Track 1 Animal models of obesity P1–P26
Eur. J. Biochem.
0 Department of Medicine, University of Melbourne, Royal Melbourne Hospital , Parkville, VIC, 3050 , Australia
1 SmithKline Beecham , Saint GrA?egoire , France
2 A Quignard-Boulange
3 DeA?partement de Biochimie MeA?dicale, Centre MeA?dical Universitaire , CH-1211 GeneA?ve 4 , Switzerland
4 DeA?partement de et Physiologie, Centre MeA?dical Universitaire , CH-1211 GeneA?ve 4 , Switzerland
5 Istituto di Morfologia Umana Normale, FacoltaA? di Medicina, Universita di Ancona , 60020 Ancona , Italia
6 Department of General and Environmental Physiology, University of Naples , Via Mezzocannone 8, I-80134, Napoli , Italy
7 ACERO, Rowett Research Institute , Bucksburn, Aberdeen AB2J 9SB , UK
8 Institute for Nutrition Research, University of Oslo , P.O. Box 1046 Blindern, N-0316 Oslo , Norway
9 Dept. of Biochemistry, Silesian Medical Academy , Zabrze , Poland
10 A Kasperczyk
Cholesterol: A cell size dependent signal which regulates glucose meatabolism and gene expression in adipocytes Enlarged fat cells from obese rodents or humans exhibit modi?ed metabolic capacities, which could be involved in the metabolic complications of obesity at the whole body level. We show here that SREBP-2 (Sterol regulatory Element Binding Protein-2) and its target genes (LDL receptor, HMG-CoA reductase) are induced in the adipose tissue of several models of rodent obesity, suggesting a cholesterol imbalance in enlarged adipocytes. To investigate the potential consequences of such alterations on adipocyte function, we have manipulated cholesterol distribution in fat cells. We show that treatment of adipocytes with Methyl-beta-cyclodextrin, which mimics the membrane cholesterol reduction of hypertrophied adipocytes, induces a dose dependent decrease in maximal insulin response of glucose oxidation. We also produced cholesterol depletion in 3T3-L1 adipocytes cultured in the presence of mevastatin, leading to the activation of SREBP-2 and its target genes (HMG-CoA reductase, synthase and LDL receptor). The analysis of 40 adipocyte genes by Real-time ?uorescent RT-PCR (Taqman1) showed that the response to cholesterol depletion implicated genes involved in cholesterol traf?c (Caveolin 2, Scavenger Receptor-B1, and ATP Binding Cassette 1) but also genes encoding adipocytes-derived secretion products (TNF alpha, angiotensinogen and Interleukin-6) and proteins involved in energy metabolism (Fatty acid synthase, Glut4, UCP3). Thus cholesterol depletion in adipocytes partly reproduced altered gene expression pro?le characteristic of the obese state. Taken together, these data demonstrate that altering cholesterol balance in adipocytes profoundly modi?es adipocytes metabolism in a way resembling that seen in hypertrophied fat cells from obese rodents or humans. This is the ?rst evidence that intracellular cholesterol might serve as a link between fat cell size and adipocyte metabolic activity.
INTRODUCTION: We have recently characterized a P2-receptor for
extracellular nucleotides in human preadipocytes, which is involved in regulation of
estrogen synthesis (Ref. 1). Here we present evidence for a role of nucleotides
in the regulation of lipogenesis in rat adipocytes.
METHODS: Adipocytes from epididymal fat pads of male Wistar rats were
isolated and glucose uptake (Ref. 2) as well as lipogenesis (Ref. 3) were
measured essentially as described. Expression of P2X1-receptor-mRNA was
analyzed by RT-PCR and Southern blot.
RESULTS: In rat adipocytes 50mM ATP stimulates the incorporation of 3H-label
from D-[3-3H] glucose in triglycerides. This effect is observed in the presence
as well as in the absence of adenosine deaminase or the adenosine receptor
antagonist CGS15943, excluding involvement of adenosine receptors.
Incorporation of 3H-label in triglycerides was veri?ed by thin-layer chromatography.
ATP alone or in the presence of insulin has no net effect on glucose transport
as measured by 2-deoxy-glucose uptake. However, the stimulation of
lipogenesis by insulin is augmented by ATP. To further characterize the receptor
mediating the effects of ATP, its ligand pro?le was determined:
bg-methyleneATP > diadenosine tetraphosphate ATP ? ADP ? AMP > adenosine stimulate
lipogenesis, whereas UTP, UDP, CTP, GTP, and ITP have no effect. Suramin and
PPADS, antagonists at several P2-receptors, do not inhibit the effect of ATP.
Up to now only the mRNA for the P2X1-receptor could be detected in rat
CONCLUSION: Our results indicate that extracellular nucleotides stimulate
lipogenesis in adipocytes without modulation of glucose transport. This effect
is mediated by a P2-receptor with broad ligand selectivity. The
pharmacological pro?le does not ?t to that of any cloned P2-receptor, including the
P2X1receptor, whose expression in adipocytes was shown. Therefore, at least one
more P2-receptor subtype must exist in adipocytes. The results presented here
Animal models of obesity
Adipose tissue requires vascularization for its proper functions, including
secretion of a number of proteins. The mechanisms involved in the regulation
of vascularization during the normal or pathological development of adipose
tissue are not well understood. To document this question, we have
investigated the production of vascular endothelium growth factor (VEGF), a potent
angiogenic factor, in adipose cells from young genetically obese Zucker (fa=fa)
rats during the development of obesity.
VEGF production was measured in inguinal adipose tissue from obese (fa=fa)
and non-obese (Fa=fa) rats aged 7 to 56 days. VEGF protein was assessed by
Western blotting and enzyme immunoassay (Quantikine M, R&D SYSTEMS),
in adipose tissue and in isolated adipocytes. VEGF mRNA was determined by
northern blotting. In addition, VEGF secretion was measured in culture media
of adipose cells cultured for 24 h. Primary adipocytes cultures were also used
to study the hormonal regulation of VEGF production by insulin (30 nM) and
angiotensine II (AII) (10 nM).
In both genotypes, adipose tissue VEGF protein content increased steadily
with age, from 4 to 120 pg=pad and 6 to 720 pg=pad in (Fa=fa) and (fa=fa)
rats, respectively. In fa=fa rats, adipose tissue VEGF was 2-fold higher than in
lean rats, starting at 14 days of age. At weaning (30 days) the genotype effect
was markedly accentuated (6-fold effect). The same pattern was observed
when isolated adipocytes were used, pointing out adipose cells as major
producer of VEGF. These genotype and developmental effects were re?ected
at the mRNA level, suggesting transcriptional regulation. The VEGF secretion
rate increased sharply around the weaning period and was markedly higher
in fa=fa than in Fa=fa rat adipose cells. Treatment with AII or insulin increased
the abundance of VEGF in adipocytes by a factor 2 and 3, respectively.
