduce with food deprivation (DelGiudice et
al., 1987). Also the domestic dog has stable or
reduced TG concentrations due to fasting (de
Bruijne et al., 1981). The fasting-induced
reduction in blood TG levels may be a typical
feature to canids, as also TG levels of a
mustelid, the American badger, increase during
fasting (Harlow & Seal, 1981).
6.3.3 Effects of fasting on protein metabolism
As the wild raccoon dog can spend up to five
months in superficial winter sleep in the
northernmost areas of its geographical
distribution (Siivonen, 1972), it is possible that
its fat stores become severely depleted in late
winter. As the result the animal would be
forced to enter the phase III of fasting and to
start mobilizing its muscle proteins for energy.
In fact, proteins are the fuel, whose depletion
limits survival during starvation due to their
essential role in nervous tissue, muscle
contractility and as enzymes (Cahill, 1976).
However, the concentrations of the most
measured aa (19/26, 73 %) were lower in the
fasted raccoon dogs than in the fed animals
throughout the fast or during a particular
fasting phase (III). The Taa levels were lower
in the fasting animals from the fourth wk
without food to the end of the fast. When Taa
were grouped to essential and nonessential aa,
the fasted raccoon dogs had lower essential aa
concentrations during the whole fast. On the
contrary, they had lower nonessential aa levels
73
in denning ursids (Nelson et al., 1983b, 1984;
Franzmann & Schwartz, 1988; Hellgren et al.,
1990; Hissa et al., 1994) and in fasting gray
wolves (DelGiudice et al., 1987). As their
blood urea levels decrease simultaneously, U/C
ratios of these species reduce during fasting.
On the contrary, there are no changes in blood
creatinine levels or U/C ratios of the fasting
American badger (Harlow & Nelson, 1990),
whereas in the northern elephant seal, both
blood urea-N and creatinine concentrations
reduce during food deprivation (Costa & Ortiz,
1982). Although the blood U/C ratio decreases
in several mammalian species during fasting
(black-tailed
prairie
dog
Cynomys
ludovicianus: Pfeiffer et al., 1979; gray wolf:
DelGiudice et al., 1987), so far only a few
species (i.e. ursids and the raccoon dog) have
been demonstrated to reach ratios ≤ 10.
It is possible, if unproven, that the raccoon
dog could be able to recycle urea-N for de
novo synthesis of aa and proteins (see for the
black bear: Lundberg et al., 1976; Wolfe et al.,
1982; Nelson et al., 1983b). According to the
results of Nelson et al. (1973, 1975), urine
continues to be formed in overwintering black
bears, but the animals are able to recycle ureaN. The wall of their urinary bladder reabsorbs
urea and water making urination unnecessary.
This phenomenon of reabsorption has also been
observed in the dog with low urine flows
(Levinsky & Berliner, 1959). Ursid urea has
been suggested to diffuse across their gut
epithelium into the gut lumen, where it could be
degraded by bacterial urease to NH3 and CO2
(Nelson et al., 1975). The exploration of this
hypothesis in the raccoon dog naturally requires
further studies. The NH3 produced in the gut
could be transported via the portal circulation
to the liver, which could utilize it for the
production of Gln to be released into
circulation for the use of other tissues (III).
Abumrad et al., 1990) that the liver becomes a
net releaser of Gln during fasting. The release
of a sufficient amount of Gln from the liver
could lead to lower requirements of muscle
proteolysis. For instance, in dogs which starved
for four days, the amount of Gln released from
the liver was sufficient to meet the metabolic
demands of the kidneys and the gut for this aa
(Abumrad et al., 1990).
The concentrations of urea, which is the
primary end product of protein catabolism in
mammals (Mathews & van Holde, 1996), were
30-60 % lower in the fasted raccoon dogs
throughout the fast (III). Conversely, Asikainen
et al. (2002) have not found any differences in
blood urea concentrations between fed (n=5)
and fasted raccoon dogs (n=5) after eight wk of
fasting. Suppressed blood urea or urea-N
concentrations
have,
however,
been
documented in bears during their winter sleep
(black bear: Nelson et al., 1983b; Palumbo et
al., 1983; Franzmann & Schwartz, 1988;
European brown bear: Hissa et al., 1994,
1998b). Furthermore, gray wolves (DelGiudice
et al., 1987), American badgers (Harlow &
Seal, 1981) and northern elephant seals (Costa
& Ortiz, 1982) show a fasting-induced
reduction in their blood or urine urea, urea-N
or total-N concentrations. On the contrary, the
king penguin experiences elevated blood urea
levels during its seasonal fast (Cherel et al.,
1988b). This difference may be due to the fact
that uric acid instead of urea is the major end
product of avian nitrogen metabolism, and the
uric acid levels of penguins are not increased
until the phase III of fasting. The continuously
lower levels of plasma urea and Arg together
with occasionally reduced Orn and Cit
concentrations of the raccoon dogs may
indicate that their urea synthesis was downregulated (III).
Besides the raccoon dogs, increased blood
creatinine concentrations are also encountered
74
kidney glycogen stores. After four wk without
food, the raccoon dogs were resistant to
fasting-induced hypoglycemia probably via
stimulation
of
hepatic
and
renal
gluconeogenesis. Glycerol may serve as an
important substrate for glucose production in
fasting raccoon dogs. Previously, de Bruijne &
de Koster (1983) have shown that the rate of
canine glycogenolysis is highest during the first
three days of fasting. The dog is, however, able
to maintain normoglycemia due to high rates of
gluconeogenesis, possibly using glycerol as a
substrate. Glycerol has been suggested to be an
important substrate for gluconeogenesis also in
black bears (Ahlquist et al., 1976).
In contrast to the glucose concentrations,
the insulin levels of the fasted raccoon dogs
were lower than the levels of the fed animals
after 2-6 wk of fasting (III). The lowered
insulin levels were probably required for
efficient WAT mobilization and glucose sparing
(Cahill, 1976). They may also have roles in
several other aspects of the response to fasting
such as in the stimulation of gluconeogenesis
and ketogenesis (Kaloyianni & Freedland,
1990) providing energy for the glucose- and
ketone-dependent
tissues and in the
suppression of MR (Rothwell et al., 1983)
leading to energy preservation. Lowered blood
or pancreatic insulin levels have been
previously observed in fasting dogs (de Bruijne
et al., 1981) and gray wolves (DelGiudice et
al., 1987) and in hibernating golden-mantled
ground squirrels (Bauman et al., 1987). On the
contrary, insulin concentrations of the black
bear do not respond to winter sleep (Palumbo
et al., 1983; Herminghuysen et al., 1995). In
this respect the smaller raccoon dog may use
more numerous metabolic pathways to recruit
TG hydrolysis in WAT (III).
Although glucagon is able to stimulate
glycogenolysis, gluconeogenesis (Cherrington
et al., 1981), lipolysis (Carlson et al., 1993)
Although protein degradation and whole
body or skeletal muscle protein contents were
not determined from the fasting raccoon dogs,
it can be stated with certainty that they had not
entered the phase III of fasting during a twomonth food deprivation. The lowering of their
circulating aa concentrations may have resulted
from the reduced absorption of aa due to
decreased protein intake, but there were several
other parameters such as 3-MH, urea and U/C
ratios together with the constant rate of weight
loss clearly indicating a steady level of protein
catabolism during fasting.
6.3.4 Effects of fasting on glucose and
weight-regulatory hormones
6.3.4.1 Glucose,
concentrations
insulin
and
glucagon
The fasted raccoon dogs experienced a decline
in their plasma glucose levels during the first
two wk of food deprivation, and they had
lower glucose concentrations than the fed
animals after four wk of fasting (III).
Thereafter the glucose levels of the fed and the
fasted raccoon dogs did not differ from each
other confirming previous results (Asikainen et
al., 2002). Palumbo et al. (1983) have
observed a similar pattern of changes in blood
glucose concentrations of denning black bears.
Furthermore, constant blood glucose levels
have been measured from fasting gray wolves
(DelGiudice et al., 1987), whereas the
European hedgehog (Cherel et al., 1995) is
hypoglycemic during hibernation. Normoglycemia may be one factor enabling the rapid
arousal of overwintering raccoon dogs and
bears from their dens when required (III).
