(Received for publication, November 7, 1995; and in revised form, December 21, 1995)
From the
Regulation of obese gene (ob) expression in ob/ob and db/db mice and in cultured rat adipocytes
was examined. It has been demonstrated that exogenous human OB protein
(leptin) treatment reduces food intake and weight gain, as well as
insulin, glucose, and corticosterone levels in ob/ob mice. In
the present report we show that leptin treatment down-regulates
endogenous adipose ob mRNA. However, treatment of isolated rat
adipocytes with 100 ng/ml human or murine leptin had no direct effect
on expression of endogenous ob mRNA, suggesting that leptin
may be able to down-regulate its own expression by an indirect,
non-autocrine mechanism. Glucocorticoids increased both ob mRNA levels and secreted leptin levels in vitro.
Conversely, agents that increase intracellular cAMP, such as
-adrenergic agonists or Bt
cAMP itself, decreased ob mRNA expression and leptin secretion. Therefore, increased
glucocorticoid levels and decreased sympathetic neural activity may
contribute to the elevated ob mRNA expression observed in
genetically obese, hyperglucocorticoid rodents. Furthermore, leptin
might regulate its own expression through a feedback mechanism
involving the hypothalamic pituitary axis.
Obesity is a major risk factor for a number of human diseases
including cardiovascular disease, hypertension, and
non-insulin-dependent diabetes. Although obesity in humans is
apparently a polygenic disorder, numerous rodent models of obesity
exist as single gene mutations. Several of these which have recently
been identified include the agouti gene(1, 2) , the fat gene(3) ,
and the ob (obese) gene (4) . The ob locus on chromosome 6 involves two mutations in the gene for a
secretory protein, which result in either premature termination
(nonsense mutation at Arg-105) in the original C57BL/6J ob/ob strain or complete absence of message in the
SM/Ckc-+ob
/ob
strain(4) . The human homolog has been
cloned(4, 5) , and there appears to be no evidence of
obesity-associated mutations in human OB comparable with the ob mouse (5) nor any linkage between OB mutations and susceptibility to non-insulin-dependent
diabetes(6) . The ob mutation was originally thought
to involve a diminished satiety signal or factor based on parabiosis
experiments in both ob/ob and db/db mice(7) ,
which suggested that ob/ob mice did not express the satiety
signal while db/db mice were defective in the signal
transduction pathway. Recent experiments have supported this by
demonstrating that exogenous recombinant mouse Ob protein and human OB
protein decrease food intake and weight gain in the ob/ob, but
not in the db/db,
mouse(8, 9, 10, 11) . It has
recently been suggested that the ob gene product be referred
to as leptin(9) .
Despite the evidence that leptin acts as a
satiety factor, human obesity (5) and other forms of rodent
obesity that involve different genetic loci, such as the A mouse(12) , or non-genetic
lesions in the ventramedial hypothalamus (13, 14) are
paradoxically associated with elevated levels of leptin expression. For
this reason, we investigated the regulation of ob expression
in ob/ob and db/db mice, and in 24 h cultures of
mature rat adipocytes to determine what obesity associated factor(s)
might regulate leptin expression.
We have previously reported that treatment of ob/ob and db/db mice with recombinant human leptin induced weight loss and reduced food consumption, and lowered glucose, insulin, and glucocorticoid levels in the ob/ob mice only(11) . In the treated ob/ob mice, corticosterone levels were reduced 60% from 247 ± 9 to 95 ± 18 ng/ml (p < 0.05, n = 6) while insulin levels were reduced from 75 ± 28 ng/ml to <1.4 ng/ml, and glucose levels were reduced from 214 ± 16 mg/dl to 118 ± 7 mg/dl. Untreated lean Ob/? mice had glucose, insulin, and glucocorticoid values of 144 ± 12 mg/dl, <1.5 ng/ml, and 31 ± 6 ng/ml, respectively(11) . In the db/db mice, neither glucose (676 ± 18 mg/dl), insulin (<1.5 ng/ml), nor corticosterone (318 ± 17 ng/ml) levels were altered significantly with treatment (11) .
In the present study, Northern analysis of total RNA from epididymal fat pads from each group of mice showed that ob mRNA levels were reduced 65% in treated ob/ob mice (ob mRNA/18 S RNA: control, 1.23 ± 0.14; treated, 0.44 ± 0.06, p < 0.05) but were unchanged in db/db mice (Fig. 1, A and B). Although many factors secondary to weight loss or reduced food consumption may have contributed, it is possible that the reduction in glucocorticoids and insulin or the administration of exogenous leptin itself may have acted to down-regulate endogenous ob mRNA expression. The observation that leptin down-regulates its own expression in ob/ob mice, but not in db/db mice that have a presumed central defect in its signal transduction, would suggest that leptin does not act directly at the adipocyte but instead through a centrally regulated mediator. However, these data do not rule out the possibility that a putative adipocyte leptin receptor is also defective in db/db mice.