In conclusion, the adipose cell is an important source of VEGF, which may
contribute to the control of local vascularization processes. In obese rats, the
early increase in VEGF production by adipose cells could facilitate indirectly fat
cell hypertrophy. Whether this overproduction plays a role in hyperplasia
which develops later in life remains to be elucidated.
Central leptin insensitivity is a secondary feature of diet-induced obesity in rats
INTRODUCTION: We have developed a rat model of diet-induced obesity and
have found that after 1 week of consuming a 60% high-fat diet (HF), rats
develop increased adiposity, hyperphagia, and high plasma leptin levels
compared to rats fed a chow diet (3% fat). These abnormalities persist over
time as the rats become progressively fatter. The aim of this study was to
determine whether these rats are insensitive to centrally-administered leptin
and whether this occurs early in the development of diet-induced obesity.
METHODS: An intracerebroventricular cannula was placed into the lateral
ventricle of the brain of rats fed either a chow or HF diet for 1 week (1 w) or 4
weeks (4 w). Rats (n ? 9 ? 10) were injected with either 4ml saline or murine
leptin (3mg). Body weight and energy intake were measured 20 hours after
1 w Saline 1 w Leptin 4 w Saline 4 w Leptin
RESULTS: Results are shown in the table above as mean (SEM).
After 1 w of feeding, chow rats responded to leptin by reducing their body
weight gain by 5 g (p < 0.05) and energy intake by 16 kcal (p < 0.05)
compared to saline. The HF rats also responded to leptin by reducing their
energy intake by 41 kcal compared to saline (p < 0.05). After 4 w, the chow
rats again reduced their body weight gain by 6 g (p < 0.05) and energy
intake by 23 kcal (p < 0.05) after leptin treatment. In HF rats, however,
leptin did not signi?cantly reduce either body weight or energy intake
compared to saline.
CONCLUSION: These results suggest that rats respond normally to centrally
administered leptin after 1 w of HF feeding but become insensitive by 4 w.
Therefore it appears that leptin insensitivity is secondary to the development
of diet-induced obesity.
Targeted disruption of the b3-adrenoceptor in mice suppresses the
occurrence of brown adipocytes in white adipose tissue
INTRODUCTION: Brown adipocytes are multilocular mitochondria-rich
thermogenic cells, de?ned by their unique expression of uncoupling protein
UCP1. While mainly located in brown adipose tissue (BAT), brown adipocytes
have been found in typical white adipose (WAT) depots. In mice, occurrence
of these so-called `ectopic' brown adipocytes in WAT is increased by intense
sympathetic stimulation during cold acclimation or by b3-adrenoceptor
(b3AR) agonists. The b3-AR agonist-induced appearance of `ectopic' brown
adipocytes in WAT varies considerably from one strain to another and
correlates with b3-AR agonist-induced resistance to diet-induced obesity.
METHODS: Our model of b3-AR de?cient (b3KO) mice initially generated on a
mixed genetic background was backcrossed towards 2 puri?ed backgrounds
(129Sv=ev and C57B1=6J). In this work we used our b3KO model to study the
role of the b3-AR in the cold-induced emergence of `ectopic' brown
adipocytes in WAT, as assessed by morphological studies and detection of UCP1 by
immuno-histochemistry, Northern blot and Western blot.
RESULTS and CONCLUSION: Our results suggest that, depending on genetic
background, the b3-AR is essential for cold-induced emergence of multilocular
cells expressing UCP1 in WAT. This emergence of `ectopic' brown adipocytes
correlates with the capacity to maintain body temperature during
coldacclimation. Our results further suggest that the origin of `ectopic' brown
adipocytes differs from that of classical brown adipocytes. Target genes of
b3AR involved in the recruitment of `ectopic' brown adipocytes are either
downstream or independent of the cold-inducible coactivator PGC-1 since
the expression levels of the latter are increased in WAT and BAT of
coldacclimated b3KO and wild-type mice to the same extent.
High fat diet induces mitochondrial uncoupling protein 1 in white adipose tissue in mice
T Praz? a?k1, M Hora?kov a?1, P Flachs1, P Brauner1, and J Kopecky?1
1Institute of Physiology and Centrum of Integrated Genomics, Academy
of Sciences of the Czech Republic, V??den?ska? 1083, Prague 4, Czech Republic
Mitochondrial UCP1 is typically expressed only in brown adipose tissue (BAT).
In small mammalian species, newborns and hibernators, BAT is essential for
nonshivering thermogenesis and control of total energy balance. Brown fat
is activated and its UCP1 content increased by cold exposure of the organism
or feeding high fat-containing diet (HF diet). Some brown adipocytes are
interspersed in typical white fat depots and pharmacological induction of
UCP1 in white fat can reduce adiposity. Aim of this study was to ?nd out
whether feeding mice HF diet could induce UCP1 in their white fat depots.
Male C57BL6=J mice were randomly assigned standard or HF diet at 4 wk of
age. Standard diet contained 25, 9, and 66% calories as protein, fat and
carbohydrate, respectively. These values for HF diet were 13,60, and 27,
respectively. Sun?ower oil was the main lipid constituent of the HF diet
(Kopecky? et al. 1996 Am. J. Physiol. 270:E768). After 2 wk, animals were
sacri?ced and interscapular brown fat (BF), subcutaneous dorsolumbar (DL)
and epididymal (EWAT) white fat were dissected. UCP1 content was estimated
using immunoblotting in the membranous fraction (100 000 g) isolated from
tissue homogenates. UCP1 mRNA was quanti?ed using real-time RT-PCR in
total RNA isolated from tissues.
Feeding HF diet resulted in about 1.8-fold elevation of UCP1 content in both
BAT (65 8 vs 119 16 mg UCP1=mg membrane protein, in standard and HF
diet fed mice; n ? 12, p ? 0.02) and DL (1.47 0.21 vs 2.75 0.53 mg
UCP1=mg membrane protein; n ? 11, p ? 0.04). The level of UCP1 mRNA
in DL was 4-fold higher in HF than in standard diet fed mice (n ? 4, p ? 0.02).
The corresponding increase observed in BAT (1.2-fold) was not statistically
signi?cant. In EWAT, no UCP1 gene expression could be detected, regardless
of the type of the diet.
Feeding HF diet is associated with up-regulation of UCP1 gene in both,
interscapular BAT and DL. This induction is more pronounced in the latter fat
depot. It remains to be clari?ed whether feeding HF diet results in recruitment
of new brown adipocytes in subcutaneous white fat.