The documented reduction in the blood
glucose concentrations of the raccoon dogs
during the first month of fasting could have
resulted from the depletion of their liver and
75
and ketogenesis (Beylot, 1996), fasting did not
elevate the glucagon concentrations of the
raccoon dogs (III). This may have enabled their
constant rate of protein catabolism during
fasting (Peterson et al., 1963). Neither have
Palumbo et al. (1983) observed any significant
changes in blood glucagon concentrations of
denning black bears, and also the dog is able to
maintain normoglycemia without hyperglucagonemia (de Bruijne et al., 1981). On the
contrary, glucagon concentrations of king
penguins increase during seasonal fasting
(Cherel et al., 1988b). Food deprivation,
however, increased the glucagon-insulin ratios
of the raccoon dogs (III; see also Cahill, 1976
for humans), manifesting a possible role for
glucagon in their response to fasting.
al., 1979; American badger: Harlow & Seal,
1981; gray wolf: DelGiudice et al., 1987; king
penguin: Cherel et al., 1988b; European brown
bear: Hissa et al., 1994; arctic fox: Fuglei,
2000). Also the domestic dog shows decreasing
blood T3 levels during fasting (de Bruijne et al.,
1981). As metabolism of raccoon dogs can
decrease by 25 % during the winter sleep
(Heptner et al., 1974), the observed
suppression of T3 levels may function as an
adaptation to reduce energy requirements (III).
Apparently it is not the peripheral
monodeiodination of T4 to T3 but the secretion
activity of the thyroid gland that is downregulated in food-deprived raccoon dogs.
6.3.4.2 Cortisol
concentrations
Humans (Kolaczynski et al., 1996) and
laboratory rodents (Hardie et al., 1996)
experience a rapid drop in their leptin levels
with fasting, and this decrease is not directly
proportional to the amount of lost fat. Ghrelin
(Tschöp et al., 2000) and GH concentrations
(Roth et al., 1963), on the other hand, increase
due to food withdrawal. In contrast to studies
performed with nonseasonal mammals, the
concentrations of leptin, ghrelin or GH were
not affected by fasting in the raccoon dog (I).
In fact, the leptin and GH levels of the fed and
the fasted animals were virtually identical
during the whole two-month period without
food. Although the fasted raccoon dogs had
lower ghrelin levels during food deprivation,
this difference was present already prior to the
fast, and thus resulted from the heterogeneity
of the study groups and not from fasting.
Food deprivation lowers circulating leptin
concentrations of some seasonal mammals
(golden hamster: Schneider et al., 2000; sheep:
Marie et al., 2001; pup of Antarctic fur seal:
Arnould et al., 2002). However, the fasting
periods in these experiments have been
and
thyroid
6.3.4.3 Leptin, ghrelin and GH concentrations
hormone
The plasma cortisol concentrations were lower
in the fasted raccoon dogs during 4-8 wk of the
fast (III). Also the European ground squirrel
(C. citellus) has lowered blood cortisol levels
but only during the first half of its seasonal
hibernation (Shivatcheva et al., 1988). In
contrast, black bears experience elevated
cortisol concentrations during the winter sleep,
which has been suggested to play a role in the
stimulation of ursid lipolysis (Harlow et al.,
1990; Hellgren et al., 1993). Glucocorticoids
may play important roles also in the fat and
protein utilization of northern elephant seals
(Ortiz et al., 2001a,b) and king penguins
(Cherel et al., 1988b) showing increased blood
cortisol and corticosterone concentrations due
to fasting.
The fasted raccoon dogs had lower
circulating T3 and T4 concentrations during 2-6
and 2-4 wk of fasting, respectively (III). This
confirms previous data obtained from several
seasonally fasting animals (black bear: Azizi et
76
relatively short (12 hr-5 days). Different results
have been obtained in studies with long-term
food deprivation. Nieminen et al. (2001a) have
not found any influence of fasting on plasma
leptin levels of raccoon dogs during a total
three-wk fasting period, although leptin
concentrations of blue foxes tended to
decrease. Wild arctic foxes, on the other hand,
have stable leptin concentrations during a 13day fast (Fuglei et al., unpubl.). Neither are
leptin concentrations of Steller sea lions
(Eumetopias jubatus) affected by complete
fasting (subadults: 14 days; pups: 2.5 days) or
food-restriction (subadults: 28 days; Rea &
Nagy, 2000). Furthermore, postweaned pups of
the northern elephant seal have constant leptin
concentrations after a 35 day fast (Ortiz et al.,
2001b).
It could be argued that also the leptin levels
of the raccoon dogs decline during short-term
fasting, but thereafter the concentrations
rebound to normal levels, when food
deprivation proceeds. The leptin concentrations
of the raccoon dogs were, however, measured
after three days of fasting (on Nov 29th 2000)
and they were identical to the levels of the fed
group (I). Studies conducted on patients with
anorexia nervosa have also shown that leptin
levels of undernourished humans are on a very
low level and do not rebound until a weight
gain of a few kg (Hebebrand et al., 1997).
These findings indicate that leptin levels of
humans and many wild mammals differ in their
response to fasting. The main function of leptin
in the raccoon dog does not seem to be an
indicator of body fat stores (I), which is
supported by abundant data obtained from
other seasonal carnivores (mink: Nieminen et
al., 2000; Steller sea lion: Rea & Nagy, 2000;
blue fox: Nieminen et al., 2001a; northern
elephant seal: Ortiz et al., 2001a,b; Antarctic
fur seal: Arnould et al., 2002). In contrast, the
relatively nonseasonal dog has gradually
decreasing leptin concentrations during
prolonged fasting (Ishioka et al., 2002).
As the raccoon dog experiences extensive
seasonal and annual fluctuations in its food
abundance, it is likely that the species has
evolved endogenous circannual rhythms in its
metabolism in response to unpredictable
seasonal changes in food availability. The
wintertime food scarcity poses a real threat to
its survival and for this reason the raccoon dog
has evolved to sleep through the coldest part of
the winter with its considerable fat reserves.
Our data shows that a long period of
wintertime fasting is a normal physiological
phenomenon for the species not affecting the
levels of leptin, ghrelin or GH (I). In fact the
concentrations of several weight-regulatory
hormones follow the same seasonal patterns in
animals with a total fast or with voluntarily
reduced wintertime food intake (I; III).
6.3.5 Endocrine response to fasting in canids
vs. ursids
The response to fasting seems to be a relatively
well-preserved phenomenon among canids
(Table IV). On the contrary, the raccoon dog
and the black bear differ somewhat in their
responses, although the both species utilize
winter sleep as an adaptational strategy to
overcome the cold season. Compared to deep
hibernators, Tb and MR decrease only
moderately in over-wintering raccoon dogs and
bears (Heptner et al., 1974; Nelson et al.,
1983b; Watts & Cuyler, 1988; Asikainen et al.,
2002). The bears are, however, anorectic in the
winter and even after the denning period and do
not eat, drink, urinate or defecate for 3-7
months (Nelson et al., 1983a).
Though the winter sleep of the raccoon dog
can last for several months in the northernmost
areas of its geographical distribution, the
seasonal rest is facultative with occasional
77
arousal and foraging during the warmer periods
(Siivonen, 1972). As a consequence, the
raccoon dog probably eats snow, urinates and
defecates during the winter (J. Asikainen, pers.
commun.). On the contrary, the bear fulfills its
water requirements with metabolic water
derived from fat mobilization (Nelson et al.,
1983a). Its bladder transports water and Nwastes back into the circulation at a rate about
equal to their entry into the bladder and thus no
urination occurs.
Moreover, bears deliver and nurse their cubs
during the winter rest, but the raccoon dog
female gives birth in spring after arousal
(Siivonen, 1972). There are also clear
differences in the weight loss between the
species, the rate being almost twice as high in
the smaller raccoon dog (III) than in the black
bear (Nelson et al., 1973). As there are so
many differences in the general patterns of the
winter rests between these species, it is
understandable that also the endocrine
responses to fasting have their own special
characteristics.
dogs do not experience an anorectic phase after
arousal from winter lethargy.
