From the
Genetic studies in mice have identified the ob gene
product as a potential signaling factor regulating body weight
homeostasis and energy balance. It is suggested that modulation of ob gene expression results in changes in body weight and food
intake. Glucocorticoids are shown to have important metabolic effects
and to modulate food intake and body weight. In order to test the
hypothesis that these metabolic effects of glucocorticoids are linked
to changes in the expression of the ob gene, ob mRNA
levels were evaluated in rats treated with different
glucocorticosteroids at catabolic doses and correlated to the kinetics
of changes in body weight gain and food intake. Results from time
course experiments demonstrate that adipose tissue ob gene
expression is rapidly induced by glucocorticosteroids. This induction
is followed by a concordant decrease in body weight gain and food
consumption. These data suggest that the catabolic effects of
corticosteroids on body weight mass and food intake might be mediated
by changes in ob expression. Modulation of ob expression may therefore constitute a mechanism through which
hormonal, pharmacological, or other factors control body weight
homeostasis.
Obesity, which can be defined as a body weight more than 20% in
excess of the ideal body weight, is a major health problem in Western
societies, since it is associated with an increased risk for
cardiovascular disease, diabetes, and an increased mortality
rate(1) . Obesity is the result of a positive energy balance, as
a consequence of an increased ratio of caloric intake to energy
expenditure. The molecular factors regulating food intake and body
weight balance are incompletely understood. Five single-gene mutations
resulting in obesity have been described in mice, implicating genetic
factors in the etiology of obesity(2) . In the ob mouse
a single gene mutation, obese, results in profound obesity,
which is accompanied by diabetes(3) . Cross-circulation
experiments have suggested that ob mice are deficient of a
blood-borne factor regulating nutrient intake and energy
metabolism(4) . Using positional cloning technologies, the mouse ob gene, and subsequently its human homologue, have been
cloned recently(5) . Amino acid sequence analysis revealed a
high degree of homology between mouse and human ob, indicative
of an important regulatory function of this protein. The ob gene is exclusively expressed in white adipose tissue, suggesting
that the protein can best be imagined as a fat-derived satiety factor. ob mice either have a non-sense mutation, resulting in the
production of a non-functional gene product (C57Bl/6J ob/ob), or carry a genomic mutation resulting in the
complete absence of ob mRNA
(SM/Ckc-+
The aim of the present work was to
identify factors regulating ob gene expression and to evaluate
whether changes in ob gene expression are correlated with
changes in food intake and body weight. Since pharmacological doses of
glucocorticoids have a strong catabolic action, the effects of high
doses of glucocorticoids on the expression of the ob gene were
studied and correlated to changes in body weight and food intake. The
rat was chosen as a model, since body weight and adipose tissue mass
keep increasing throughout its entire lifespan, thereby resembling the
human situation of adult onset obesity. Furthermore, the relative short
lifespan of this animal permits the study of factors affecting body
weight homeostasis in a relatively short time period. The results from
our studies demonstrate that administration of pharmacological doses of
glucocorticoids induces adipose tissue ob gene expression.
This induction is accompanied by reduced food intake and decreased body
weight gain in these animals. These data indicate that modification of ob gene expression is subject to hormonal/pharmacological
regulation, leading to the modulation of caloric intake and body mass
gain.
To study the effects of pharmacological doses of
glucocorticoids on body weight, food intake, and ob gene
expression, 80-day-old male rats were treated once daily during 20 days
with 100 µg of hydrocortisone/g of body weight Sham-treated control
rats exhibited a significant, steady gain in body weight throughout the
treatment period, attaining approximately 110% of the initial body
weight after 20 days (Fig. 1). Administration of hydrocortisone,
however, completely prevented this gain in body weight and resulted in
a slight decrease in body weight at the end of the treatment period (Fig. 1). This difference in body weight gain between control and
treated animals became only gradually apparent; the first 2 days of
treatment body weights did not differ significantly from controls, and
only thereafter a gradually more pronounced difference was observed.
Compared with untreated animals, hydrocortisone-injected animals
consumed 10-15% less food throughout the entire treatment period (Fig. 2), indicating that a reduction of food intake may, at
least in part, account for the lower gain in body weight after
hydrocortisone treatment.