Ucoupling protein 1 stimulates mitochondrial biogenesis in adipocytes in vivo
P Brauner1, G Barbatelli2, M Rossmeisl1, P Flachs1, MC Zingaretti2, M Marelli2,
P Janovska? 1, M Hora?kova?1, I Syrovy?1, S Cinti2, and J Kopecky?1
1Department of Adipose Tissue Biology and Centrum of Integrated Genomics,
Institute of Physiology, Acad. Sci., V??den?ska? 1083, Prague 4, Czech Republic;
2Institute of Anatomy, University of Ancona, Italy
INTRODUCTION: Uncoupling protein 1 (UCP1) acts as protonophor in
mitochondria. It is present in multilocular adipocytes (MA) in brown fat
where it mediates thermogenesis. Some pharmacological treatments that
reduce obesity induce UCP1 in MA interspersed in traditional white fat
depots. Expression of UCP1 in white fat of transgenic (aP2-Ucp1) mice
prevents obesity, possibly by increasing thermogenesis and depressing fatty
acid synthesis (Rossmeisl et al., 2000, FASEB J.). Pharmacological induction of
MA in white fat (Himms-Hagen et al., 2000, Am. J. Physiol.), as well as ectopic
expression of UCP1 in HeLa cells (Li et al., 1999, J. Biol. Chem.), stimulated
mitochondrial biogenesis. It was tested here whether UCP1 could induce
conversion of unilocular (``white'') adipocytes into MA, and=or activate
mitochondrial biogenesis in vivo.
METHODS: Male control C57BL=6J and heterozygous aP2-Ucp1 mice were
used. Subcutaneous dorsolumbar (DL) and epididymal (EWAT) white fat of
5wk-(young) and 7- to 9-mo-old (old) mice was analysed
immunohistochemically, by transmission electron microscopy, and by immunoblotting. Gene
expression was analysed by Northern blots or real-time RT-PCR.
RESULTS: In control mice, MA expressing UcP1 and its mRNA were detected
only in DL of young mice. In transgenic mice, UCP1 was present in both fat
depots. The transgene enhanced UCP1 content of MA in DL fat of young
animals and induced UCP1 in unilocular adipocytes. In both genotypes and fat
depots, UCP1 expression declined sharply during ageing. In both fat depots,
the levels of the transcripts for subunit IV of mitochondrial cytochrome oxidase
and UCP2 were higher (up to 3-fold) in transgenic than in control mice. Both
mitochondrial density in cytoplasm and mitochondrial area increased by 35%
in unilocular cells in DL of transgenic mice (n ? 8 ? 10; p ? 0.02 in both cases).
CONCLUSIONS: Obesity resistance resulting from mitochondrial uncoupling
in white fat is not associated with conversion of unilocular into multilocular
adipocytes in old animals. However, UCP1 in vivo could induce mitochondrial
Role of mitochondrial UCP2 in control of lipogenesis in white fat
K Bardova?1, R Mikulova?1, P Flachs1, I Syrovy?1, M Rossmeisl1, and J Kopecky?1
1Department of Adipose Tissue Biology and Centrum of Integrated Genomics,
Institute of Physiology, Academy of Sciences of the Czech Republic, V?iden?ska?
1083, 142 20 Praha, Czech Republic
INTRODUCTION: Uncoupling protein 2 (UPC2) is a recently discovered
component of inner mitochondrial membrane with wide tissue distribution.
UCP2 probably mediates proton leak across membrane, by this function it
might in?uence ef?cacy of oxidative phosphorylation, synthesis of ATP and
consecutively all metabolic pathways that require energy in the form of ATP.
Our previous work has indicated that mitochondrial uncoupling in white fat in
vivo could decrease in situ fatty acid (FA) synthesis, presumably due to
limitation of ATP supply by mitochondria (Rossmeisl et al., FASEB J., 2000).
The aim of this study was to assess whether mitochondrial ATP production
may contribute to the amelioration of FA synthesis that occurs in white fat
METHODS: Experiments were performed on adult male C57BL=6J mice. After
l2- to 24-hour fasting, the following parameters have been measured in
fragments of subcutaneous and epididymal white fat: activity of FA synthesis
(incorporation of 3H2O into saponi?able FA), gene expression (Northern
blotting), endogenous consumption of oxygen (Wahrburg apparatus), and
tissue concentrations of adenine nucleotides (HPLC).
RESULTS: In response to fasting, FA synthesis decreased in both tissues,
however, a maximal (4-fold) depression was observed in the epididymal fat
after 12 hours. Depression of FA synthesis was associated with
down-regulation of lipogenic genes (FA synthase, acetyl CoA carboxylase, and pyruvate
carboxylase) and increased expression of mitochondrial uncoupling protein 2
(UCP2). All these changes were higher in epididymal than in subcutaneous
white fat. Only in the former tissue, a signi?cant decrease of ATP=ADP ratio
(2.78 0.33 vs 1.70 0.14; n ? 5) has been observed. Fasting resulted in
about 2-fold decrease of respiration in both tissues.
CONCLUSIONS: Fasting is accompanied by depression of mitochondrial
respiration in adipose tissue, resulting in lower synthesis of ATP. Limited
supply of ATP may contribute to depression of FA synthesis. The role of
UCP2 in this process requires further clari?cation.
Food-induced 5-HT release in the lateral hypothalamus adrenalectomized normal and monosodium glutamate-obese rats of
RB Guimara?es1, RCT Mori1, MM Telles1, FLC Sardinha1, and EB Ribeiro1
1Physiology Department - Federal University of Sa?o Paulo, Sa?o Paulo, SP, Brazil
INTRODUCTION: 5-HT reduces food intake and stimulates energy
expenditure. The hormones of the hypothalamus-pituitary-adrenal axis (HPA) have
been suggested to in?uence 5-HT and disturbances of both the serotonergic
and the HPA systems have been implicated in obesity. In this study we used
brain microdialysis to evaluate the effect of bilateral adrenalectomy on the
feeding-evoked release of 5-HT in the lateral hypothalamic area (LHA) of
normal (N) and monosodium glutamate (MSG)-obese rats, a
hypophagic=hypometabolic obesity model. Food-induced changes in circulating
corticosterone (B) levels were also measured.
METHODS: Animals received a guide cannula aimed at the LHA and were
either adrenalectomized (ADX) or sham operated. Two days later a
microdialysis probe was introduced and perfused with arti?cial CSF. After an
overnight fast, 20 min microdialysis samples were collected before (basal) and up
to 2 hours (samples F1 to F6) after the presentation of a palatable food mash
(powdered chow:water, 1:2). SHAM rats received the same food amount
consumed spontaneously by the ADX group. 5-HT and 5-HIAA levels were
determined by HPLC. Plasma B was determined by RIA in F1, F2, and F6.
RESULTS: 5-HT release was signi?cantly increased by food intake in both N
SHAM (in F4, F5, and F6) and MSG SHAM (in F5 and F6) groups. The maximal
increment over basal release was of 119 59% in N and 111 37% in MSG
rats. Adrenalectomy completely abolished the serotonin release induced
by food intake. Fasting B levels were signi?cantly higher in MSG SHAM
(384 67 ng=ml) than in N SHAM rats (68 21). In F1, plasma B decreased
in MSG SHAM (58%) while it increased in N SHAM rats (280%) and levels
were similar between the groups and remained unaltered until F6.