After two wk of re-feeding, the BMs and
adiposity (BMIs) of the fasted raccoon dogs
had increased by 6 %. Their blood TG and FFA
concentrations were lower than in the animals
fed throughout the winter, indicating transport
of lipids to the depleted fat stores (III). Also
the circulating TL levels of the fasted raccoon
dogs decreased, but not until March. Our
findings confirm the previous results of Nelson
et al. (1973) showing decreased blood TG
levels in black bears after arousal from winter
sleep. Also American badgers have reduced
blood FFA concentrations after re-feeding
(Harlow & Seal, 1981).
Although the glucose concentrations of the
fasted raccoon dogs were stable, their insulin
levels increased twofold due to re-feeding
presumably stimulating the restoration of sc fat
stores (III). Feeding probably activated the
hypothalamus-pituitary-thyroid axis (Hugues et
al., 1983; Tveit & Larsen, 1983), as indicated
by the increased blood T3 levels and T3T4 ratios
of the raccoon dogs (III). Also the black bear
(Azizi et al., 1979), the American badger
(Harlow & Seal, 1981) and the arctic fox
(Fuglei, 2000) have elevated blood thyroid
hormone levels after re-feeding.
Although the levels of weight-regulatory
hormones did not respond to fasting (I), the
vernal leptin and GH concentrations were
occasionally lower and the ghrelin levels higher
in the fasted animals after the fasting had
ceased (II). These findings could be connected
to the differences in the appetite, BM loss and
lipolytic rates between the study groups during
the spring (II-III). As high leptin
(Pelleymounter et al., 1995) and GH
concentrations (Andres et al., 1991) can reduce
food intake of mammals and high ghrelin levels
increase the appetite (Tschöp et al., 2000), the
lower vernal leptin and GH concentrations
6.3.6 Influence of re-feeding on energy
metabolism
In addition to the three seasonal phases of
metabolism (hyperphagia, winter rest and
normal activity) observed in bears and raccoon
dogs, bears experience a fourth annual
physiological state (Nelson et al., 1983a),
which may be absent in the raccoon dog. After
leaving their dens in spring, bears are usually
anorectic for 2-3 wk. Though they are active
during this “walking hibernation”, their
metabolism seems to remain in the state of
winter rest. In contrast to bears, the raccoon
dogs started eating immediately after the
cessation of their experimental winter sleep (I;
III), and it is thus probable that wild raccoon
78
6.4
together with the higher ghrelin levels may be
associated with the higher appetite of the fasted
raccoon dogs compared to the fed animals (IIIII). Moreover, the lower leptin and GH
concentrations and the higher ghrelin levels
may be connected to lower rates of vernal fat
mobilization, as leptin (Reidy & Weber, 2000)
and GH (Richelsen, 1997) are known to have
lipolytic actions, whereas high ghrelin levels
suppress mobilization of fat (Tschöp et al.,
2000).
In conclusion, the clear seasonal changes in
the circulating lipid concentrations of the
raccoon dogs emphasize the importance of fat
metabolism in the seasonal adaptation of the
species (III). Insulin seems to play a key role in
permitting the efficient WAT mobilization
during the seasonal rest. Although the leptin,
ghrelin or GH concentrations did not change in
response to fasting, the leptin and GH levels
clearly increased and the levels of ghrelin
decreased during the winter (I). This indicates
that they may work in synergy to stimulate
wintertime fat mobilization and to provide the
animals with a satiety signal during their
seasonal rest. Suppression of the activities of
the thyroid gland and the adrenal cortex (III)
may be important factors limiting muscle
proteolysis
during
nutritional
scarcity
(Goldberg et al., 1980; Simmons et al., 1984;
Angerås & Hasselgren, 1985). Furthermore,
the high wintertime GH concentrations (I) may
also belong to the physiological mechanisms
preventing protein wasting (Vann et al., 2000)
and inducing sleepiness in the raccoon dogs
(Lachmansingh & Rollo, 1994). A long period
of wintertime fasting is a normal physiological
phenomenon for the species only pronouncing
the seasonal changes in the weight-regulation
observed in the fed animals (I; III).
Melatonin in body weight-regulation
6.4.1 Effects of melatonin on BM, BMI and
food intake
Exogenous melatonin treatment had only
modest effects on the autumnal BM and BMI
gains of the raccoon dogs (I-III). Melatonin,
however, decreased the BMIs of the animals in
Nov (I; III). Continuous melatonin treatment
introduced in July has previously decreased
BMs of male raccoon dogs in Dec (Xiao,
1996). In contrast to changes in BMs,
melatonin induced a clear stimulation in the
voluntary food intake of the raccoon dogs in
the autumn and a suppression in the appetite
during the winter (I; III). The vernal BMs and
voluntary food intake were not influenced by
exogenous melatonin, but a clear increase in the
BMIs of the MEL females was observed in
July-Aug 2001 (II-III). Previously Xiao (1996)
did not find any melatonin-induced effects on
BMs of male raccoon dogs, when the implants
were administered in March and BMs were
followed from April to Aug.
Melatonin treatment during LD has been
shown to stimulate food intake and BM gain as
fat in several seasonal mammals (golden
hamster: Wade & Bartness, 1984; garden
dormouse: Le Gouic et al., 1996; tundra vole:
Mustonen et al., 2002b). Our results show that
continuous melatonin treatment, if applied early
enough, increases the fat mass of the raccoon
dog in autumn and suppresses it in early winter
(I-III), thus advancing the natural BM cycle of
the species (Korhonen, 1987). A melatonininduced advancement of the seasonal pattern of
weight gain and loss has been previously
observed in the red deer (Suttie et al., 1992).
The increasing endogenous melatonin secretion
after the summer solstice (Xiao, 1996) probably
triggers the autumnal accumulation of fat and
BM gain in the raccoon dog (II-III), as
79
suppression could partly explain the higher
voluntary food intake observed in the MEL
group (I). On the other hand, the leptin
concentrations of the rats were not influenced
by one-month melatonin or LL treatments (IV).
Chronic melatonin administration through
drinking water has previously reduced blood
leptin concentrations of rats (Rasmussen et al.,
1999; Wolden-Hanson et al., 2000). In
contrast, plasma leptin levels of the mink
(Mustonen et al., 2000) and WAT and BAT
leptin gene expression of the garden dormouse
(Ambid et al., 1998) increase due to exogenous
melatonin. It is probable that the influence of
melatonin on leptin synthesis and release is
species- and season-specific.
Both leptin peaks observed during the
autumnal study period were advanced by one
wk in the MEL raccoon dogs (I). Also their
first GH peak was advanced by melatonin in a
similar way. These findings are in concordance
with results of Xiao (1996), who has observed
in male raccoon dogs that melatonin treatment
in July advances physiological changes such as
the autumn moult and testicular recrudescence
that occur normally during SD. The raccoon
dogs of the studies I-III also showed
melatonin-induced advancements in the
maturation of winter pelage and in the onset of
reproductive activity (Asikainen et al., 2003).
Similar results have been previously obtained
from other canids and mustelids (mink: Allain
& Rougeot, 1980; DiGregorio & Murphy,
1987; silver fox: Forsberg & Madej, 1990;
ferret: Nixon et al., 1995). According to the
results of study I, besides advancing the moult,
testicular activity and estrus, autumnal
melatonin treatment seems to advance
metabolic changes connected to the wintering
of the raccoon dog. In concordance with these
results, Suttie et al. (1992) have observed in
red deer that exogenous melatonin treatment
from early summer is able to advance the
suggested previously for the mink (Valtonen et
al., 1995). The animals may receive
photoperiodic cues for BM gain before midAug, explaining the observed lack of
melatonin-induced effects on their BMs and
BMIs during the early autumn 2000 (I; III).
The BMs, BMIs and food intake of the rats
were not affected by the one-month exposure
to melatonin or LL (IV-V), which is known to
suppress their melatonin synthesis (Klein &
Weller, 1970). According to Peschke et al.