Genetically obese ob mice display a marked increase
in body and adipose tissue mass and a pronounced hyperphagia. The
cloning of the mouse and human ob genes and the demonstration
that the ob gene is exclusively expressed in white adipose
tissue and markedly increased in C57Bl/6J ob/ob mice
suggest that the level of expression of this gene correlates with the
size of the adipose depot. The ob gene product may therefore
function as part of a pathway regulating body fat mass and food intake.
An increase in the ob signal may act directly or indirectly on
the central nervous system to inhibit food intake and/or regulate
energy expenditure as part of a homeostatic mechanism to maintain body
weight balance. It is therefore conceivable that the level of ob expression is inversely correlated with food intake, energy
expenditure, and the onset of obesity.
In an attempt to understand
the regulation of the ob gene and the impact of its regulation
on body weight and food consumption, we studied the effects of
different corticosteroids at pharmacological doses, which provoke
severe metabolic perturbations and a reduction in food consumption. Our
results demonstrate that glucocorticoids induce ob expression
in rat adipose tissue, whereas a gain in body weight and food intake
decrease concomitantly. Although the existence of a correlation does
not prove causality, several lines of evidence argue in support of a
causal relationship between the induction of ob gene
expression, on the one hand, and the decrease in food intake and body
weight, on the other hand. First, the induction of ob gene
expression is very rapid and nearly maximal within 24 h after a single
injection of corticosteroids. By contrast, the changes in body weight
follow much more gradually, the difference with sham-treated controls
only becoming significant after 3 days of treatment. Taking into
account that a 16-h overnight fast reduces the body weight of rats by
approximately 7.5% (fed, 376 ± 12 g; fasted, 350 ± 10 g),
it appears that the effects of corticosteroids on body weight changes
are much more gradual and lag behind the induction of ob gene
expression. Second, the induction of ob expression by
corticosteroids is independent of food intake since it is observed
regardless of whether animals are fed or fasted. Third, it is unlikely
that the alterations in ob expression are secondary to the
decrease in food intake and body weight, since ob mRNA levels
are increased in hyperphagic C57Bl/6J ob/ob mice.
Finally, in contrast to normal mice, genetically obese ob/ob mice are dramatically resistant to
glucocorticoid-induced weight loss(10) , indicating that the
presence of a functional ob gene product is required to
transmit the glucocorticoid-induced weight loss. Therefore, it seems
that the induction of ob expression after corticosteroid
treatment precedes and probably provokes the observed alterations in
food intake and body weight. In this respect it is interesting to note
that plasma corticosterone levels are elevated in obese C57Bl/6J ob/ob mice(11, 12, 13) , which
may, at least in part, explain the increase in ob mRNA levels
observed in these mice(5) .
Corticosteroids seem to exert a
dual metabolic action on gain in body weight and feeding efficiency,
strictly depending on the dose used(9) . Administration of high
doses of glucocorticoids, such as in this study, have a dramatic
catabolic action, resulting in a marked decrease in food intake and
body weight. In contrast, administration of lower doses of
corticosteroids has an anabolic activity, which is accompanied by
increased appetite in humans and stimulation of food intake in
laboratory animals. However, in contrast to their catabolic effects, it
is unlikely that the anabolic effects of glucocorticoids, observed
after administration of low doses, are mediated through changes in ob gene expression. Indeed, although ob/ob mice do
not express a functional ob gene product, adrenalectomy
reduces food intake and normalizes energy
balance(14, 15, 16) , whereas corticosteroid
replacement therapy restores food intake in these adrenalectomized ob/ob mice(17) . This unequivocally proves that the
anabolic action of steroid hormones is ob-independent.
The
effects of corticosteroids on ob gene expression may be due to
a direct action of these hormones on ob gene transcription.
Alternatively, these hormones may have indirect effects. Administration
of high doses of glucocorticoids may, for instance, influence the
plasma concentrations of other hormones that regulate food intake, such
as dehydroepiandrosterone or corticotropin-releasing hormone.
Alternatively, high doses of glucocorticoids increase gluconeogenesis,
predispose to diabetes, and may therefore increase plasma glucose
concentrations. According to the glucostasis theory, decreased plasma
glucose concentrations would be a signal, triggering food intake. Thus,
glucocorticoids may act by increasing plasma glucose concentrations,
which in its turn may induce ob gene expression resulting in a
reduction of food consumption. In this case factors involved in glucose
metabolism, such as glucose itself, glucagon, and insulin, would be
expected to be important modulators of ob gene expression.