CONCLUSION: The data showed that the obese rats had a normal 5-HT
response to food. The maintenance of the same B levels as those of N rats was
probably important, due to the stimulatory effect of physiological levels of
glucocorticoids on 5-HT transmission. The abolition of the response by ADX
may rely on the reported stimulation of 5-HT1A receptors and of CRH
production, the latter being an anorexigenic factor that may mediate
serotonin effects. Financial support: FAPESP - Sa?o Paulo, Brazil
Contrasting hypothalamic responses to increased adiposity and food intake in sheep
ZA Archer1, SM Rhind2, PA Findlay1, CE Kyle2, L Thomas1, and CL Adam1
1ACERO, Rowett Research Institute, Bucksburn, Aberdeen AB21 9SB, UK;
2MLURI, Craigiebuckler, Aberdeen AB15 8QH, UK
Reproductive neuroendocrine activity in sheep is stimulated by increased food
intake but not by chronically increased adiposity. It is unknown how
hypothalamic mechanisms respond to these distinctive components of nutritional
Castrate sheep with oestradiol implants were (1) fed similar amounts to
maintain constant low or high adiposity for 4 weeks, determined by
maintaining body condition scores 2 or 3, respectively (LBC or HBC); or (2)
maintained at intermediate adiposity (BC 2.5) with low food intake or allowed
high ad libitum intake to increase adiposity from BC 2 to 2.5 over 4 weeks (LI
or HI). Blood samples were taken to determine pulsatile luteinising hormone
(LH) secretion (by inference hypothalamic LHRH) then brains removed for
gene expression study by in situ hybridisation. LBC and HBC sheep had similar
LH secretion. Gene expression in the hypothalamic arcuate nucleus (ARC) was
higher in LBC than HBC sheep for neuropeptide Y (NPY), agouti-related
protein (AGRP) and leptin receptor (OB-Rb), but similar for
proopiomelanocortin (POMC). LH output was greater in HI than LI sheep. ARC gene
expression was higher in HI than LI sheep for AGRP but similar for NPY,
OB-Rb and POMC.
Thus increased food intake, but not high adiposity, stimulated LHRH=LH
secretion, but adiposity had a profound in?uence on hypothalamic gene
expression irrespective of intake. This model shows that the hypothalamus
can differentiate between steady-state nutritional feedback provided by
adipose mass and dynamic changes in feedback provided by increased food
Changes in skeletal muscle mitochondrial proton leak in rats fed high fat diet
INTRODUCTION: We have previously shown that young rats fed high fat diet
do not develop obesity, since they are able to increase energy expenditure and
lipid oxidation in response to increased lipid intake. As for lipid oxidation, an
increase has been found both in liver and skeletal muscle from rats fed high fat
diet. Since it has been shown that skeletal muscle mitochondrial proton leak
accounts for 17 ? 21% of resting metabolic rate in rats, we considered of
interest to investigate whether changes in skeletal muscle mitochondrial
proton leak could be involved in the increased energy expenditure induced
by high fat feeding. Measurements were made in subsarcolemmal and
intermyo?brillar mitochondria, since it is known that these two skeletal
muscle subpopulations are functionally distinct and differentially regulated
in various physiological conditions.
METHODS: Young (25 days old) Wistar rats were fed a low fat or high fat diet
for ?fteen days. At the end of the experimental period, rats were killed and
hindquarter skeletal muscles were removed and subsequently used for the
preparation of isolated subsarcolemmal and intermyo?brillar mitochondria.
Kinetics of the proton leak were measured by titrating state 4 respiration
in the presence of succinate 10 mM ? rotenone 3.75 mM ? oligomycin
(4 mg=ml) ? nigericin (80 ng=ml) by sequential additions of malonate.
Mitochondrial oxygen consumption was measured polarographically with a Clark
electrode, while mitochondrial membrane potential was measured with
safranine O in a dual-wavelength spectrophotometer.
RESULTS: Intermyo?brillar mitochondria exhibited higher inner membrane
proton permeability compared to subsarcolemmal ones. In addition, high fat
feeding induced a signi?cant decrease in proton leak kinetics only in
CONCLUSION: Our results show that inner membrane permeability of the
two mitochondrial populations from skeletal muscle is remarkably different,
thus in?uencing their relative contribution to resting metabolic rate. On the
other hand, skeletal muscle involvement in the response to high fat feeding
occurs through an increased lipid oxidation but not through increased
mitochondrial proton leak.
A neural substrate of the adipostat
Nina Pronchuk1, and F William Colmers1
1Department of Pharmacology, University of Alberta, Edmonton, AB, Canada
INTRODUCTION: Central regulation of energy balance, including appetite,
energy expenditure and related physiology has been postulated to reside in
hypothalamic nuclei. One nucleus implicated in this is the paraventricular
nucleus (PVN). We set out to identify the neurons and responses that might be
involved in regulation of energy balance in the PVN, using the orexigen
Neuropeptide Y (NPY).
RESULTS: Coronal slices containing the PVN were prepared from 21 ? 42
dayold rats, and whole-cell recordings made of neurons in numerous subdivisions
of the nucleus. A subpopulation of the medial parvocellular PVN (mpPVN)
neurons received a prominent GABAA-mediated inhibitory postsynaptic
current (IPSC) when the slice was stimulated lateral to the neuron. Application of
NPY reversibly reduced these synaptic responses. The action of NPY was
presynaptic. Use of receptor-selective agonists and antagonists showed that
the pharmacology of the NPY response is complex. At least 3 receptors, Y1,
Y5 and Y2, can mediate this response to NPY, consistent with feeding data.
Furthermore, the IPSC is enhanced by melanocortin agonists, and the effects
of these are blocked by pretreatment with AGRP, which is co-localized with
NPY in arcuate PVN projections. Finally, in fa=fa rats, the actions of NPY on this
response are blunted, consistent with the reduced food intake response to icv
NPY in these rats.
CONCLUSION: Based on these results, the IPSC impinging on a subset of
mpPVN neurons integrates countervailing signals regarding energy balance,
and may form part of the adipostat.
Supported by MRC=PMAC and Lilly Canada.
Does melanin-concentrating hormone (MCH) stimulate food intake of obese Zucker rat?
A Burlet1, O Marrion1, and B Fernette1
1EA3114, Biologie Cellulaire, UHP, 54000 Nancy, France
Melanin-concentrating hormone (MCH) is restrictively synthesized in the
neurons of the lateral hypothalamus of the rat brain but these neurons project
in numerous brain sites, namely hypothalamic paraventricular (PVN) and
ventromedian (VMN) nuclei. MCH increases food intake when it is
administered into the lateral ventricles of the Long Evans rats. We have tested the
response of the obese Zucker rat to the injection of MCH to determine
whether the hyperphagia of this rat can be due to a greater sensitivity to
the orexigenic effect of this peptide. Injections have been performed ?rstly in
the adult rats then in the 2-week-old rats (2-w-old rats).