(1987), BM and food intake of the laboratory
rat follow seasonal rhythms being highest in
spring and summer and lowest in autumn and
winter. For this reason, it could be assumed
that melatonin treatment would induce a
decrease in their BMs, adiposity and appetite,
whereas LL would have opposite effects. In
fact, Rasmussen et al. (1999) and WoldenHanson et al. (2000) have induced a reduction
in BMs of rats with chronic melatonin
treatment through drinking water, whereas
melatonin did not affect food intake or total
body adiposity of their animals. The lack of
melatonin- and LL-induced effects in our
experiment may be due to the subacute
exposure (IV-V). Similar results have also been
obtained with rats by Dark et al. (1980) after
eight wk of melatonin or LL exposure. The
lack of influence may derive from the
degeneration of the photoperiodic machinery
for body weight-regulation of the laboratory
rat.
6.4.2 Effects of melatonin
regulatory hormones
on
weight-
Autumnal
melatonin
treatment
acutely
decreased the plasma leptin concentrations of
the raccoon dogs measured three wk after the
implantation (I). As low leptin levels are
hypothesized to lead to a state of positive
energy balance (Friedman, 1997), this
80
increase by prenatal melatonin exposure (Díaz
et al., 2000). Ghrelin is secreted primarily by
the stomach (Kojima et al., 1999), which is also
a potential location of melatonin production
and binding (Bubenik et al., 1993). Melatonin
binding sites are also located on another ghrelin
production site, the hypothalamic ARC
(Weaver et al., 1988; Kojima et al., 1999). It
remains to be established whether melatonin
affects ghrelin concentrations peripherally,
centrally or by both mechanisms. Apparently
the effects of melatonin on ghrelin levels are
species- as well as season-specific.
During the vernal study period the MEL
male raccoon dogs had lower leptin
concentrations than those of the SHAM males
at the end of March (II). The opposite was
observed in the females, as the leptin levels of
the MEL group were higher than those of the
SHAM group in early March i.e. the time when
most of the MEL females were pregnant. As
increased leptin levels are encountered during
pregnancy of several mammalian species
(human: Butte et al., 1997; baboon: Henson et
al., 1999; big brown bat: Kunz et al., 1999;
sheep: Ehrhardt et al., 2001), the higher leptin
concentrations of the MEL females (II) could
have been an indicator of the advancement of
their mating season (Asikainen et al., 2003).
Also the vernal rise in the ghrelin levels of
the females as well as the drop and the
subsequent rise in their GH concentrations
advanced by 2-3 wk due to melatonin (II). In a
previous study vernal melatonin treatment has
slowed down testicular regression of male
raccoon dogs, stimulated the growth of their
underfur and inhibited the growth of guard
hairs (Xiao, 1996). The raccoon dogs of the
present experiment were implanted with
melatonin twice, in Aug 2000 and in Feb 2001
(I-III). It could be assumed that the autumnal
implantation advanced the autumn moult and
seasonal rhythms of reproduction and weight-
seasonal rise and drop of their blood IGF-I
concentrations.
The MEL raccoon dogs had periodically
higher GH concentrations than the SHAM
animals during Nov-Dec (I). A stimulatory
effect of melatonin on blood GH levels has
been previously observed in humans (Smythe &
Lazarus, 1974; Valcavi et al., 1987). On the
contrary, the GH concentrations of the rats
were not influenced by melatonin or LL
treatments after a four-wk exposure (IV).
Previously GH release from rat pituitary cells
has been inhibited with melatonin (Griffiths et
al., 1987) or it has not responded to the
treatment (Valcavi et al., 1987).
The seasonal rhythm of plasma ghrelin
concentrations of the raccoon dogs was not
affected by melatonin during the autumn and
early winter (I). Instead, melatonin seemed to
accentuate the seasonal changes in the ghrelin
levels by increasing the autumnal levels and by
decreasing the concentrations during the
winter. When the whole ghrelin data of the
raccoon dogs were pooled together, the MEL
group had higher ghrelin concentrations than
the SHAM group. On the contrary, the
melatonin treatment of the rats induced a 35 %
reduction in their ghrelin concentrations, and
this suppression was independent of the
prevailing photoperiod (IV).
It can be speculated that the increasing
endogenous melatonin secretion in the autumn
(Xiao, 1996) could function as a signal that
enhances ghrelin secretion of the raccoon dog
leading to seasonal hyperphagia. This is
supported by previous results showing
increased ghrelin concentration and food intake
in tundra voles after exogenous melatonin
treatment (Mustonen et al., 2002b). The
ghrelin-induced stimulation of appetite is
probably mediated by hypothalamic NPY
(Nakazato et al., 2001; Shintani et al., 2001),
concentrations of which have been shown to
81
(IV). The physiological significance and the
mechanism behind this interaction require
further studies to be revealed.
regulation, whereas the vernal implantation
delayed the spring moult and induced the
accumulation of fat in the autumn 2001 (I-III;
Asikainen et al., 2003). Sheep and goats also
require LD treatment before a melatonininduced advancement of their reproductive
cycles can be demonstrated (Arendt, 1995).
The gender differences in the vernal responses
of weight-regulatory hormones to melatonin
may be associated with reproduction and with
the advancement of the mating season by
autumnal melatonin treatment (II).
Accumulation and mobilization of fat are
crucial for the survival of the raccoon dog
through the winter, as WAT provides metabolic
energy and thermal insulation for the animals
during the coldest part of the year (Korhonen,
1987). According to the results of the raccoon
dog experiment (I), leptin, ghrelin and GH may
play roles in the autumnal accumulation of fat,
participate in the physiological transition from
autumnal anabolism to wintertime catabolism
and stimulate the mobilization of fat during the
winter rest. In addition, the levels of these
peptides do not seem to be regulated by
temporally fluctuating food availability, but
they appear to follow seasonal rhythms
entrained by photoperiod via the darkness
hormone melatonin. The clear melatonininduced effects on the weight-regulatory
hormones in comparison to the lack of effects
of a total fast on the leptin, ghrelin and GH
levels emphasizes the importance of
photoperiod in the control of seasonal weightregulation of the species (I-II).
The laboratory rat, on the other hand, does
not experience cold seasons but lives in
continuously favorable circumstances. Perhaps
for this reason photoperiod and melatonin have
no subacute influence on its BM, adiposity,
energy intake or leptin and GH concentrations
(IV-V). However, the blood ghrelin levels of
the rat clearly respond to exogenous melatonin
6.4.3 Effects of melatonin and LL on
intermediary metabolism
6.4.3.1 Liver carbohydrate metabolism
Because the liver functions as the centre of the
intermediary metabolism (Harris, 1986), some
metabolic effects of melatonin are most
probably mediated by it. In fact, melatonin
binding sites have been located in the liver
(Acuña-Castroviejo et al., 1994) and Poon et
al. (2001) have shown that melatonin may
affect glucose metabolism directly through
hepatic receptors. The melatonin-induced
effects on the liver and kidney energy
metabolism were minor in the raccoon dogs
(Nevalainen, 2002; unpubl. data), whereas the
rat responded to the both melatonin and LL
exposures (V).
One could speculate that the lack of
melatonin-induced effects on the raccoon dogs
was caused by the long time interval between
the melatonin implantation and sampling, being
two months for the males and six for the
females (Nevalainen, 2002; unpubl. data). The
diurnal plasma melatonin concentrations were,
however, significantly higher in the MEL group
during the whole vernal study period (II-III),
and for this reason, melatonin-induced changes
in liver metabolism could have been possible.
Moreover, liver glycogen content and enzyme
activities of wild raccoon dogs have been noted
to fluctuate between seasons (Asikainen et al.,
unpubl.). Besides BM, BMI, appetite and
leptin, ghrelin and GH concentrations (I-III),
also the annual rhythms of liver metabolism
could have been under photoperiodic control.
However, only the liver lipase esterase
82
(liver or kidneys) to release free glucose from
glucose-6-phosphate into the circulation
(Harris, 1986). High activity rates could be
expected to be encountered during fasting in
association with stimulated glycogenolysis and
gluconeogenesis.
Autumnal
melatonin
treatment has previously increased liver G-6Pase activities of female minks probably as an
adaptation to wintertime food scarcity
(Nieminen et al., 2001b). On the contrary,
hepatic G-6-Pase activities of female tundra
voles have decreased by exogenous melatonin
(Mustonen et al., 2002b). The activities of the
rats could have been unresponsive to melatonin
and LL, as the animals were in good nutritional
status and continued to be fed ad lib (V).
activities of the raccoon dogs responded to
melatonin (Nevalainen, 2002).
