 |
INTRODUCTION |
Leptin is an adipocyte-derived hormone of 167 amino acids (1). It
has potent weight-reducing effects in vivo (2-4). In ob/ob mice, the gene encoding leptin is mutated, resulting
in morbid obesity and associated abnormalities, including hyperphagia, hypothermia, diabetes, and infertility.
The leptin receptor, OB-R,1
is a member of the cytokine receptor family (5). It is encoded by the
diabetes (db) gene, mutation of which also
results in morbid obesity and other abnormalities similar to that in
ob/ob mice. OB-R is alternatively spliced into at least five
transcripts from a single gene. These transcripts encode proteins that
are called the long (OB-Rb), short (OB-Ra, -c, and -d), and soluble
(OB-Re) forms of the leptin receptor. With the exception of the soluble
leptin receptor, receptor isoforms differ from each other by the
alternative use of a unique terminal coding exon (6). OB-Rb is
essential in mediating leptin's weight-reducing and other biological
effects (6, 7).
OB-R is expressed in both the nervous system and peripheral tissues.
The relative levels of expression of different receptor isoforms vary
among different tissues, providing a possible mechanism of regulating
leptin's biological activity at various leptin target sites (8). OB-Rb
is enriched in the hypothalamus, the site of leptin's action on food
intake and body weight. Leptin activation of OB-Rb within this brain
region results in the inhibition of neuropeptide Y/agouti-related
protein neurons and activation of pro-opiomelanocortin/cocaine- and
amphetamine-regulated transcript neurons (9). These neural
circuits in turn mediate leptin's biological effect centrally. OB-Rb
can also activate signal transduction in a variety of peripheral
tissues, including the adipose tissue, T cells, endothelial cells, and
the pancreatic
cells (10-14). Direct leptin signaling at these
additional sites may contribute to the many biological effects of leptin.
Although OB-Rb is essential in mediating leptin's biological effects,
other receptor isoforms may still be necessary for leptin to exert its
full spectrum of in vivo functions. Among the short forms of
the leptin receptor, OB-Ra is most abundantly expressed (15). It is
enriched at the choroid plexus and brain microvessels, sites of
blood-cerebrospinal fluid barrier and blood-brain barrier, suggesting
that it may be involved in the transport of leptin across these
barriers to reach the hypothalamus. One of the mutant OB-R alleles in
rats, fak/fak, does not
have this form of the leptin receptor. They have reduced levels of
leptin in the cerebrospinal fluid, supporting a role of the short form
leptin receptor in leptin transport to the hypothalamus (16, 17).
Previously, we have shown that the secreted form of the leptin
receptor, OB-Re, circulates in mouse plasma and is capable of binding
to leptin (18). The level of both OB-Re and leptin increased by up to
40-fold during late stages of mouse pregnancy, suggesting that the
soluble leptin receptor may modulate leptin's biological activity
in vivo (19). In this report, we demonstrate that, in leptin
nonresponsive ZDF rats, OB-Re expression is increased by more than
20-fold. We also show that adenovirus-mediated overexpression of the
soluble leptin receptor causes increases of circulating leptin without
affecting leptin expression. The elevation of circulating leptin
results from delayed clearance in the presence of overexpressed OB-Re.
Finally, we show that in ob/ob mice, leptin's effect on food intake and body weight is enhanced when its soluble receptor is overexpressed.
 |
EXPERIMENTAL PROCEDURES |
Construction of Adenoviruses Encoding the Soluble Leptin
Receptor--
The cDNA encoding the soluble leptin receptor was
polymerase chain reaction-amplified and subcloned into a shuttle vector pAdCMV. The shuttle vector containing the soluble leptin receptor was
first transfected into 293T cells to verify the correct expression of
the encoded protein. It was then recombined with the adenovirus backbone vector pJM17 by cotransfection into 293 cells. Viruses were
plaque-purified on soft agar and amplified to a titer of 1012 virus particles/ml before being used for injection.
Adenoviruses encoding leptin (Ad-CMV-leptin) (20) and
-galactosidase
(Ad-CMV-
-Gal) (21) were kindly provided by Dr. C. B. Newgard
(University of Texas Southwestern Medical Center, Dallas, TX).