In conclusion, we have shown that in vivo glucocorticoid
treatment induces ob gene expression. Our data suggest that
modulation of ob expression is inversely correlated with
alterations in food intake and body weight. Knowledge of the regulation
of ob gene expression and factors implicated therein will
therefore be important in the prevention and treatment of obesity.
We thank D. Cayet for excellent technical assistance
and acknowledge the interesting discussions with Drs. M. Briggs, R.
Heyman, R. Mukkerjee, J. Rosen, and J.-C. Fruchart.
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
-ob
/ob
)
(5). ob mRNA levels are highly increased in C57Bl/6J ob/ob mice, suggesting that the level of expression
of this gene signals the size of the adipose depot in an attempt to
maintain body weight homeostasis.
Animals and Treatments
Eighty-day-old male rats
received once daily subcutaneous injections with the indicated
corticosteroids at a dose and for the period of time indicated. Control
animals received saline only. Rats were group-housed and accustomed to
a 12:12 h day-night illumination cycle. Animals were allowed free
access to standard rat chow. Body weight (per animal) and food
consumption (per treatment group) were measured at regular intervals
throughout the experiment. At the end of the experiment, animals were
killed between 9 and 10 a.m. by exsanguination while under ether
anesthesia. Epididymal fat pads were removed immediately and frozen in
liquid N.
RNA Analysis
Total cellular RNA was prepared by
the acid guanidinium thiocyanate/phenol-chloroform method(6) .
Northern and dot blot hybridizations of total cellular RNA were
performed as described previously(7) . A mouse ob cDNA
fragment spanning nucleotides +50 to +659 was cloned from
adipose tissue by reverse transcription and polymerase chain reaction
amplification (sense primer: 5`-CCA AGA AGA GGG ATC CCT GCT CCA GCA
GC-3`; antisense primer: 5`-CCC TCT ACA TGA TTC TTG GGT ACC TGG TGG
CC-3`)(5) . The resulting polymerase chain reaction fragment was
cloned into pBluescript KS, and sequence analysis revealed complete
identity to the reported mouse ob cDNA sequence(5) . A
-actin cDNA clone was used as a control probe(8) . All
probes were labeled by random primers (Boehringer Mannheim). Filters
were hybridized to 1.5
10
cpm/ml of each probe as
described(7) . They were washed once in 0.5
SSC and 0.1%
SDS for 10 min at room temperature and twice for 30 min at 65 °C
and subsequently exposed to x-ray film (X-Omat AR, Kodak).
Autoradiograms were analyzed by quantitative scanning densitometry
(Bio-Rad GS670 densitometer) as described(7) .
Figure 1:
Treatment with hydrocortisone reduces
gain in body weight. Adult male rats received for the indicated number
of days once-daily subcutaneous injections of hydrocortisone (100
µg/g of body weight). Control animals received saline only. Body
weights were recorded at regular intervals and are expressed as a
percentage of pretreatment (day 0) body weight. Values represent the
mean ± S.D. of 4 animals/group.
Figure 2:
Treatment with hydrocortisone reduces food
intake. Adult male rats (n = 4/group) were treated as
described under Fig. 1. Total food consumption of each treatment group
was measured at regular intervals and is expressed as a percentage of
the food intake of a group of sham-treated
controls.
Since the ob gene product has
been suggested to be a factor modulating food intake and energy
utilization, the regulation of adipose tissue ob mRNA
expression by hydrocortisone was determined next. Treatment with
hydrocortisone increased ob mRNA levels more than 2-fold, an
effect that was already maximal after 2 days (Fig. 3). ob mRNA levels remained elevated throughout the entire treatment
period. This induction was specific, since -actin mRNA levels
remained constant throughout the entire treatment period (Fig. 3).
Figure 3:
Kinetics of induction of adipose tissue ob mRNA by hydrocortisone. Adult male rats (n = 4/group) were treated as described under Fig. 1. Adipose
tissue was isolated, RNA was extracted, and ob and -actin
mRNA levels were measured as described under ``Materials and
Methods.'' Values represent the mean ± S.D. of 4 animals
and are expressed in relative absorbance units (R.A.U.) taking
the pretreatment values as 100%. Statistically (analysis of variance, p < 0.05) significant differences are observed between
values followed by different letters (
-actin, not
significant).
