(Received for publication, November 6, 1995; and in revised form, January 30, 1996)
From the
Mutation of the obese (ob) gene results in severe
hereditary obesity and diabetes in the C57BL/6J and related strains of
mice. In this study we examined the expression of the ob gene
in a dietary model in which moderate obesity develops in response to
fat (58% of calories from fat) without mutation of the ob gene, and in four genetic models of obesity in mice: ob/ob, db/db, tubby, and fat.
Several white and brown adipose depots were examined (epididymal,
subcutaneous, perirenal, and interscapular). Northern blot analysis
shows that levels of ob mRNA are increased in all adipose
depots examined in every model of obesity. The average fold increases
were 12.0 ± 2.1 (ob/ob), 4.8 ± 1.5 (db/db), 2.8 ± 0.1 (tubby), 2.4 ± 0.3 (fat), and 2.1 ± 0.2 (high fat diet-induced A/J).
Moreover, we found that the expression of the ob gene could be
manipulated by pharmacologically blocking the development of
diet-induced obesity. Supplementation of a high fat diet with a
-adrenergic receptor agonist (CL316,243) prevented
obesity, but not hyperphagia associated with high fat feeding (body
weights of high fat-fed A/J mice = 34.0 ± 1.0 g; high fat
plus CL316,243-fed mice = 26.8 ± 0.5 g; n = 10). CL316,243-treated, high fat-fed animals contained
levels of ob mRNA in all adipose depots that were equal to or
less than levels in low fat-fed mice (average levels in high fat plus
CL316,243-fed mice relative to low fat-fed mice: 0.93 ± 0.09).
Inasmuch as fat cell size, but not number, was increased in a previous
study in diet-induced obese A/J mice, these results indicate that
expression of the ob gene serves as a sensor of fat cell
hypertrophy, independent of any effects on food intake.
The recent cloning of the ob gene has led to renewed
interest in the genetic basis of obesity(1) . This autosomal
recessive mutation is most often expressed on the C57BL/6J (B/6J)
background strain and is associated with severe obesity,
hyperinsulinemia, hyperglycemia, hypercorticosteronemia, and
infertility. Early studies by Coleman and Hummel (2) and
Coleman (3) led to the proposal that ob may encode a
diffusable signaling factor, while the product of a separate obesity
mutation, db (``diabetic''), residing at a different
chromosomal location, has been postulated to receive the signal from
the diffusable factor; perhaps encoding a receptor for ob(4) . The cloning and sequence analysis of the ob locus suggests that the ob gene indeed encodes a
protein that is secreted from adipocytes(1) . In the ob/ob mouse, a nonsense mutation in the coding region appears to prevent
the production of a functional OB protein, and a marked up-regulation
of ob mRNA has been observed in adipose tissue of ob/ob mice(1) . From these data, and because it is widely
believed that obesity in the ob/ob mouse is caused by
hyperphagia(5) , it has been proposed that this protein
normally serves as a feedback regulator of satiety(1) . Support
for this hypothesis has been provided by several recent studies in
which ob/ob or db/db mice were administered
recombinant OB protein(6, 7, 8) . Decreased
food intake and body weight were observed in ob/ob mice, but
the OB protein had no effect in db/db mice(7, 8) . However, in order to demonstrate
that the OB protein truly serves a sensor function, the expression of ob protein must be shown to be modulated through alteration of
fat mass. We now report that the expression of the ob gene can
be increased by diet and adipocyte hypertrophy, and reduced by
stimulation of -adrenergic receptors
(
AR), (
)independent of effects on food
intake. These data suggest that changes in ob gene expression
do not predict changes in food intake.
As previously reported(1, 17) , ob gene expression was elevated in obese, diabetic ob/ob and db/db mice (Fig. 1). In the ob/ob mice, the
increase ranged from 10- to 14-fold, while the overexpression in db/db mice was 4- to 8-fold. These values are somewhat less
than reported by other investigators but consistent with their
findings. Based upon the early studies by Coleman (2, 3) he proposed that ob encodes a
diffusable signaling factor and that the db locus might encode
the receptor for ob. Thus, persistently up-regulated
expression of ob mRNA in the db/db diabetic mouse
would be consistent with this model, in which db would be
incapable of receiving the signal from ob. Support for this
original hypothesis has recently been obtained from studies in which
recombinant OB protein was administered to ob/ob and db/db mice(6, 7, 8) . In these studies,
decreased food intake and body weight were observed in ob/ob mice, but the OB protein had no effect in db/db mice(7, 8) . These latter results support the
notion that the db locus is down-stream in the pathway from ob. However, we and others have also observed significantly
increased expression of the ob gene in other genetic models of
obesity that do not possess mutations in the ob locus. For
example, the levels of expression and nucleotide sequence of the ob gene from the Zucker ``fatty'' (fa/fa) rat have
been examined(18, 19) . While similar increases in ob mRNA are observed in obese fa/fa rats, unlike ob/ob mice the ob gene from the Zucker fa/fa is not mutated. More recently, increased expression of ob in overweight humans without mutations of the ob gene has
also been reported(20) . We examined ob expression in
several white and brown adipose tissues from ``tubby'' (tub/tub) and ``fat'' (fat/fat) mutant
mice(21) , including gonadal, subcutaneous, perirenal, and
interscapular. The fat locus has recently been identified as
carboxypeptidase E(22) , but the nature of the mutation in tubby has not yet been reported. These two animal models
develop obesity more gradually with age and do not possess the severe
hypercorticism that is such a prominent feature in ob/ob or db/db mice (21) . ()As shown in Fig. 2, ob mRNA was significantly increased in adipose
tissue from tubby and fat mice as compared with
genetically lean (``+'') littermates, although the
degree of overexpression is less dramatic than in ob and db mice. Table 1presents the quantitative comparisons
for individual adipose depots. The results demonstrate that, similar to ob and db animals, a high degree of adiposity in tubby and fat mice is associated with elevated levels
of ob expression.