Animals--
Male Zucker Diabetic Fatty (ZDF) rats and Zucker
lean rats at 9 weeks of age were obtained from Dr. Roger Unger's
laboratory (University of Texas Southwestern Medical Center). Lean rats
were divided into two groups. The experimental group received
AdCMV-OB-Re (OB-Re) virus, whereas the control group received
AdCMV-
-Gal virus. After virus injection, rats were fed with high fat
diet containing 20% fat (Teklad, Madison, WI). Female C57Bl/6J
ob/ob mice at 5 weeks of age were purchased from The Jackson
Laboratories (Bar Harbor, ME). Mice received virus injection similarly
as performed in rats. All animals used were housed and cared by the
staff in the Animal Resource Center of University of Texas Southwestern.
Adenovirus Infusion--
Adenoviruses encoding OB-Re or
-galactosidase were injected into the jugular vein of rats weighing
between 250 and 300 g. Each rat received ~1 × 1012 total virus particles in 0.5 ml of PBS. For expression
in mice, each mouse received ~1 × 1011 virus
particles in 0.1 ml of PBS via the tail vein.
Leptin Treatment of ob/ob Mice--
Three days after adenovirus
injection, one group of ob/ob mice was implanted
subcutaneously with Alzet osmotic pumps (model 1002D, Alza Corp., Palo
Alto, CA). The pumps delivered 12.5 µg of mouse recombinant leptin
(Sigma Chemical Co., St. Louis, MO) per day continuously for 12 days.
Food intake and body weight were measured daily.
Plasma Preparation--
Blood samples were collected from the
tail vein into Eppendorf tubes coated with EDTA. Plasma was prepared by
low speed centrifugation (5000 × g, 5 min) and used
for measurement of glucose, free fatty acids, triglyceride, insulin,
the soluble leptin receptor, and leptin.
Northern Blot Analysis of Leptin Expression--
Mice that
received adenovirus injection were sacrificed under sodium
pentobarbital anesthesia. Epididymal fat was dissected immediately,
washed with phosphate-buffered saline, and snap-frozen in liquid
nitrogen. Total RNA was extracted from tissues with TRIzol reagent
(Life Technologies Inc., Gaithersburg, MD) following the
manufacturer's instructions. Northern blotting of leptin was performed
as described previously. Probes were derived by reverse transcriptase-polymerase chain reaction amplification using
primers specific for leptin. Primer sequences were: forward primer,
5'-TGAGTTTGTCCAAGATGGAC-3'; reverse primer, 5'-GCCATCCAGGCTCTCTGG-3'.
Product size was expected to be 190 base pairs. Probe was labeled with
[
-32P]dCTP (PerkinElmer Life Sciences) with a random
primer labeling kit (New England BioLabs).
Soluble Leptin Receptor Assay--
Plasma from ZDF rats and lean
rats was diluted in PBS and incubated with leptin-Sepharose resin
overnight. Leptin beads were washed with PBS three times. After boiling
in 2× SDS sample buffer for 5 min, resin suspension was loaded
directly onto an 8% SDS-PAGE gel and blotted with an anti-leptin
receptor polyclonal antibody as described previously (18). 1-µl
plasma samples from AdCMV-OB-Re-treated rats or mice were run on
SDS-PAGE directly to detect the levels of the soluble leptin receptor
and leptin by Western blotting.
Leptin Assay--
Circulating levels of leptin in plasma of rats
and mice overexpressing the soluble leptin receptor were detected by
Western blotting. Leptin levels in animals that did not receive
AdCMV-OB-Re virus were measured by a rat leptin RIA kit (Linco
Research, St. Louis, MO).