In contrast to the raccoon dogs, the liver
energy metabolism of the rats was clearly
influenced by the one-month melatonin
treatment and 24L exposure (V). Exogenous
melatonin did not consistently affect their liver
glycogen content (see also Fabiś et al., 2002),
but it increased the activity of glycogen
phosphorylase (V), the regulatory enzyme of
glycogenolysis (Harris, 1986). This indicates a
melatonin-induced stimulation of glycogen
synthesis and turnover rate in the liver (V).
These results differ from data obtained
previously from tundra voles (Mustonen et al.,
2002b) and rats (Mazepa et al., 2000), in which
melatonin had no effects or it increased liver
glycogen stores, respectively.
The 24L treatment, on the other hand,
decreased the liver glycogen contents of the
rats (V). According to Kaminsky et al. (1984),
the hepatic glycogen stores of laboratory rats
are minimal at the end of photophase, when
animals have fasted and at the beginning of
scotophase, when they are active. Liver
glycogen concentrations increase around
midnight, when digested carbohydrates are
deposited as glycogen. Rats are normally active
during scotophase (Hirsimäki, 1996), but if the
animals are maintained in LL, their activity
rhythms disrupt and dissociate into several
periods (Chesworth et al., 1987). It is thus
possible that the rats maintained in LL had
several activity peaks and they continuously
utilized the glucose obtained from the food, and
for this reason could not restore their liver
glycogen stores (V).
The liver G-6-Pase activities of the rats were
not affected by melatonin or LL. Neither have
Daniels et al. (1995) found any influence of
melatonin on carbon tetrachloride-suppressed
microsomal G-6-Pase activities of rat livers. G6-Pase activity indicates the ability of the tissue
6.4.3.2 Liver lipid metabolism
The activity of liver lipase esterase, indicating
the mobilization of lipids, decreased with
exogenous melatonin in three out of four rat
study groups (V). This may indicate that their
liver TG were not hydrolyzed but transported
and stored elsewhere in the body. In fact,
several studies on seasonal mammals have
demonstrated the fat storing effect of melatonin
(golden hamster: Wade & Bartness, 1984;
garden dormouse: Le Gouic et al., 1996). Also
the MEL male raccoon dogs had lower lipase
esterase activities in their livers than the SHAM
males (Nevalainen, 2002).
According to Asikainen et al. (unpubl.),
liver lipase esterase activities of wild raccoon
dogs are lower during the autumnal
accumulation of fat than during the rest of the
year. For this reason, the suppression of the
enzyme activities due to melatonin (Nevalainen,
2002) can be interpreted as an establishment of
the autumnal lipase esterase activity levels by
artificially shortened daylength. A similar
suppression of lipase esterase activity by
melatonin has also been observed in livers of
83
male minks preparing themselves for the winter
by storing sc fat (Nieminen et al., 2001b). On
the contrary, the hepatic lipase esterase
activities of tundra voles are not influenced by a
one-month melatonin treatment (Mustonen et
al., 2002b). The exposure to LL increased the
liver lipase esterase activities in the control
male rats, but decreased them in the melatonintreated males (V). Previously 24L has
decreased liver lipogenesis in chickens, while
exogenous melatonin had the opposite effect
(Osei et al., 1989).
the male rats by LL. The kidney lipase esterase
activities increased due to melatonin in the 24L
maintained rats, but decreased in the 12L:12D
kept females. Moreover, LL increased renal
lipase esterase activities in the melatonintreated rats. Previously, a similar one-month
melatonin exposure has decreased lipase
esterase activities, but increased glycogen
stores and G-6-Pase activities in the kidneys of
tundra voles (Mustonen et al., 2002b).
According to these results, the melatonininduced effects on kidney energy metabolism
seem to be species-specific. Renal actions of
melatonin may be mediated by the Mel1a
receptor subtype localized on the basolateral
membrane of proximal tubules (Song et al.,
1997).
In summary, both exogenous melatonin and
24L treatments seem to have a strong
regulatory effect on rat liver and kidney energy
metabolism (V). The LL exposure stimulates
the carbohydrate metabolism of the liver,
whereas its lipid metabolism remains stable. On
the other hand, exogenous melatonin elevates
the utilization of hepatic carbohydrates but
suppresses mobilization of fat. The observed
differences in the renal energy metabolism
suggest that changes from the normal
circulating melatonin concentrations to either
direction increase the utilization of kidney
glycogen. Furthermore, the suppression of
hepatic lipid mobilization of the male raccoon
dogs by melatonin (Nevalainen, 2002) provides
further support for the hypothesis that
melatonin promotes the utilization of
carbohydrates instead of fat (John et al., 1990).
6.4.3.3 Kidney energy metabolism
Melatonin binding sites have been localized in
kidneys of birds and mammals (duck A.
platyrhynchos: Song et al., 1992; human: Song
et al., 1995), and several experiments have
shown that melatonin can influence the
functions of mammalian kidneys. Melatonin
treatment affects e.g. water consumption, urine
production, its Na+ and K+ concentrations,
circulating
antidiuretic
hormone
levels
(Richardson et al., 1992), blood pressure
(Kawashima et al., 1987), plasma renin
activities (Acuña et al., 1984) and glomerular
filtration rates (Tsuda et al., 1995). Moreover,
urine volume of the raccoon dog decreases due
to exogenous melatonin (Xiao, 1996). As
melatonin has such diverse effects on renal
function, it presumably affects kidney energy
metabolism, as well.
Exogenous melatonin treatment did not
affect the kidney glycogen content or glycogen
phosphorylase, G-6-Pase or lipase esterase
activities of the male or the female raccoon
dogs (Nevalainen, 2002; unpubl. data). On the
contrary, both the melatonin and LL treatments
decreased the kidney glycogen stores and G-6Pase activity levels of the rats (V). The
glycogen phosphorylase activities were not
influenced by melatonin, but they decreased in
6.4.4 Effects of melatonin on glucose, insulin,
thyroid hormone, lipid and aa levels
The role of melatonin in mammalian glucose
and insulin regulation is poorly understood.
The plasma glucose concentrations of the
84
indicate increased metabolic output perhaps
counteracting the changes in circulating
melatonin levels to either direction. Exogenous
melatonin has previously increased T3T4 ratios
of birds (John et al., 1990).
The melatonin-induced effects on the plasma
lipids and on the parameters of nitrogen
metabolism of the raccoon dogs were modest
and mainly inconsistent (III). The only clear
effect was observed in the FFA concentrations,
whose autumnal drop and the subsequent rise
to higher wintertime levels were advanced in
the MEL group. Moreover, the FFA levels of
the MEL females decreased rapidly in Aug
2001, while those of the SHAM females
remained stable. The observed decline in the
FFA concentrations probably indicates their
advanced autumnal transportation to WAT.
Unfortunately, there is a lack of studies
focusing on the effects of melatonin on blood
lipid levels of seasonal mammals. Nieminen et
al. (2001b) have treated minks with exogenous
melatonin in late summer and observed a
decrease in their plasma polar lipid levels, but
this could not be reproduced with the raccoon
dogs (III). Future studies will be required to
unravel the possible role for melatonin in the
regulation of plasma lipid profile of wild
carnivores.
Studies conducted on laboratory rodents
show a melatonin-induced elevation in
circulating PL levels and a suppression in FFA
and Chol concentrations (Aoyama et al., 1986;
Esquifino et al., 1997; Hoyos et al., 2000;
Mazepa et al., 2000). According to Fabiś et al.
(2002) melatonin is able to increase blood total,
free, esterified and HDL-Chol levels, but it
does not affect plasma TG and PL levels or
liver TG and Chol concentrations of rats. The
plasma lipid levels could not be determined
from the rats in the experiments IV-V, as there
were insufficient volumes of plasma left after
the other measurements. However, their liver
raccoon dogs (III) or the rats (V) were not
affected by exogenous melatonin. Neither had
LL any influence on the glucose levels of the
rats. Previously several experiments have
provided data for hyperglycemic effects of
melatonin (John et al., 1990; Poon et al., 2001;
Fabiś et al., 2002). On the other hand, WoldenHanson et al. (2000) have not found any effects
of melatonin on blood glucose concentrations
of rats, confirming our results (III; V). Neither
had melatonin treatment any influence on the
plasma insulin levels of the raccoon dogs (III)
or the rats, but the LL exposure increased
plasma insulin concentrations of the female rats
(V). This result is in concordance with the data
of Gorray et al. (1979), who observed that Px
induces hypersecretion of insulin from rat
pancreatic islets. Chronic melatonin treatment
has previously decreased blood insulin
concentrations of rats (Rasmussen et al., 1999;
Wolden-Hanson et al., 2000). There also exist
contradictory data showing that melatonin
increases blood insulin levels (Fabiś et al.,
2002) or has no influence on pancreatic insulin
release (Frankel & Strandberg, 1991).