Corticosteroids are known to exert dual metabolic
actions, reflected by a bitonic dose-response curve for body weight
gain(9) . In order to evaluate the dose-dependent effects of
hydrocortisone on body weight and ob gene expression adult
rats were treated once daily during 20 days with three different doses
of hydrocortisone (1, 10, or 100 µg/g of body weight),
respectively, corresponding to doses at the top, the middle, and the
bottom of the descending arm of the body weight gain-corticosteroid
dose-response curve(9) . Treatment of adult rats for 20 days
with different doses of hydrocortisone resulted in a dose-dependent
reduction in body weight gain (Fig. 4A), which was
accompanied by a dose-dependent induction of ob mRNA levels in
adipose tissue (Fig. 4B).
Figure 4:
Dose-dependent effects of hydrocortisone
on body weight gain (A) and adipose tissue ob mRNA
levels (B). Adult male rats (n = 4
animals/group) received during 20 days once-daily subcutaneous
injections of hydrocortisone at the indicated doses. Control animals
received saline only. PanelA, body weights were
recorded at the beginning and end of the experiment and are expressed
as a percentage of pretreatment (day 0) body weight. PanelB, at the end of the experiment adipose tissue was
isolated, RNA extracted, and ob and -actin mRNA levels
measured as described under ``Materials and Methods.'' Values
are expressed in relative absorbance units (R.A.U.) taking the
controls as 100%. Values represent the mean ± S.D. Statistically
(analysis of variance, p < 0.05) significant differences
are observed between values followed by different letters (
-actin,
not significant).
Finally, the effects of the
synthetic glucocorticoids dexamethasone and triamcinolone, which are
relatively pure type II corticosteroid receptor agonists and produce a
more pronounced monotonic negative dose-response curve of body weight
gain(9) , were analyzed and compared with hydrocortisone.
Treatment of adult male rats during 4 days with triamcinolone or
dexamethasone also resulted in a marked decrease in body weight, which
was paralleled by a similar decrease in food consumption (Fig. 5A). Concomitantly, ob mRNA levels
increased after all three glucocorticoids tested (Fig. 5B). Northern blot hybridization analysis
indicated that the ob cDNA probe hybridized to an mRNA of
approximately 4.5 kilobases, a size similar to mouse adipose tissue ob mRNA(5) . Furthermore, ob mRNA levels
already increased 2.2-fold in rat adipose tissue within 24 h after one
single injection of dexamethasone (Fig. 5C), thereby
indicating that the induction of ob gene expression by
corticosteroids is a very rapid event.
Figure 5:
Influence of different corticosteroids on
body weight, food consumption (A), and adipose tissue ob mRNA levels (B and C). Panels A and B, adult male rats (n = 3 animals/group) were
treated for 4 days with vehicle (CON), hydrocortisone (HC, 100 µg/g of body weight/day), triamcinolone (TRIAM, 20 µg/g of body weight/day), or dexamethasone (DEXA, 3.7 µg/g of body weight/day). Body weights and food
consumption were recorded at the end of the experiment and are
expressed as a percentage of the controls (PanelA).
Adipose tissue was isolated, RNA extracted, and ob and
-actin mRNA levels measured as described under ``Materials
and Methods'' (PanelB). Values are expressed in
relative absorbance units (R.A.U.) taking the controls as
100%. Values represent the mean ± S.D. Statistically (analysis
of variance, p < 0.05) significant differences are observed
between values followed by different letters (
-actin, not
significant). PanelC, adult male rats (n = 3 animals/group) were sacrificed 24 h after a single
injection of dexamethasone (DEXA; 3.7 µg/g of body weight)
or vehicle (CON). Total RNA (10 µg) extracted from
individual animals was pooled and subjected to electrophoresis,
transferred to a nylon membrane, and hybridized consecutively to
labeled ob (toppanel) or
-actin (bottompanel) cDNA as described under
``Materials and Methods.'' The positions of the 18 and 28 S
rRNA bands are indicated on the right of the toppanel.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.