Antibodies--
Polyclonal antibodies against leptin and the
soluble leptin receptor were purchased from Santa Cruz Biotechnology
(Santa Cruz, CA) or as described previously (18, 22).
 |
RESULTS |
Soluble Leptin Receptor Is Elevated in Leptin Nonresponsive ZDF
Rats--
Leptin mRNA levels in the adipose tissue of genetically
obese db/db mice and mice with hypothalamic lesions are
increased by about 20-fold compared with lean controls. The level of
circulating leptin is also elevated in plasma in these mice (23). In
db/db mice, mutation of the leptin receptor results in a
truncation of the long form leptin receptor and loss of leptin
signaling (6, 7). Similar increases of both leptin mRNA and protein also occur in ZDF rats, which have a single amino acid mutation within
the extracellular domain of the leptin receptor (Gln-269
Pro) (24). Because leptin signaling is absent in
db/db mice or ZDF rats, elevated leptin levels in these
mutant animals raises the possibility of a feedback control mechanism
regulating leptin expression.
We asked if the expression of the soluble form of leptin receptor is
changed when leptin signaling is absent, such as in db/db mice or ZDF rats. Previously, we have established an assay to measure
the level of circulating leptin receptor with leptin-Sepharose beads,
which is a useful method for concentrating available receptor protein
for detection (18). The protein level of other forms of the leptin
receptor is too low to be detected directly. Plasma from Zucker lean or
ZDF rats was prepared and incubated with leptin-Sepharose resin to
determine the circulating soluble leptin receptor level. The soluble
leptin receptor present in plasma was estimated by its ability to bind
an excess amount of added immobilized leptin that was coupled to
Sepharose beads. Bound receptor protein is then separated by SDS-PAGE
gel and detected with a polyclonal antibody. This method, however,
cannot be used to concentrate and measure membrane-bound receptors,
because leptin binding is very sensitive to the presence of detergents
(data not shown).
To compare the relative abundance of the soluble leptin receptor in
Zucker lean and ZDF rats, a fixed amount of plasma from lean Zucker
rats and varying amounts of plasma from ZDF rats were used to incubate
with leptin-Sepharose beads. The amount of receptor present in each
sample did not saturate the binding capacity of the leptin resin used,
because controls with a high level of recombinant receptor protein
recovered from leptin-Sepharose resin or loaded directly to SDS-PAGE
give rise to receptor signals with indistinguishable intensity (data
not shown). We also measured the amount of leptin present in each
sample using leptin radioimmunoassay (RIA). Fig. 1A shows the amount of leptin
present in each plasma sample in Zucker lean and ZDF rats. There is
about a 10-fold elevation of leptin in plasma from ZDF rats compared
with lean rats. Fig. 1B is a Western blot of bound soluble
leptin receptor from each plasma sample after incubation with the
leptin-Sepharose beads. Samples containing 2-5 µl of plasma from ZDF
rats generated signal density that is about equal to that from 100 µl
of plasma from Zucker lean rats, indicating that the plasma
concentration of soluble leptin receptor in ZDF rats is elevated by at
least 20-fold. Although the plasma levels of the soluble leptin
receptor from rats of the same genotype vary, a similar -fold increase
of the soluble leptin receptor was found in ZDF rats compared with
Zucker lean rats. This result demonstrates that, in the absence of
leptin signaling, expression of both leptin and its soluble receptor is
elevated in plasma.

View larger version (53K):
[in this window]
[in a new window]
|
Fig. 1.
Circulating levels of both leptin
(A) and its soluble receptor (B) are
elevated in leptin nonresponsive ZDF rats. Leptin level was
measured with RIA, whereas the amount of its soluble receptor in each
sample was determined by leptin-Sepharose pull-down assay. To compare
the relative amount of the soluble leptin receptor present in plasma of
Zucker lean or ZDF rats, different volumes of plasma from ZDF rats were
incubated with leptin-Sepharose resin as indicated. When receptor
signal was compared with that from 100 µl of plasma from a Zucker
lean rat, an increase of the soluble leptin receptor by at least
20-fold was noted in ZDF rats.