Figure 1:
A, chronic effects of exogenous human
leptin on endogenous murine ob mRNA expression in ob/ob and db/db mice. RNA was isolated and hybridized to P-labeled murine ob probe as described under
``Materials and Methods.'' RNA loading was evaluated by
quantitation of 18 S RNA. B, graph quantitates ob mRNA relative to 18 S RNA. Data are presented as relative ratios
± S.E. (n = 6). C, effect of 24-h
incubation of rat adipocytes on endogenous ob mRNA levels: lane 1, control; lane 2, 25 nM dexamethasone; lane 3, 100 ng/ml human leptin; lane
4, 100 ng/ml mouse leptin. These data represent a single
experiment.
In order to examine direct (autocrine regulation at the adipocyte) versus indirect effects of leptin on ob mRNA regulation, we treated rat adipocytes in vitro with either 25 nM dexamethasone or 100 ng/ml human or mouse leptin for 24 h. Northern analysis (Fig. 1C) shows that leptin treatment did not alter endogenous ob mRNA levels but that 25 nM dexamethasone up-regulated ob expression dramatically. Therefore, the ability of exogenous human leptin to down-regulate endogenous expression of ob mRNA in the ob/ob mouse argues for an indirect mechanism, possibly secondary to reduced glucocorticoid levels. It should be noted, however, that our measurements are of steady-state mRNA levels, and in the absence of nuclear run-on experiments we cannot distinguish transcriptional regulation from effects on mRNA stability. The recent report that treatment of rats with dexamethasone and hydrocortisone increased adipocyte ob mRNA expression levels is consistent with these in vitro results (18) .
The development of a sensitive RIA allowed us to examine a number of agents to investigate their role in regulating expression of leptin. The limit of detection for this assay is approximately 0.5-1 ng/ml. Cross-reactivity between rat and mouse leptin was confirmed by showing that rat serum diluted in parallel with the mouse leptin standard (data not shown). However, the absolute level of cross-reactivity has not been determined quantitatively with recombinant rat leptin standard. The anti-mouse leptin antibody also cross-reacted with secreted rat leptin in a Western blot. These results are reasonable based on the high degree of conservation of the protein sequence between rat and mouse leptin (96% at the protein level(13) ) and suggest that this assay is suitable for measuring relative, if not necessarily absolute, changes in rat leptin levels.
Because of our initial in vitro observation that dexamethasone up-regulated ob mRNA expression, we examined a number of steroids, including
glucocorticoids, mineralocorticoids, estrogens, and androgens, for
their ability to regulate leptin expression and secretion (data not
shown). Preliminary experiments indicated that glucocorticoids such as
dexamethasone, hydrocortisone, and corticosterone had a pronounced
3-4-fold stimulatory effect on leptin expression (as measured by
RIA). Testosterone and aldosterone were relatively ineffective (data
not shown), while 17-estradiol increased leptin release
approximately 2-fold. Fig. 2shows a dose response for
stimulation of leptin secretion by dexamethasone, hydrocortisone, and
17
-estradiol. Both glucocorticoids reach the same maximal response
(3-3.5-fold increase over basal) with the synthetic
glucocorticoid being more potent (EC
: dexamethasone, 3.3
nM; hydrocortisone, 24.7 nM). These values are
consistent with other gluccorticoid-induced functions in adipocytes,
such as inhibition of preadipocyte proliferation(19) .
Dexamethasone and hydrocortisone increased ob mRNA expression (Fig. 3A) as well as leptin secretion (Fig. 3, B and C).
Figure 2:
Dose response for stimulation of rat
leptin secretion by dexamethasone and hydrocortisone. Adipocytes were
incubated serum-free for 24 h in culture in the presence of increasing
doses of steroid as described under ``Materials and
Methods.'' Conditioned medium was collected from beneath the
adipocyte layer and assayed for leptin levels by RIA. ,
dexamethasone;
, hydrocortisone;
, 17-
estradiol.
Values represent single points or averages of two separate experiments
± S.E.
Figure 3:
Effect of glucocorticoids, insulin, and
BtcAMP/
-adrenergic receptor agonists on rat ob mRNA and secreted leptin levels. Rat adipocytes were incubated as
described in the legend for Fig. 2: lane 1, control; lane 2, 25 nM insulin; lane 3, 25 nM dexamethasone; lane 4, 100 nM hydrocortisone; lane 5, 0.5 mM Bt
cAMP; lane 6, 10 µM isoproterenol + 100 µM ascorbate; lane 7, 10 µM ICI 201,651. After
24 h, cells were recovered for RNA isolation, and conditioned medium
was used for RIA and Western analysis of leptin levels. A,
Northern blot.