A suppressive influence of melatonin on the
thyroid activity has been recorded in several
studies conducted on rodents (Vriend et al.,
1979; Vriend, 1983). However, in certain
experimental conditions also a stimulatory
effect has been demonstrated (Vriend et al.,
1982). T4 concentrations of seasonal carnivores
have shown both positive (mink: Mustonen et
al., 2000) and negative responses (silver fox:
Forsberg & Madej, 1990) to exogenous
melatonin. The MEL raccoon dogs had higher
plasma T4 levels in early Jan and higher T3
concentrations in late Jan, whereas the SHAM
group had higher T3T4 ratios in early Jan (III).
Also the T4 concentrations of the 12L:12D
maintained female rats increased by melatonin
(V). Moreover, the T3T4 ratios of the male rats
increased by melatonin and LL. This may
85
this reason lowered circulating leptin levels
could be required for successful ovulation in
female raccoon dogs. The plasma leptin levels
of the SHAM females increased during the first
half of gestation in late March-early May (II).
Pregnancies of several other mammals are also
characterized by increasing blood leptin
concentrations (human: Butte et al., 1997;
baboon: Henson et al., 1999; big brown bat:
Kunz et al., 1999; sheep: Ehrhardt et al.,
2001). During the second half of gestation the
leptin concentrations of the SHAM females
decreased simultaneously with their falling
progesterone levels (II). There was also a
positive correlation between the leptin and
progesterone levels during the reproductive
season. A similar relation has also been
observed during the human menstrual cycle
(Hardie et al., 1997).
After giving birth, the leptin concentrations
of the SHAM females decreased (II) in concert
with results of others (human: Butte et al.,
1997; baboon: Henson et al., 1999; little and
big brown bat: Kunz et al., 1999). There were
no differences in the leptin concentrations
between the lactating and non-lactating female
raccoon dogs (II; see also Butte et al., 1997 for
women), although it has been observed in
several mammalian species that blood leptin
levels decrease during lactation (little and big
brown bat: Kunz et al., 1999; rat: Woodside et
al., 2000; sheep: Ehrhardt et al., 2001). The
lack of influence may derive from the small
number of blood samples obtained from
lactating raccoon dogs (n=5), as they were not
sampled for three wk after parturition (II).
lipid data are in concert with the results of
Fabiś et al. (2002), showing no influence of
melatonin on the liver TG or Chol levels (V).
As there is a strong inconsistency concerning
the effects of melatonin on lipid metabolism,
these interactions require further studies to be
clarified.
6.5 Seasonal reproduction
regulatory hormones
and
weight-
6.5.1 Leptin and reproduction of the raccoon
dog
Both the male and the female raccoon dogs
experienced high leptin concentrations in Nov
2000-Jan 2001 (I) simultaneously with pubertal
rises in their testosterone, estradiol and LH
concentrations (Asikainen et al., 2003). This
finding supports a current hypothesis of leptin
functioning as a permissive metabolic gate for
the onset of mammalian puberty (Cheung et al.,
1997). Reproductive maturation of humans
(Farooqi et al., 1999) and mice (Chehab et al.,
1996) fails to occur in the absence of leptin,
and exogenous leptin treatment can advance
the timing of puberty in rodents (Ahima et al.,
1997; Chehab et al., 1997). The role of
endogenous leptin in the onset of mammalian
puberty is, however, far from being understood.
For instance, Suter et al. (2000) have found an
increase
in
nocturnal
blood
leptin
concentrations before the onset of puberty in
male rhesus monkeys, whereas diurnal leptin
levels of monkeys of Mann et al. (2000) have
not shown any association with rises in
testosterone and LH concentrations.
The leptin levels of the SHAM female
raccoon dogs decreased before estrus occurring
during the second half of March (II). Leptin has
been previously shown to inhibit early follicular
development (Kikuchi et al., 2001) and
ovulation (Duggal et al., 2000) in rodents. For
6.5.2 Connection of leptin-LI to energy
metabolism of the spawning burbot
The BMs and liver weights of the burbots were
highest before spawning, the relative liver
masses accounting for 8.5 % of BM (VI-VII).
86
the prespawning season. The plasma leptin-LI
concentrations of the burbots were relatively
low before spawning (VI).
During reproduction the BMs of the burbots
were relatively high, but the absolute and
relative liver weights, hepatic lipase esterase
activities and testosterone and estradiol
concentrations were already reduced (VI-VII).
The liver weights had decreased due to the
mobilization and transportation of hepatic fats
into the gonads (see also Karhapää, 1978;
Singh & Singh, 1984). In fact, the liver fat
stores of the DS burbots had decreased to one
half of that of the BS fish (VII). The low
plasma FFA levels indicate their efficient βoxidation in tissues, whereas the decrease in
plasma PL concentrations was probably due to
their transportation to the gonads (see also
Lund et al., 2000).
The liver glycogen stores and glycogenolysis
remained relatively high during spawning (VI).
The muscle glycogen stores, on the other hand,
were stable but glycogen phosphorylase
activities elevated, indicating higher turnover
rate of muscle carbohydrates. This probably
derived from the high locomotor activity levels
of the spawning burbots (Müller, 1973). The
activity of muscle glycogenolysis was lower
before and after spawning supporting this
assumption (VI). The leptin-LI concentrations
of the burbot plasma remained low during
reproduction. Previously, leptin-LI levels of
burbots fasted for two wk have been on the
same level (2.3 ± 0.16 ng ml-1; Nieminen et al.,
2003) as those measured in the spawning fish
of the study VI. For this reason, it seems
unlikely that burbot reproduction requires
above-fasting concentrations of leptin-LI to
proceed (see also Schneider et al., 2000 for the
golden hamster).
The postspawning lowering of the liver Chol
levels (VII) may be connected to the
downregulation of steroidogenesis (Vance,
About 40 and 2 % of their liver fresh weight
were fat and glycogen, respectively. In
comparison, even 70-80 % of liver fresh weight
can be fat in some chondrichthyes species
(Patent, 1970). Another gadoid fish, the
Atlantic cod (Gadus morhua), has an even
smaller capacity for hepatic glycogen storage
than the burbot and depends on its fat reserves
as a major energy supply during periods of
negative energy balance (Kamra, 1966).
The liver glycogen and lipid stores of the
burbots were mobilized rapidly before
spawning, as indicated by the high liver
glycogen phosphorylase and lipase esterase
activities and blood FFA levels (VI-VII).
Hepatic fat was presumably transported to the
gonads during gametogenesis (Karhapää, 1978;
Brown & Murphy, 1995). As about 95 % of
the liver lipids of the burbots were TG (VII),
their hepatic fat represents a readily
metabolizable depot rather than structural
lipids. In some other fish species muscle and
mesenteric fat can be important sites of
mobilizable lipids, and their hepatic fat can
mainly consist of PL (Sheridan, 1988).
The feeding activity of the burbot is high
before reproduction (McCrimmon & Devitt,
1954), and probably for this reason, their
plasma TG, FFA and Chol concentrations were
elevated (VII). Chol may be synthesized rapidly
during gonadal development, as it functions as
an important component of cell membranes
(Vance, 1998). It is also the precursor of
steroid hormones, the levels of which were
maximal before spawning (VI). High estradiol
concentrations probably participated in the
production of vitellogenin (Matty, 1985). The
relatively high T3 concentrations together with
the lower T4 levels (VI) indicate efficient
conversion of T4 to T3, which may have
resulted from high energetic requirements or be
connected to increased lipolysis (Matty, 1985)
or protein synthesis (Plisetskaya et al., 1983) of
87
observed
correlations
among
ghrelin,
testosterone and estradiol concentrations in the
raccoon dogs and the rats support this
hypothesis. There were large interindividual
differences in the timing of estrus of the MEL
females and probably for this reason the
possible connections of leptin, ghrelin and GH
with their reproductive processes remained
unidentified (II).