In the adult rats, MCH was injected into the brain through bilateral chronic
cannulae implanted in several groups of obese (fa=fa, n ? 12) or lean (FA=FA,
n ? 12) Zucker male rats maintained in free moving conditions to measure
food intake 1, 2, 4, 6 hrs after the injection, and also during the light and dark
phases of the day. MCH injection was alternated with injection of
cerebrospinal ?uid (vehicle) in the same rat. Three sites of injection were tested: the
lateral ventricles (icv, 5 or 10 mg MCH in 4 ml), the hypothalamic PVN (5 mg in
0.25 ml per nucleus) and VMN (5 mg in 0.25 ml per nucleus). The antagonistic
properties of a-MSH and CRF were also determined. In the young rats, 1 and
5 mg MCH in 3 ml were successively injected in the ventricles of 16 ? 20 obese
(fafa) or lean (FAFA) Zucker rats, and also in 16 ? 20 lean rats, heterozygous for
the fa gene (faFA).
MCH increased food intake of adult lean rat when it was injected into the
ventricles or above PVN, where it could stimulate food intake during 24 hrs. It
was less ef?cient when it was injected above VMN of FA=FA rat. On the
contrary, MCH totally failed to increase food intake of the obese Zucker rat
when injected into the ventricles or above hypothalamic PVN, but restrictively
increased food intake when injected above VMN. The direct injection of
corticotropin releasing factor (CRF) into VMN prevented the orexigenic effects
of MCH injection in both genotypes.
Five mg MCH increased food intake when injected into the ventricles of
obese 2-w-old rats but 1 mg MCH signi?cantly reduced food intake. On the
contrary of the adult response, 1 or 5 mg MCH totally failed to increased food
intake of the lean young rats, regardless of their genotype, FAFA or faFA.
We conclude to that multiple brain sites are involved in the orexigenic effects
of MCH; these vary with the genotype and the age of the rats. (Danone Grants)
A follow-up program to study the effect of dietary management and behaviour modi?cation among obese adult females
WH Mehelba1, YM Ghanem1, and NA Elsayed1
1Association for Study of Obesity for Women and Community Health, 212
Abdelsalam Aref st., Loran Alexandria, Egypt
The aim of this work was to study the characteristics of obese females, outline
the aetiological factors of their obesity, study the effect of the initial weight
loss on health status and to measure the effectiveness of dietary management
alone versus combined dietary and behavioural modi?cation on weight loss
To accomplish this aim, one hundred females aged 25 ? 54 years were
chosen. They ful?lled the following criteria: Body Mass Index not less than 30
not more than 40, free from any medical problems that might interfere with
weight loss and able to attend all follow up sessions outlined for them. In the
?rst interview, a complete medical check up was done after which females
were randomly classi?ed into two groups: DG group which was kept only on
dietary management, and DBG group which was kept on combined dietary
management and behavioural modi?cation. Dietary management was the
same for both groups. Behavioural procedures were contracting method, self
monitoring, and life style education program. A follow up schedule of 12
weekly visits for weight loss followed by a recall visit after 6 months to check
weight loss maintenance was outlined for both groups. A dieting test of 30
questions was used in the interview session as an assessment tool, as a learning
tool in the education program during follow up sessions for combined dietary
and behaviour group, and as an evaluation tool in the recall visit.
The results of this work was presented in 3 parts: part A: results of interview
visit(characteristics of the sample, part B: the effect of initial weight loss on
health status. part C: the effectiveness of dietary management versus
combined dietary and behavioural management on weight loss and maintenance,
commitment of females, and dietary habits and behaviour ``score of dieting
The present study concluded that weight loss had a positive impact on
health status regardless of the method of management. Combined dietary
and behavioural management had a better effect than dietary management
alone on commitment of females to the schedule of visits dietary habits and
behaviour, and maintenance of lost weight.
A b3-adrenergic agonist improves insulin-dependent glucose uptake by adipocytes in lean and overweight (cafeteria) rats
MJ Moreno-Aliaga1, O Lamas1, A Marti1, and JA Mart??nez1
1Department of Physiology and Nutrition, University of Navarra, 31008
INTRODUCTION: The b3-adrenoceptor agonist (AR) Trecadrine has been
shown to have potential anti-diabetic and anti-obesity properties in rodents.
It is also known that b3-AR agonists promote glucose transport into skeletal
muscle and adipose tissue in rats. However, it is not well understood whether
b3-AR agonists affect the insulin-dependent and=or independent glucose
METHODS: Control and cafeteria-fed rats were treated with placebo or
Trecadrine during 35 days. Adipocytes were isolated from epididymal fat by
collagenase digestion and cultured with DMEM containing 5 mM glucose in
the absence or presence of 1.6 nM insulin. Statistical differences and
interactions were evaluated using a factorial two way ANOVA (Overweight: Ow
x Trecadrine: T).
RESULTS: Animals fed with a cafeteria
diet showed an increase in body and
epididymal fat weights as compared to
controls, which were prevented by
Trecadrine treatment. Insulin-dependent
glucose uptake was impaired in
adipocytes of the overweight rats in relation
to lean animals (p ? 0.003). Trecadrine
treatment induced an increase in
insulin-stimulated glucose uptake as compared to the non-treated animals
(p ? 0.003). In fact, Trecadrine treatment was able to restore to control
values the impairment in insulin-mediated glucose uptake induced by the
CONCLUSIONS: The decrease in the insulin-stimulated glucose uptake levels
observed in diet-induced overweight rats suggests the development of some
insulin resistance in the adipose tissue of these animals, which is prevented by
Effect of high-fat diet on body mass and energy balance in the bank voles (Clethrionomys glareolus)
Macronutrient diet selection and energy balance in brown adipose tissue ablated transgenic UCP-DTA mice
WL Peacock1, and JR Speakman1,2
1Aberdeen Centre for Energy Regulation & Obesity (ACERO), Zoology
Department, Aberdeen University, Aberdeen AB24 2TZ, Scotland, UK;
2ACERO, Rowett Research Institute, Aberdeen, AB21 9SB, UK
The object of our study was to investigate the effects of a high-fat diet on the
body mass, resting metabolic rate (RMR) and body composition of the bank
vole (Clethrionomys glareolus); a small mammal which responds to shortened
photoperiod by a decrease in body mass.
Bank voles fed a standard maintenance diet (fat ? 5.4% by mass, 12.3% by
energy) from weaning to age 3 ? 5 months were either maintained on this diet
(control) or switched to a high-fat diet (fat ? 13.5% by mass, 28.2% by
energy) for ?ve weeks. Body mass, food intake, RMR, body composition,
using total body electrical conductivity (TOBEC), and apparent energy
assimilation ef?ciency (AEAE) were measured for all voles before and ?ve weeks after
the high-fat diet had been introduced to the experimental group.