Figure 1:
Overexpression of ob mRNA in
genetically obese ob/ob and db/db mice. Northern
blots containing 40 µg of total RNA from epididymal white adipose
tissue (EWAT), or interscapular brown adipose tissue (IBAT) were prepared as pools from two or three animals as
described under ``Experimental Procedures'' and hybridized
successively with P-labeled probes for the mouse ob coding region and glyceraldehyde 3-phosphate dehydrogenase (gapdh). The position of the ob or gapdh mRNA is
indicated by the arrowhead. A, comparison of
genetically lean (+) C57BL/6J mice and obese (ob) mice.
In the data shown, the level of ob mRNA is increased 14-fold
in the ob/ob mutant (average of two experiments = 12.0
± 2.1-fold increase). B, comparison of genetically lean
(+/+) C57BL/KsJ mice and diabetic (db/db) mice. In
the data shown, the level of ob mRNA is increased 4-fold in
EWAT and 8-fold in the IBAT of the db/db mutant. The data
shown are representative of two
experiments.
Figure 2:
Overexpression of ob mRNA in tubby and fat mutant mice. A, comparison of
genetically lean (+) C57BL/6J mice and tubby (tub) mice. B, comparison of genetically lean (+) C57BL/KsJ mice and
fat (fat) mice. Northern blots containing 30 µg of total
RNA from the indicated adipose tissue depots were prepared as described
under ``Experimental Procedures'' and hybridized successively
with P-labeled probes for the mouse ob coding
region and cyclophilin (cyclo): epididymal white adipose
tissue (EWAT), interscapular brown adipose tissue (IBAT), subcutaneous (SQ), and perirenal (PR). The position of the ob or cyclo mRNA is
indicated by the arrowhead.
Unlike these mutant strains of mice which
develop severe obesity on laboratory chow, which is quite low in fat
(4.5% of calories from fat, according to manufacturer, Purina), most
human obesity is thought to occur in response to high fat
diets(23, 24) . For this reason we have been studying
the physiological and biochemical features of diet-induced obesity in
genetically ``lean'' normal mice. We have previously reported
that A/J mice will develop moderate obesity on a high fat
diet(11) . Attempts to develop effective treatments for obesity
have included several reports that AR agonists are
effective in reducing body weight and adipose tissue mass following
acute treatment of ob/ob mice or diet-induced obese rats (13, 25, 26, 27) . Therefore we
decided to test whether chronic supplementation of the high fat diet
with a highly selective
AR agonist, CL
316,243(12) , could prevent diet-induced obesity in A/J mice
and whether the effect could be sustained over prolonged treatment. The
body weights of animals on the three diets are shown in Fig. 3.
Compared with animals fed the low fat diet, the high fat-fed animals
exhibited significant increases in total body weight even within the
first 2 weeks. This trend continued throughout the 16 weeks of the
experiment. However, animals consuming the high fat diet supplemented
with the
AR agonist were resistant to the
obesity-inducing effects of the fat in the diet (Fig. 3). In
fact, they tended to weigh even less than animals consuming the low fat
diet.
Figure 3:
Obesity induced by high fat feeding and
its prevention by a AR agonist. Thirty A/J male mice
were obtained from The Jackson Laboratories at 4 weeks of age. Groups
of 10 mice were randomly assigned to a low fat diet (
), high fat
diet (
), or high fat diet containing 0.001% CL316,243 (
).
Animals were weighed at biweekly intervals through the 16-week period
on the diets.
We next examined the levels of ob mRNA in epididymal,
subcutaneous, and perirenal white adipose tissue and interscapular
brown adipose tissue from these animals (Fig. 4). Levels of ob mRNA in high fat-fed obese mice increased 1.48 ±
0.07-fold in epididymal, 2.45 ± 0.32-fold in subcutaneous, and
1.78 ± 0.13-fold in perirenal depots compared to low fat-fed
control animals. In interscapular brown adipose tissue from high
fat-fed animals some samples showed a modest elevation, but overall
there was no significant increase (1.07 ± 0.19-fold) compared
with low fat-fed mice. Most likely the levels of ob expression
observed in this brown adipose depot are derived from small amounts of
white adipocytes present in the tissue that we were unable to separate
by visual inspection alone. By contrast to these increases seen in
adipose tissue from the high fat-fed mice, these same adipose tissue
regions from mice consuming the high fat diet supplemented with the
AR agonist expressed levels of ob mRNA that
were equal to, or slightly less than, that observed in animals fed the
low fat diet (0.93 ± 0.09-fold). Thus, these data suggest that
we were able to manipulate the expression of the ob gene by
pharmacologically blocking the development of diet-induced obesity,
despite the consumption of a calorically rich diet.