The ghrelin-LI concentrations of the burbot
plasma did not change in association with
spawning, but the concentrations increased
after
reproduction
(VI).
As
ghrelin
administrations increase appetite of the goldfish
(Unniappan et al., 2002), and the burbots
consume large amounts of food after spawning
(McCrimmon & Devitt, 1954), the rise in
ghrelin-LI levels may be associated with
stimulation of appetite in the postspawning fish
(VI). It is also possible that the observed
increases in the levels of leptin- and ghrelin-LI
are seasonal events perhaps facilitating the
recovery of the fish from the exhaustion of
reproduction.
1998). In contrast, the hepatic G-6-Pase
activities increased after reproduction (VI).
This
probably
indicates
increased
gluconeogenesis, which leptin has been shown
to stimulate in mammals (Rossetti et al., 1997).
The leptin-LI concentrations of the burbots
rose after reproduction (VI). In contrast to
mammals with increasing leptin levels with
excessive fat stores and re-feeding (Maffei et
al., 1995; Kolaczynski et al., 1996), this rise
was associated with low energy reserves (VIVII). When the whole burbot data were
analyzed together, there was an inverse
correlation between the plasma leptin-LI levels
and BMs, relative liver weights, liver glycogen
contents, phosphorylase activities, and liver
TG, Chol and TL contents. This phenomenon
of decoupling of blood leptin concentrations
from body energy reserves has been previously
observed in several wild mammals (little brown
bat: Kronfeld-Schor et al., 2000; mink:
Nieminen et al., 2000; raccoon dog and blue
fox: Nieminen et al., 2001a).
6.5.3 Roles
reproduction
of
ghrelin
and
GH
in
6.5.4 Gender differences in the levels of
weight-regulatory hormones
The ghrelin and GH concentrations of the
SHAM female raccoon dogs were high before
the mating season (II). As observed in the
leptin concentrations, also their ghrelin and GH
levels decreased before estrus. Moreover, the
GH levels of the male raccoon dogs were high
between late Feb and early March and
decreased before the mating season, as
observed in the females. As GH is known to
influence e.g. timing of puberty and production
of sex steroids (Scanes & Harvey, 1995) and
ghrelin can regulate LH and testosterone
secretion of mammals (Furuta et al., 2001;
Tena-Sempere et al., 2002), the possible
participation of these peptides in raccoon dog
reproduction cannot be excluded (II). Also the
Previous studies have documented that women
and female rodents have higher circulating
leptin concentrations than males (Frederich et
al., 1995; Saad et al., 1997). At first, this was
supposed to derive from the higher body fat
content of the females, but the sexual
dimorphism remained after corrections for body
adiposity. When the raccoon dog data were
analyzed together (Aug 16th 2000 - March 27th
2001, i.e. the time when the both sexes
participated in the study), there were no gender
differences in their leptin concentrations.
Neither were there any sex differences in the
leptin levels of the rats (IV). However, the male
raccoon dogs had occasionally higher leptin
88
not display sexual dimorphism. The rats (IV)
and the burbots (VI) had no gender differences
in their ghrelin or ghrelin-LI levels either.
However, when the raccoon dog data between
Aug 16th 2000 and March 27th 2001 were
analyzed together, the female raccoon dogs had
higher ghrelin concentrations than the males.
This finding is supported by results of
Ryökkynen et al. (2003), in which female
minks had higher ghrelin concentrations than
males. Also a recent paper on humans by
Barkan et al. (2003) showed elevated blood
ghrelin levels in women compared to men,
confirming the results on carnivores.
The male raccoon dogs had higher GH
concentrations than the females at one
measurement and vice versa, whereas the GH
levels of the female rats were higher than those
of the males. Jansson et al. (1985) have
previously described sexual dimorphism in the
GH secretory pattern of rats. The male
secretion is characterized with episodic bursts
at 3-4-hr intervals with low or undetectable
levels between peaks, whereas secretion is
more continuous in females. This secretory
rhythm may have led to the higher mean GH
concentration of the female rats.
As the gonads of the raccoon dogs are
quiescent for several months of the year (Xiao,
1996; Asikainen et al., 2003), and sex steroids
may affect the production of e.g. leptin
(Shimizu et al., 1997; Luukkaa et al., 1998),
the possible sex differences in the leptin levels
were studied also during the reproductive
season (Jan 25th-March 27th 2001). Although
the female raccoon dogs had higher body
adiposity than the males during this period,
there were no gender differences in their leptin
concentrations. Neither were the ghrelin nor
GH levels affected by sex.
concentrations than the females and vice versa.
The higher leptin levels of the females were
mainly observed in mid-winter.
In contrast to the data with the raccoon
dogs and the rats, the female burbots had
higher plasma leptin-LI levels than the males
(VI; Nieminen et al., 2003). This gender
difference could not have derived from their
higher body adiposity, as there were no
differences in the relative liver weights or liver
fat contents between the sexes (VI-VII). The
leptin-LI concentrations of the burbots could
have been influenced by sex steroids as
observed previously in humans and laboratory
rodents. Estradiol is able to elevate leptin
production of rats (Shimizu et al., 1997),
whereas testosterone decreases blood leptin
levels of men (Luukkaa et al., 1998). In fact,
the leptin-LI concentrations of the male burbots
correlated negatively with their testosterone
levels (VI) confirming the previous results on
men (Behre et al., 1997). It is possible that the
high testosterone levels could have decreased
the leptin concentrations of BS and DS male
burbots (VI). When the testosterone levels
decreased after spawning, their leptin
concentrations were permitted to rise. On the
other hand, leptin has been shown to inhibit rat
testosterone secretion in vitro (Tena-Sempere
et al., 1999). For this reason it can be
speculated that low leptin levels of the BS and
DS burbots were a prerequisite for their high
testosterone
concentrations
and
the
postspawning rise in their leptin levels
suppressed the synthesis of testosterone (VI).
Leptin has been previously proposed to have a
function in fish reproduction, as it is able to
increase in vitro LH release in the European
sea bass (Peyon et al., 2001).
Until recently, it was believed that blood
ghrelin levels (Tschöp et al., 2001; Bellone et
al., 2002; Shiiya et al., 2002) and ghrelin
mRNA expression (Gualillo et al., 2001b) do
89
LI concentrations of the burbot (Nieminen et
al., 2003). It must be emphasized that the
teleost fish represent a large and diverse group
of vertebrates inhabiting aquatic environments,
and they probably have their own
characteristical weight-regulatory systems very
different from those in terrestrial mammals.
After the discovery of ghrelin (Kojima et al.,
1999), its primary function was considered to
be the stimulation of GH secretion. The results
of this thesis showed a negative correlation
between the ghrelin and GH concentrations and
between the ghrelin-leptin ratios and GH levels
of the raccoon dogs, whereas the ghrelin and
GH concentrations of the rats were not
interrelated (IV). Other experiments have also
demonstrated that circulating ghrelin and GH
concentrations do not always reflect each other
(rat: Tolle et al., 2002; mink: Ryökkynen et al.,
2003). Neither does ghrelin require GH for its
metabolic actions, as it is able to increase food
intake and BM gain of GH-deficient dwarf rats
(Tschöp et al., 2000; Nakazato et al., 2001;
Shintani et al., 2001). The leptin concentrations
of the raccoon dogs correlated positively with
their GH levels, but there was no correlation
between the leptin and GH levels of the rats.
Leptin has been previously shown to have a
positive influence on GH secretion (rat: Carro
et al., 1997; pig: Barb et al., 1998) supporting
the raccoon dog data.