Both groups exhibited an initial decrease in body mass of approximately
0.5 g (3.3%) over the ?rst 7 days. This was due to a signi?cant decrease in fat
mass. Over the following 21 days the control group regained body mass to its
original level, whereas voles fed the high-fat diet showed no subsequent
increase and kept their mass relatively constant at the reduced level. The
increase exhibited by the control group was again due to a change in fat mass.
Mean food intake (g=day) was 5.3% lower and energy intake (kJ=day) was
3.2% higher in the high-fat diet group from day 7 to 27, however these
differences failed to reach signi?cance. The experimental group had a higher
AEAE and when this was taken into account, the total energy assimilated by
these animals was signi?cantly higher than the controls. Furthermore, voles
fed the high-fat diet consumed more than twice as much fat per day as the
control animals. There were no signi?cant differences in RMR within or
between groups over the ?ve-week period.
Bank voles were resistant to fat gain on a high fat diet, consistent with
predictions based on their response to photoperiod. They achieved this
resistance despite assimilating more energy than control animals fed the low
fat diet and taking in twice as much fat. Resistance, however, did not occur by
modi?cation of RMR, implicating differences in activity levels.
Patterns of circulating leptin in sheep
CL Adam1, PA Findlay1, L Thomas1, and M Marie1
1ACERO, Rowett Research Institute, Bucksburn, Aberdeen AB21 9SB, UK
Sheep are widely used in neuroendocrine research and may also serve as
animal models of obesity and bodyweight regulation. Circulating
concentrations of leptin have previously been correlated with adiposity and
photoperiod, but factors affecting short-term dynamics of leptin secretion are
Serial blood samples were taken over 48 h from sheep after 16 weeks in
short days (16 h dark, 8 h light) with ad libitum food (SD), and then after 16
weeks in long days (16 h light, 8 h dark) with either ad libitum food (LD) or
restricted food approximating to SD intake (LDR). During the second 24 h of
sampling, half of the sheep were food deprived and half had the twice-daily
meal times shifted forwards by 4 h.
Plasma leptin was higher in LD than SD, associated with higher food intake,
body weight and adiposity, but tended to be lower in LDR than SD, associated
with slightly lower food intake, body weight and adiposity. In both
photoperiods, there was no evidence for a consistent nocturnal rise or circadian
rhythm of plasma leptin, but clear evidence of post-prandial peaks of low
amplitude (15 ? 36%) 2 ? 8 h after meals. Shifting the meal times shifted the
time of day that leptin peaks were seen. Food deprivation caused a fall in
plasma leptin, starting from the time of the ?rst missed meal, to basal levels
within 24 h. There was positive association of plasma leptin with plasma
insulin, and negative association with NEFA.
Thus plasma leptin concentrations in sheep do not show a circadian rhythm;
they are sensitive to short-term changes in energy balance both between
meals and during fasting, as well as to long-term changes in food intake and
J Prinzler1, S Klaus1, and S Ortmann1
1German Institute of Human Nutrition, Arthur-Scheunert-Allee 114 ? 116,
14558 Bergholz-Rehbru?cke, Germany
Speci?c expression of the diphteria-toxin A chain (DTA) under the control of
the uncoupling protein-1 promoter (UCP) in the brown adipose tissue (BAT) of
transgenic mice (UCP-DTA) abolishes BAT function and leads to severe obesity
and insulin resistance1. It has been demonstrated for adult UCP-DTA-mice that
the increased body mass is not due to hyperphagia but to a reduction in
energy expenditure (EE) and core body temperature (Tb)2. The aim of this
study was to determine an effect of the ablation of BAT on macronutrient
selection of growing UCP-DTA mice. Further the in?uence of the individual
composition of macronutrients effects on growing rate, body weight and
parameters of the energy budget, i.e. Tb, activity and EE was examined.
Male mice (n ? 30) were fed a three-choice macronutrient diet of either
pure fat, carbohydrate (CHO), or protein after weaning. Food consumption
and body weight were measured three times a week, EE (indirect calorimetry)
and Tb (telemetry) every two weeks for two consecutive days. Body
composition was determined by carcass-analysis and Soxhlet.
UCP-DTA mice (n ? 16) exhibited a lower weaning body mass than control
litter mates (n ? 14; 9.8 1.0 g vs. 11.8 1.2 g; p < 0.01) but grew faster and
reached body weight of controls after ten weeks of life. From week twelve of
life UCP-DTA mice had a signi?cantly higher body weight compared to
controls (40.0 7.9 g vs. 33.2 2.9 g; p < 0.05). Control mice consumed
11% more energy than UCP-DTA mice. Although both groups showed a
high preference for fat UCP-DTA mice consumed signi?cantly more fat and
less CHO than control mice, (fat: 83% vs. 81%; CHO 3% vs. 6%; p < 0.05;
protein 13% vs. 13%; n.s.). From week 14 of life transgenic mice showed a
lower EE than the controls (p < 0.01) whilst Tb was not decreased. Body fat
content was doubled in UCP-DTA mice (32.6 10.8% vs. 16.5 6.6%;
p < 0.001).
In conclusion, BAT ablation had an effect on energy intake, body
composition and diet selection. When growing UCP-DTA mice were fed a free-choice
diet, they showed no hyperphagia but a reduction in EE, which facilitates the
development of obesity.
1: Lowell et al. (1993) Nature 366:740 ? 742; 2: Klaus et al. (1998) Am. J.
Physiol. 274:R287 ? R293.
A sensitive chemiluminescent ELISA for the measurement of ovine leptin
Studies on leptin de?cient rodents and humans clearly demonstrate the
importance of leptin on energy balance. To clarify its function further, it is
necessary to carry out studies in other species such as the sheep. Several
immunoassays for ovine leptin have recently been reported; most have
sensitivities between 100 and 1000 pg=ml. We have recently described a
colorimetric sandwich ELISA with a sensitivity of 1 ng=ml [
suf?cient for the measurement of leptin in plasma from adult sheep, it is not
sensitive enough to measure the hormone in cerebrospinal ?uid (CSF) or fetal
plasma. We therefore examined whether the sensitivity of our ovine ELISA
could be increased by using chemiluminescence detection.
Af?nity puri?ed antibodies raised in rabbits and chickens against
recombinant ovine leptin were used to develop a sandwich ELISA. CDP-Star (Tropix)
was utilised as a chemiluminescence substrate and the signal was measured by
The chemiluminescent procedure gave a 20-fold increase in sensitivity, with
a detection limit of 50 pg=ml. Leptin levels in ovine CSF and fetal plasma,
which were previously undetectable by colorimetric analysis, could be readily
measured with the chemiluminescent assay.
It is concluded that the increase in sensitivity with chemiluminescent
analysis is particularly valuable for quantifying low levels of leptin, as in CSF
and fetal plasma.
Thomas L, Wallace JM, Aitken RP, Mercer JG, Trayhurn P and Hoggard N
(2001). J. Endocrin. (submitted).