-Actin is shown to compare total RNA loading. B, Western blot, lanes are same order as in A; C, RIA. RIA data represent averages ± S.E. of three
separate experiments. Data are plotted as percent of control. Control
adipocytes typically secrete 12-18 ng of leptin/10
cells/24 h. Northern and Western data are representative of two
independent experiments.
Insulin treatment increased leptin secretion only 20-25% as measured by RIA (Fig. 3). Effects of insulin on ob mRNA expression have been variable, probably reflecting the difficulty in measuring small changes in RNA by Northern analysis. Therefore, the effect of insulin under these conditions appears modest. Incubation of adipocytes with 25 mM glucose had no effect on ob mRNA expression (data not shown), suggesting that ob expression may not be regulated directly by acute changes in glucose availability. A recent report by Saladin et al.(20) indicated that a single insulin injection or a hyperinsulinemic clamp of rats increased ob mRNA expression severalfold, independent of glucose concentration. In vitro treatment of adipocytes with insulin for 24 h also doubled ob mRNA. However, Murakami et al.(21) reported that in vitro insulin treatment of adipocytes increased ob mRNA only 10%. Insulin clearly increases leptin expression, but the degree would appear to be less than that of glucocorticoids.
Increases in intracellular cAMP result
in decreased expression of ob mRNA and leptin secretion in rat
adipocytes. This is demonstrated in Fig. 3, where it is shown
that dibutyryl cAMP, the non-selective -agonist isoproterenol, and
the selective
agonist ICI 201,651 (22) all
reduce ob mRNA and protein expression. This would suggest that
when lipolysis is stimulated in adipocytes, leptin expression is
reduced. This hypothesis is consistent with decreased expression of a
satiety signal under conditions of starvation. A recent report has
demonstrated that fasting of lean Ob/? mice (but not ob/ob mice) is associated with a reduction of ob mRNA levels,
which is reversed upon refeeding(23) . Pertussis toxin (50
ng/ml) and adenosine deaminase treatment (0.5 unit/ml) reduced leptin
secretion (as measured by RIA) 60 and 20%, respectively, suggesting a
role for G
in regulating leptin secretion. The effect of
adenosine deaminase was mimicked by the adenosine antagonist
8-phenyltheophylline (2 µM) and was blocked by the
adenosine agonist phenylisopropyl adenosine (10 nM) (data not
shown).
Interestingly, ICI 201,651 is less effective than
isoproterenol at lowering ob expression, even at the
relatively high concentration of 10 µM. This is similar to
the effect of another selective agonist BRL 37344 on
cAMP production in rat adipocytes and may represent a weaker coupling
of the
receptor to cAMP production(24) .
Since
agonists induce weight loss through increased
thermogenesis in rats(25) , it is interesting that they also
down-regulate expression of leptin, which similarly increases the basal
metabolic rate in rats(10) .
We have previously demonstrated that leptin treatment of ob/ob mice reduces synthesis and release of hypothalamic NPY, and that this may represent its mechanism of action on food intake and basal metabolic rate(11) . It has been suggested that glucocorticoids and insulin regulate energy balance by their central regulation of food intake through NPY and through their peripheral effects on energy storage(26) . Centrally, glucocorticoids increase NPY levels and food consumption(26) , while insulin inhibits NPY release (27) . The data presented here suggest that leptin may represent another arm of this regulatory pathway. Increased glucocorticoids directly stimulate leptin secretion from adipose tissue, which then negatively modulates NPY release in the hypothalamus(11) . Increased NPY levels would also lead to decreased sympathetic activity (28) which, based on our results, would lead to further increases in leptin secretion. This would explain the observed increased leptin expression in genetic models of rodent obesity, such as the Zucker fa/fa rat(29) , since these animals are characterized by hyperglucocorticoidism, elevated hypothalamic NPY, and reduced sympathetic activity(30) . Increased NPY is also associated with increased insulin secretion, which could further contribute to elevated leptin levels.
In summary, we have demonstrated that treatment of ob/ob mice with exogenous human leptin down-regulates endogenous murine ob mRNA expression, while similar treatment of ob-resistant db/db mice has no effect. Treatment of isolated rat adipocytes with leptin in vitro has no effect on endogenous ob mRNA levels, while glucocorticoids up-regulate expression of ob mRNA and protein secretion. Agents that increase intracellular cAMP down-regulate ob expression. These results suggest a centrally mediated mechanism of feedback regulation of ob expression involving the hypothalamic-pituitary-adrenal axis.