|
|
Overexpression of the Soluble Leptin Receptor Leads to a Parallel
Rise of Circulating Leptin without Increasing Leptin
Expression--
Because OB-Re level is elevated in plasma of ZDF rats,
we asked if it plays a role in modulating the level of total
circulating leptin. We chose to test this hypothesis by overexpressing
the soluble leptin receptor in both Zucker lean and ZDF rats. High level in vivo expression of the soluble leptin receptor was
achieved by administration of a recombinant adenovirus containing its
cDNA (AdCMV-OB-Re). When transfected into 293T cells, virally
expressed receptor protein migrated at the same position on SDS-PAGE as the soluble leptin receptor purified from plasma, suggesting that there
is correct post-translational modification (data not shown). Before
adenovirus-mediated overexpression of OB-Re, there were comparable
amounts of leptin present in plasma samples in each genotype as assayed
with RIA; the levels of the soluble leptin receptor were also
comparable in Zucker lean or ZDF rats using the leptin pull-down assay
(data not shown). Two days after adenovirus injection, the leptin
pull-down assay was repeated. Robust expression of the soluble leptin
receptor was detected in both lean and ZDF rats that received
AdCMV-OB-Re virus compared with those that received AdCMV-
-Gal virus
(Fig. 2).

View larger version (46K):
[in this window]
[in a new window]
|
Fig. 2.
Adenovirus-mediated overexpression of the
soluble leptin receptor in vivo. Adenoviruses
encoding OB-Re (+) or -galactosidase ( ) were injected via the
jugular vein into Zucker lean or ZDF rats. 50 µl of plasma from each
rat was prepared and incubated with leptin-Sepharose resin after
dilution in PBS. Bound receptor protein was detected by blotting with a
polyclonal antibody specific for the receptor. Adeno-Re,
adenovirus encoding OB-Re.
|
|
In parallel, we measured the levels of circulating leptin in these
OB-Re-overexpressing rats with RIA. Surprisingly, the leptin level was
over the upper limit of detection in both Zucker lean and ZDF rats
overexpressing the soluble leptin receptor (data not shown). These
results suggest that expression of the soluble leptin receptor leads to
a concomitant increase of circulating leptin. The robust expression of
OB-Re in vivo via adenovirus and a similar rise of
circulating leptin prompted us to test if it is possible to detect both
proteins from plasma directly. In normal rats, the amount of
circulating leptin is too low to be detectable by direct blotting of
plasma proteins. When samples from OB-Re-overexpressing rats were run
on an 8% or 16% SDS-PAGE and detected with antibodies specific for
soluble leptin receptor or leptin, strong signals were obtained for
both proteins in lanes containing samples from rats that received
AdCMV-OB-Re virus (Fig. 3). The signal
for leptin or its soluble receptor was absent in rats that received
AdCMV-
-Gal virus or were sham-operated, suggesting that the
elevation of circulating leptin is the result of overexpression of its
soluble receptor.

View larger version (45K):
[in this window]
[in a new window]
|
Fig. 3.
Parallel increase of circulating leptin after
overexpression of its soluble receptor. Plasma was prepared from
rats that received adenoviruses encoding the soluble leptin receptor
(OB-Re) or -galactosidase ( -Gal)
or that were sham-operated (Sham). 1 µl of plasma from
each sample was loaded onto an 8% or 16% SDS-PAGE gel and blotted for
the soluble leptin receptor (top) or leptin
(bottom). Leptin signal is only detectable in plasma of rats
expressing the soluble leptin receptor.
|
|
Adenoviruses that are injected via the jugular vein or tail vein
preferentially infect the liver. To verify that the soluble leptin
receptor we detected in plasma indeed comes from adenovirus-mediated overexpression of OB-Re but not from induction of endogenous soluble leptin receptor, we performed Northern blot analysis. Rats were sacrificed 2 weeks after treatment with AdCMV-OB-Re or AdCMV-
-Gal. Total RNA was prepared from liver and other tissues, and virally mediated expression of OB-Re was detected with a cDNA probe for OB-R. Fig. 4A shows that the
livers of rats that received AdCMV-OB-Re contain a high level OB-Re
mRNA, which is not detected in the livers of rats that received
AdCMV-
-Gal. AdCMV-OB-Re-treated rats also have a high level of
circulating OB-Re protein, demonstrating that it is produced from the
adenoviruses administered (Fig. 4B).

View larger version (43K):
[in this window]
[in a new window]
|
Fig. 4.