UCP1, UCP2, UCP3 and b3-AR expression in male rats: effects of cold in overweight animals
AM Rodr??guez1, P Roca1, and A Palou1
1Department of Biologia Fonamental i Cie?ncies de la Salut, Universitat de les
Illes Balears, Cra. Valldemossa, Km 7.5. E-07071, Palma de Mallorca, Spain
Physiological heat production in response to environmental stimuli, such as
cold or overfeeding, by adaptive thermogenesis is of great relevance in small
mammals. Brown adipose tissue (BAT) is fundamental in adaptive
thermogenesis, where uncoupling protein 1 (UCP1), is the main direct mediator of
this thermogenesis. Other putative uncoupling proteins have been described
more recently, such as UCP2 and UCP3. On the other hand, the b3-adrenergic
receptor (b3-AR) plays an important role in the adrenergic regulation of
thermogenesis in BAT.
In the present work, we aimed to study the effects of cold exposition in the
expression of UCP1, UCP2, UCP3 and b3-AR in the BAT of overweight male
rats, as compared to controls, in the overweight=obesity model known as
post-cafeteria, by the Northern Blot technique. Post-cafeteria male Wistar rats
were fed with cafeteria diet plus standard chow diet from lactation until day
110 of life, and then they were fed only with standard chow diet until day 180
of life, showing at that moment a stabilised 12% overweight. The animals
were killed at day 180 of life and Northern Blot determinations were made.
Half of the control and post-cafeteria rats were kept at 4 C for 24 hours before
they were killed.
There was a pronounced up-regulation of UCP1, UCP2 and UCP3 mRNAs in
response to cold, showing a synergetic effect of overweight and cold in the
induction of UCP1 and UCP2 mRNAs. In control rats, a signi?cant
downregulation of b3-AR mRNA by cold was observed, but not in overweight rats.
On the whole, the results suggest there would exist a stronger response to
cold of some thermogenic parameters, such as UCP1, in overweight rats than
in controls. The lack of down-regulation of b3-AR mRNA in response to cold in
overweight rats also suggests this. On the other hand, the results agree with
the possibility that UCP2 and UCP3 could play a role in the thermogenic
response to obesity and cold.
Circulating a-MSH directly down regulates adipocyte expression of leptin
N Hoggard1, J Duncan1, L Hunter1, J Crabtree1, and V Rayner1
1ACERO, Rowett Research Institute, Bucksburn, Aberdeen AB21 9SB, UK
Pro-opiomelanocortin (POMC) is expressed in the pituitary and is processed to
several biologically active peptides including, aMSH, b-MSH, ACTH and
alipotropin, which are then secreted into the circulation. Melanocortin
receptors for these peptides are located in the fat cells, thus adipose tissue is a target
for one or more of these peptides. Several recent studies have demonstrated
that leptin acts on the central nervous system through an a-amelanocyte
stimulating hormone (MSH)-mediated mechanism. However very little is
known about the potential peripheral effects of circulating a-MSH with
regard to obesity. This preliminary study was to clarify the function of
circulating a-MSH in the regulation of leptin.
Rat adipocytes in primary culture were treated with a-MSH. At physiological
concentrations of a-MSH (0.15 nM) leptin secretion into the medium was
inhibited by approximately 2-fold. While at higher concentrations of a-MSH
leptin secretion was virtually abolished. In vivo, plasma a-MSH was increased
2.6 fold in the leptin de?cient ob=ob mice compared with their lean
littermates (+=?), as determine by a commercial RIA.
This data suggests a possible negative feedback loop involving a-MSH and
leptin, an increase in a-MSH will lead to a decrease in leptin and in turn the
decrease in leptin will allow an increased expression of a-MSH secreted from
the pituitary, the main site of a-MSH expression.
Seasonally-inappropriate body weight and hypothalamic mRNA in the
JG Mercer1, KM Moar1, TJ Logie1, PA Findlay1, CL Adam1, and PJ Morgan1
1ACERO, Rowett Research Institute, Bucksburn, Aberdeen AB21 9SB, UK
INTRODUCTION: Body weight regulation in mammals functions at different
levels, the most familiar being defence of an appropriate body weight against
an energetic imbalance. Animals may also adjust the level of body weight that
they will seek to defend; seasonal mammals defend a sliding appropriate body
weight according to photoperiodic history. This study investigates the
expression of hypothalamic genes following the induction of seasonal and
seasonally-inappropriate body weight change.
METHODS: Weight loss or growth restriction was induced in Siberian
hamsters (Phodopus sungorus) either by transfer from long (LD; 16 h light:8 h
dark) to short days (SD; 8 h light:16 h dark), or by imposed food restriction.
Plasma leptin was measured by radioimmunoassay. Hypothalamic
neuropeptide and receptor mRNA was quanti?ed by in situ hybridization.
RESULTS: Food restriction in SDs was accompanied by increased
neuropeptide Y, agouti-related protein (AGRP) and leptin receptor (OB-Rb) gene
expression in the arcuate nucleus (ARC). Food restriction of LD hamsters to
mimic SD body weight trajectory increased AGRP and OB-Rb mRNA in the
ARC, but reduced proopiomelanocortin (POMC), cocaine- and
amphetamineregulated transcript (CART) and melanocortin 3-receptor gene expression.
This contrasted with the elevated CART and reduced OB-Rb mRNA in SD
hamsters with the same body weight trajectory and a similar low plasma leptin
CONCLUSION: Hypothalamic mRNA levels indicate that the hypothalamus
differentiates between seasonally-appropriate and inappropriate body mass.
Leptin feedback may be important.
Resting metabolic rate and morphology in mice (Mus musculus) selected for high and low food intake
JR Speakman1,3, JC Selman1, L Bu?nger 2, S Lumsden1, and WG Hill2
1Aberdeen Centre for Energy Regulation and Obesity (ACERO), University of
Aberdeen, Aberdeen, UK AB24 2TZ; 2Institute of Cell, Animal and Population
Biology, University of Edinburgh, Edinburgh, UK EH9 3JT; 3ACERO, Rowett
Research Institute, Aberdeen, UK, AB21 9SB
Resting metabolic rate (RMR) is a major contributor to total energy demands.
Individual variation in RMR may be linked to differences in total daily energy
expenditure and hence may re?ect a risk factor for development of obesity.
We investigated the relationship between resting metabolic rate (RMR) and
morphology in non-breeding mice, selected for high and low food intake,
corrected for body mass, for 38 generations. RMR was measured at 30 C
(thermoneutral) and mice were subsequently dissected into 19 components.