Figure 4:
Increased expression of ob mRNA
in diet-induced obese A/J mice and its repression by a
AR agonist. Northern blots containing 40
µg of total RNA from the indicated adipose tissue depots were
prepared from A/J animals consuming the low fat (LF) diet, or
the high fat (HF) diet alone or supplemented with the
AR agonist CL316,243 (HF+CL): epididymal
white adipose tissue (EWAT), interscapular brown adipose
tissue (IBAT), subcutaneous (SQ), and perirenal (PR). The blots were hybridized successively with
P-labeled probes for the mouse ob coding region
and cyclophilin (cyclo). The position of the ob or
cyclo mRNA is indicated by the arrowhead. The data shown are
representative of three experiments. The average fold changes in ob mRNA are described in the text.
In our studies
as well as those of other investigators, the fold increases in ob mRNA observed in either diet-induced obese A/J mice, or the
severely obese ob/ob or db/db, are consistently
greater than would be predicted based upon increase in total body
weight alone. For example, we observe a 25% increase in body
weight of A/J mice consuming a high fat diet, but there is a
50-250% increase in ob mRNA levels. Similarly, ob/ob mice generally display a 75-100% increase in body
weight(4) , but a 10-20-fold increase in ob mRNA
levels (1, 17) (Fig. 1). When we examined fat
pad weights in A/J mice raised on these three diets, the average
increase over all depots in high fat fed animals increased by 2.30
± 0.30 (n = 3), while in the presence of the
AR agonist, average fat pad weights were essentially
identical to low fat-fed animals (fold change = 1.03 ±
0.13; n = 3). These values are very similar to the
changes observed in ob mRNA levels in this study.
In a
previous study we have shown that high fat feeding in A/J mice causes
adipocyte hypertrophy, but not hyperplasia(11) . Therefore, the
differences in weight in A/J mice raised on high fat diets with and
without the AR agonist CL316,243 are most likely due
to differences in adipocyte size between the groups, but not in number.
Thus, the difference in ob mRNA in these mice, compared to
those raised on fat alone, suggests that OB is a
``sensor'' of fat cell size presumably sending a signal,
either autocrine, paracrine, or endocrine to elicit some metabolic
response; possibly to control energy partitioning and utilization.
Interestingly, A/J animals consuming the high fat diet supplemented
with the
AR agonist, CL316,243, also display increases
in brown adipose tissue content and
AR gene
expression. (
)
Recent research has emphasized the role of
leptin in satiety(6, 7, 8) . In these studies
decreased food intake and body weight were observed in ob/ob mice, but the OB protein had no effect in db/db mice.
Campfield et al.(8) also found that administration of
recombinant OB protein to diet-induced obese AKR/J mice caused
reductions in food intake and body weight, although to a more modest
degree than in ob/ob mice. To understand the relationship
between food intake and ob gene expression in our dietary
model and the effects of a AR agonist, we examined
food intake in individual animals from all three diet groups at three
times during the study period (Fig. 5). These data show that the
caloric intake of animals on either of the high fat diets was
equivalent and greater than that consumed by animals fed the low fat
diet. However, despite this greater caloric density of the high fat
diet, animals supplemented with CL316,243 actually weighed less than
low fat fed animals. Therefore, our experiments appear to highlight a
disassociation between ob gene expression and caloric
consumption. In summary, we have shown that A/J mice consuming a high
fat diet produce more leptin and become moderately obese, while mice
consuming the high fat diet supplemented with CL316,243 do not become
obese or show increases in ob gene expression, despite the
fact that they consumed as much, or more, food than animals on high fat
alone. It is possible that treatment with CL316,243 may serve to mimic
effects of OB protein involved in enhancing metabolic and/or
thermogenic activity, as recently speculated(28) . Therefore,
because food intake in animals fed high fat diets with and without the
-agonist was similar, these results indicate that
there is no apparent obligatory effect of OB protein on satiety.
Figure 5: Food intake. Food intake measurements over a 24-h period were made for individual animals at three different times during study (2, 6, and 16 weeks on the diets). Each group of 10 animals was housed individually and grams of food consumed over 24 h were measured. Caloric content of each diet was calculated based on 5.55 kcal/g for the high fat diets and 4.07 kcal/g for the low fat diet. #, significantly different from low fat-fed animals at p < 0.05. *, significantly different from low fat-fed and high fat-fed animals at week 6 and from low fat-fed animals at week 16, p < 0.005.