Liver-specific expression of adenoviruses
encoding the soluble leptin receptor. Rats that received
adenoviruses encoding the soluble leptin receptor (Re) or
-galactosidase ( -Gal) were sacrificed, and
total RNA was prepared from liver and other tissues. A,
OB-Re mRNA was detected in the liver of rats that received OB-Re
virus but not control virus ( -Gal).
B, the expressed protein was also readily detected by
Western blotting of plasma protein with an antibody recognizing the
leptin receptor.
|
|
To estimate the -fold increase of the soluble leptin receptor and
leptin after adenovirus treatment, we performed serial dilution analysis of both proteins in plasma. Plasma samples from AdCMV-OB-Re virus-treated ZDF rats were diluted and compared with undiluted sample
before adenovirus injection. Antibody against leptin or its soluble
receptor was used to detect the amount of each protein present in all
samples. Fig. 5 shows that, compared with
the undiluted plasma sample obtained prior to virus treatment, a rise
of about 25-fold for leptin and about 100-fold for its soluble receptor was achieved.

View larger version (51K):
[in this window]
[in a new window]
|
Fig. 5.
Titration of the -fold increase of leptin and
its soluble receptor. Plasma (1 µl) from ZDF rats with
overexpression of the soluble leptin receptor was undiluted, or diluted
1:5, 1:10, 1:25, 1:50, 1:100, and 1:200 in PBS. Total protein in
diluted samples was adjusted by adding plasma from lean rats that did
not overexpress the soluble leptin receptor. These samples were loaded
side by side with plasma from two control ZDF rats that received
AdCMV- -Gal virus ( ). Receptor and leptin signals were compared
after Western blotting to estimate the -fold increase of both leptin
and its soluble receptor after overexpression of OB-Re. The signal in
all lanes above the 49-kDa marker arose from IgG in plasma that reacted
with horseradish peroxidase-conjugated goat anti-rabbit secondary
antibody.
|
|
Soluble Leptin Receptor Does Not Affect Leptin
Expression--
Hyperleptinemia induced by its soluble receptor may be
explained by the following three possibilities: increased leptin
synthesis, increased leptin stability by binding to its overexpressed
soluble receptor or other proteins, or a combination of both. We first tested whether the soluble leptin receptor affects leptin expression at
the transcriptional level by Northern blot analysis. In both ob/ob and db/db mice, expression of leptin is
increased by up to 20-fold at the mRNA level (24). To test whether
the soluble leptin receptor plays a role in leptin expression in
animals with normal leptin function, we overexpressed the soluble
leptin receptor in wild type mice for 3 days and compared the leptin
mRNA levels in mice that received AdCMV-OB-Re virus or
AdCMV-
-Gal control virus. This time point was chosen, because
adenovirus-mediated overexpression reaches a peak level 2-3 days after
virus injection.
Total RNA was prepared from the white adipose tissue of mice treated
with AdCMV-OB-Re or AdCMV-
-Gal and blotted with a cDNA probe
specific for leptin. As shown in Fig. 6,
mice that received OB-Re virus expressed various amounts of the soluble
leptin receptor. In all cases, the levels of circulating leptin were
also elevated. However, the leptin mRNA is not increased in adipose
tissue in response to OB-Re overexpression. We also examined other
tissues to test whether they express leptin after adenovirus-mediated OB-Re overexpression. No leptin signal was detected from all other tissues examined (data not shown).

View larger version (56K):
[in this window]
[in a new window]
|
Fig. 6.
Overexpression of the soluble leptin receptor
does not lead to increase of leptin expression. A,
adenoviruses encoding OB-Re or -galactosidase
( -Gal) were injected into the tail vein of
10-week-old C57Bl/6J mice. Receptor expression and the level of
circulating leptin were simultaneously monitored by Western blotting of
plasma samples with receptor- or leptin-specific antibodies.