High food intake mice had signi?cantly greater body masses (by 8% in males
and by 9.6% in females) and signi?cantly elevated RMR (by 17% in males and
by 51% in females) compared to the low intake mice. Body mass, sex and
strain together explained over 56% of the observed variation in RMR. The
effects of strain and sex on the RMR were statistically removed and three
separate analyses performed to investigate the relationship between RMR and
organ morphology: employing individual regression analysis with each tissue
component as a separate predictor against RMR, individual regression analysis
with residual organ mass against residual RMR (i.e. with strain, sex and body
mass effects removed) and pooling of some organ masses into functional
groupings to reduce the number of predictors. Independent of the analysis
method, liver mass was the most signi?cant morphological trait linked to
differences in RMR. Liver mass was 19% larger in the high intake strain in
males and 46% larger in the high intake strain females. The effects of strain on
RMR were consistent with the anticipated effect from the strain differences
in the size of the liver.
Several strategies have been described for weight reduction but a truly
effective treatment remains to be discovered. In this context, some authors
have proposed that dietary medium-chain triglycerides (MCT) could be useful
in obesity treatment, as an alternative to traditional hypoenergetic diets. The
aim of this work is to study the changes of body weight, adipose depots and
protein body mass under two weight-loss strategies: hypoenergetic versus ad
libitum MCT dietary treatment.
Diet-induced overweight rats were divided into three groups: Control group
was fed on a standard rodent diet, the second group (40%R) received a 40%
restricted diet (60% of Control group intake), and the third group was fed ad
libitum on a diet containing MCT as lipid source, during 23 days. Body weight
and adipose depots from several locations were measured. Serum leptin was
determined by ELISA, total body composition by conductivity, tisular protein
content by the Lowry method, enzyme proteolytic Cathepsin A activity by
spectrophotometry and urinary 3-Methylhistidine excretion by HPLC.
Initial body weight (g)
Final body weight (g)
Total body fat (g)
Serum leptin (ng=mL)
Urinary 3-Methyl-His (mg=kg=day)
a,b,c: data in the same row not sharing a common letter are statistically different, at least
Leptin levels were correlated with the retroperitoneal fat depot (r ? 0.68;
P ? 0.01), which was lower in the 40%R group, as compared to the MCT
group. No signi?cant differences between treated groups were found in
hepatic protein content and proteolytic activity.
In conclusion, both treatments, energy restriction and dietary MCT, could
be useful for body weight and fat store reduction but MCT treatment seems to
be more ef?cient sparing muscle protein.
In?uence of hypercholesterolemic diet on activity selected enzymes metabolic paths and lipid peroxidation in serum and liver
INTRODUCTION: It is known that obesity is one of the agent of
atherosclerosis that lipid peroxidation plays important role in pathophysiology.
Hypercholesterolemic diet (HCD), used as a model for study atherosclerosis,
can be recognized as a state of very good alimentation for organism which can
induce obesity. It is still not precisely known if modi?cation of diet e.g. HCD
can in?uence on cell membrane. The aim of our study was if HCD can modify
cell membrane by lipid peroxidation.
METHODS: 12 rabbits were divided into two groups ? one of them were fed
with standard diet (SD) and the second were additionally received 2 g of pure
cholesterol per day (HCD). The study lasted 3 months. The venous blood was
taken before study and every month of experiment. The liver was taken after
decapitation. We estimated activity of selected enzymes of metabolic paths:
Steen B Pedersen1, Jens M Bruun1, Kurt Kristensen1, and Bj?rn Richelsen1
1Department of endocrinology and metabolism C, Aarhus Amtssygehus, Aarhus
University Hospital, Aarhus, Denmark
BACKGROUND: Ovariectomy (OVX) of rodents has been associated with
increased body weight which can be normalized by estrogen substitution,
part of this effect are explained through changes in food-intake, however,
other mechanisms might also be involved.
AIM: To study the effects of OVX (with or without estrogen treatment) on
body weight, body composition, food intake, UCP1, UCP2 and UCP3
expression in brown adipose tissue (BAT), white adipose tissue (WAT) and skeletal
METHODS: Body composition was determined by bio-impedance
measurements, UCP-expression by realtime RT-PCR.
RESULTS: Non-substituted OVX-rats increased the food-intake and became
obese during the study (5 weeks), whereas estrogen treatment decreased
food-intake, and decreased body weight gain. In addition a third group of rats
were studied (OVX rats without estrogen treatment) but they were on food
restriction, so their weight gain matched a group of control rats. However,
despite these OVX rats on reduced diet consumed the same amount of food as
the estrogen treated rats, the OVX rats on diet became signi?cantly heavier
than the estrogen treated OVX rats.
When studying the UCP-expression in BAT, the UCP1 expression was
signi?cantly decreased in OVX-rats, whereas estrogen treatment normalised
the UCP1 expression, the UCP2 and UCP3 expression in BAT was not
In WAT the UCP2 expression was signi?cantly depressed in OVX-rats, again
estrogen treatment normalized the expression. Finally, in SM neither OVX nor
estrogen did affect the UCP expression, but food-restriction of the OVX-rats
was associated with a signi?cant decrease in UCP3 expression.
IN CONCLUSION, our ?ndings indicate that estrogen, besides its well-known
effects on food-intake, also affects energy-expenditure. In addition we found
decreased UCP1 expression (in BAT) and decreased UCP2 expression (in WAT)
in estrogen de?cient rats, and these anomalies could be normalized by
estrogen treatment. Thus, we suggest that the estrogen effects on energy
expenditure might be caused by its effects on UCP-expression in BAT and WAT.
AspAT (EC 220.127.116.11), AlAT (EC 18.104.22.168), LDH (EC 22.214.171.124) sorbitol
dehydrogenase ? SDH (EC 126.96.36.199), glutaminiane dehydrogenase ? GLDH (EC
188.8.131.52) and aldolase ? ALD (EC 184.108.40.206). All estimated enzymes origin
from hepatic cell and activity in serum are much lower than in liver.
Additionally we measured concentration of malonyldialdehyde ? MDA
(product of lipid peroxidation).
RESULTS: Data are shown in table as a mean values ? enzymes in IU=l in
serum and IU=g protein in liver, MDA in mmol=l in serum and in mmol=g tissue
CONCLUSION: Increasing of activity above enzymes in serum can testi?ed
that there is failure of hepatocyte. There were no changes of aminotransferases
in serum so we can suggest that there were not enlarge hepatocytes
disintegration. Increasing activity of other enzymes in serum (as well as MDA) and
decreasing in hepatocyte allow to assume that heptocyte membrane change
their property in permeability which can be explain by lipid peroxidation
(increasing of MDA). Such changes may concern other membranes e.g.
adipocyte or cell muscle and in?uence in cell metabolism so modi?cation
diet might normalized it.
Comparison between ad libitum medium chain triglycerides feeding and energy restriction in overweight rats
Regulation of UCP1, UCP2 and UCP3 expression in brown adipose tissue, white adipose tissue and skeletal muscle in rats by estrogen
after 1 month
after 2 months
after 3 months
E Simo?n1 , AS Del Barrio1, and MP Portillo1 1Dpt . Nutrition, University of the Basque Country, Paseo de la Universidad, 7 .01006 Vitoria (Spain) 40%R Group