6.6 Interactions between weight-regulatory
hormones
When the whole raccoon dog data were
analyzed together, the leptin concentrations of
the raccoon dogs correlated positively with
their BMs and adiposity (BMIs). There was
also a positive correlation between the leptin
concentrations and the BMIs of the 24L kept
rats, which was absent in the rest of the animals
(IV). Also the ghrelin levels of the raccoon
dogs showed a positive correlation with their
BMs and BMIs, but the correlation between the
ghrelin levels and the BMIs was negative in the
control rats. Moreover, the leptin levels
correlated negatively and the ghrelin
concentrations and the ghrelin-leptin ratios
positively with the voluntary food intake of the
raccoon dogs. There was also an inverse
correlation between the leptin and ghrelin
concentrations of the raccoon dogs and the
12L:12D kept control rats (IV). These findings
fit quite well to data obtained from laboratory
rodents showing antagonism between the
functions of leptin and ghrelin via hypothalamic
NPY (Nakazato et al., 2001; Shintani et al.,
2001). Exogenous leptin has been shown to
decrease food intake, BM and adiposity of
rodents (Pelleymounter et al., 1995), whereas
the effects of ghrelin are the opposite (Tschöp
et al., 2000). Consequently exogenous ghrelin
is able to reverse the leptin-induced reduction
in the appetite of rodents, whereas leptin
treatment inhibits stimulation of appetite by
ghrelin (Nakazato et al., 2001; Shintani et al.,
2001).
Osteichthyean fish seem to be an exception,
as a positive correlation between the plasma
leptin- and ghrelin-LI concentrations was
observed in the burbots (VI; Nieminen et al.,
2003). It has been previously observed that a
two-wk fast leads to a unidirectional change
(i.e. a decrease) in the both leptin- and ghrelin-
6.7 Liver as a possible site for leptin
synthesis
Mammalian leptin is mainly synthesized by
WAT (Zhang et al., 1994). In addition to
adipose tissue, the chicken leptin is produced
by the liver, which may be due to its key role in
avian lipogenesis (Taouis et al., 1998). Leptin
expression has also been found in activated
hepatic stellate cells of rats (Potter et al.,
1998), and Johnson et al. (2000) have found
90
leptin expression in livers of various bony fish
species. They have also hypothesized that the
liver could have been the main production site
for leptin in early vertebrates.
As previously stated, the liver is the main fat
storing organ of the burbot (Karhapää, 1978).
Although its muscle tissue also contains a
considerable amount of lipids (8-12 % of dry
weight vs. 45-65 % in the liver), the muscle fat
store mainly consists of structural lipids (PL
and Chol) showing no clear seasonal variations
in size. The results of the study VII
demonstrated
measurable
leptin-LI
-1
concentrations (5-34 ng g liver ) in the livers
of the burbots without clear seasonal
fluctuations. For comparison, the leptin-LI
concentrations in the plasma were 1-4 ng ml-1
measured with the same multi-species RIA kit
(VI; Nieminen et al., 2003). There was also a
positive correlation between the plasma and
liver leptin-LI concentrations in the BS and AS
groups (VII), supporting the hypothesis of liver
being one production site for burbot leptin.
7
Although the structures of some weightregulatory hormones have remained constant
throughout the developmental history of
animals, they may have distinct physiological
purposes in different vertebrate classes.
Melatonin, for instance, has a large repertoire
of physiological functions, among which is its
ability to time seasonal body weight rhythms
and energy metabolism. By searching for the
prevalence, structure and significance of
weight-regulatory hormones in a wide range of
organisms, it is possible to unfold the evolution
of hormonal weight-regulation from its ultimate
origin to its most sophisticated forms
manifested in nature.
8
CONCLUSIONS
1. The leptin, ghrelin and GH concentrations of
the raccoon dog show clear seasonal
fluctuations, which may have functions in its
body weight-regulation.
2. Photoperiod and the darkness hormone
melatonin have a stronger regulatory effect on
these weight-regulatory hormones than the
nutritional status of the raccoon dog.
GENERAL IMPLICATIONS
Animals can maintain their body weights
remarkably constant in spite of large temporal
fluctuations in food availability. This derives
from their ability to utilize small molecules as
hormonal signals that assist them to store
energy during food abundance and to spare it
during energy shortage. Available simple
molecules, such as derivatives of amino acids
and cholesterol, were discovered to be
advantageous in the course of evolution and are
now utilized as hormones by distinct groups of
vertebrates. These messengers function
together with more complex polypeptides to
inform the individual about the accumulation
and mobilization of energy stores, thus helping
it to adapt to the changing environment.
3. A long wintertime fast is a normal
physiological phenomenon for the raccoon dog
only accentuating the seasonal changes in
weight-regulation observed in the animals fed
throughout the winter.
4. The fasting-induced decreases in the
activities of the thyroid gland and the adrenal
cortex as well as in the levels of blood insulin
are possible candidates for enabling efficient
protein conservation and lipid mobilization
during seasonal food deprivation of the raccoon
dog.
91
5. The plasma leptin and GH concentrations of
the rat do not respond to constant light nor
exogenous melatonin. Its ghrelin levels
decrease due to melatonin treatment.
concerning my manuscripts and research work in
general.
I most sincerely thank Prof Esa Hohtola and Prof
Timo Soveri for their expert reviews of this thesis. MSc
Juha Asikainen and MSc Maija Miettinen are
acknowledged for introducing me to the fascinating
world of fur bearers. At the Laboratory of Animal
Physiology, Mrs Anita Kervinen kindly helped me with
different determinations. Docents Matti Puukka, Seppo
Saarela and Sirkka-Liisa Karonen are also greatly
acknowledged for their help in laboratory analyses. Ms
Leena Koponen and Mrs Heli Asikainen took good care
of our experimental animals and were always eager to
help. MA Ulla Nieminen kindly checked the English of
this thesis and sponsored our research group with all
kinds of delicacies. I am also grateful to Mr Harri
Kirjavainen and Mr Heino Pirinen, who tried to teach
me the basics of taxidermy. Mr Kari Ratilainen
captured our burbots and Mrs Eija Ristola ordered RIA
products for us. MSc Ari Ryökkynen is acknowledged
for (un)scientific discussions and for his help in several
issues. The Interlibrary Lending and Document Supply
Service of the University library carried out a great job
by providing me with numerous articles and books with
flawed references.
My parents Leena and Lauri have given me
financial and mental support. They have always been
excited and probably also amused about my weird study
plans and way of life. Mr Kasper Heikkilä has provided
me with technical help as well as with his
companionship and endless patience during my
physical and mental absence. He has also bravely
tolerated my dear but evil pet ferrets Pena, Kössi,
Killeri and Lumipallo.
Finally, it did not feel like working at all, when
surrounded by enthusiastic people, cardamom coffee,
sweet and a great skull collection. The most memorable
moments on my journey were the times, when I was
sitting at my desktop holding new and exciting results
in my hands and started writing.
6. Constant light stimulates carbohydrate
metabolism in the rat liver. Exogenous
melatonin elevates utilization of liver
carbohydrates but suppresses mobilization of
hepatic fat. Continuous illumination and
melatonin treatment stimulate utilization of
renal carbohydrates.
7. The blood concentrations of leptin- and
ghrelin-LI of the burbot fluctuate in response to
spawning, but their roles in teleost physiology
remain unraveled.
ACKNOWLEDGEMENTS
These studies were carried out at the Department of
Biology of the University of Joensuu. I wish to express
my sincerest gratitude to the present and former Heads
of the Department, Markku Kirsi, Jussi Kukkonen and
Heikki Hyvärinen, for providing me with excellent
working facilities. This thesis was funded by the Faculty
of Science of the University of Joensuu, the Helve
Foundation, the Academy of Finland, the National
Technology Agency of Finland and the Finnish
Konkordia Fund.
I am grateful to my supervisor Prof emer Heikki
Hyvärinen, who has provided me with his endless
optimism being always accessible when I needed him.
With his remarkable insight, Heikki can easily predict
and even contribute new results before the actual
measurements have been conducted! Neither would
have these studies been accomplished without my
second supervisor PhD, D Med Sci Petteri Nieminen,
who introduced me to comparative endocrinology. Like
Heikki, Petteri is easily inspired by new research plans
and continues to study new techniques. He has been my
greatest critic refereeing my texts about a thousand
times, but also giving me continuous encouragement.
Moreover, Petteri likes to share his weird experiences
and dreams with us, which has made the working days
cheerful. I also blame him for getting me excited about
running, which is awful. Prof Jussi Kukkonen has lately
joined my supervisors and given me valuable advice
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