B, mice were sacrificed, and epididymal fat was removed from
each mouse. Total RNA was prepared and loaded onto a 1.2% formaldehyde
gel and blotted with a cDNA probe specific for leptin. The amount
of RNA loaded in each lane is shown at the bottom. Leptin
mRNA was not increased in mice that received adenoviruses encoding
the soluble leptin receptor.
|
|
The Soluble Leptin Receptor Protects Leptin from Degradation in
ob/ob Mice--
To further understand the mechanism by which leptin
levels are increased by OB-Re overexpression, ob/ob mice
were given leptin continuously via a subcutaneous miniature osmotic
pump. This method has been used successfully to achieve delivery of
leptin at controlled rates (25). ob/ob mice have no
endogenous leptin due to a mutation in its coding region (1).
Consequently, all circulating leptin will be derived from material
released from the pump. If the soluble leptin receptor increases the
stability of leptin, we would predict that it would cause an elevation
in circulating leptin. As shown in Fig.
7, the soluble leptin receptor reached a
very high level in mice that received AdCMV-OB-Re, but not in those
that received AdCMV-
-Gal. As predicted, circulating leptin is also
elevated by manyfold in mice that received AdCMV-OB-Re. The increased
leptin level in OB-Re-overexpressing mice can be completely explained by its delayed clearance.

View larger version (47K):
[in this window]
[in a new window]
|
Fig. 7.
Stability of circulating leptin in plasma of
ob/ob mice is increased in the presence of its soluble
receptor. Miniature subcutaneous osmotic pumps were implanted to
10-week-old ob/ob mice 2 days after injection of
adenoviruses encoding the soluble leptin receptor (Re) or
-galactosidase ( -Gal). Plasma samples were
prepared at 2-day intervals to monitor the circulating concentration of
leptin and its soluble receptor. Leptin signal was entirely from
material released from the pump, because ob/ob mice have no
endogenous leptin. Mice with OB-Re overexpression have a higher
circulating level of leptin (left four lanes). The
band above leptin signal in all lanes represents nonspecific
cross-reactions of the antibodies used.
|
|
Overexpressed Soluble Leptin Receptor Enhances Leptin's Effect on
Body Weight and Food Intake in ob/ob Mice--
Does the
hyperleptinemia induced by the soluble leptin receptor affect leptin's
effects on food intake and body weight? To answer this question, we
studied the response of ob/ob mice to exogenous leptin when
they are also treated with AdCMV-OB-Re or Ad-CMV-
-Gal virus. After
leptin pump implantation, both groups of ob/ob mice (with or
without OB-Re overexpression) started to lose body weight at similar
rates. Food intake was also similarly reduced (Fig.
8). Interestingly, when OB-Re is
overexpressed, a further reduction of food intake and body weight was
observed, beginning at day 7 after pump implantation. Because leptin
activity is determined by the amount of free leptin, this result
suggests that similar levels of free leptin may be present in both
groups of mice at the start of the experiment. The increased biological activity of leptin may be the result of extra free leptin that has
saturated the binding capacity of overexpressed OB-Re or that was
dissociated from its soluble leptin receptor. The absolute concentration of free leptin in the absence or presence of its soluble
receptor at each time point remains to be determined.

View larger version (85K):
[in this window]
[in a new window]
|
Fig. 8.
The soluble leptin receptor enhances
leptin's activity on food intake and body weight in ob/ob
mice. Two groups of mice (n = 4/group) were
treated with leptin via subcutaneous pumps with (OB-Re) or
without ( -Gal) overexpression of its soluble
receptor. Leptin's effect on food intake (A) and body
weight (B) was measured daily. Arrows on top of each
graph indicate the time point the pump was removed. Leptin's
effect was enhanced in the presence of its soluble leptin
receptor.
|
|
Taken together, our data demonstrate that leptin level in plasma is
determined by at least two factors, as depicted in Fig. 9. Its production is controlled primarily
by the degree of adiposity, with acute regulation by feeding state,
insulin, and other factors. Its steady-state level is also modulated by
its soluble receptor and other binding proteins. A combination of
factors regulating leptin synthesis and stability may determine the
total circulating level of leptin in different nutritional and
physiological states.

View larger version (9K):
[in this window]
[in a new window]
|
Fig. 9.
Modulation of circulating leptin levels in
plasma. Leptin expression is determined by the size of adipose
tissue, feeding state, insulin, and other factors. Its steady-state
level is also modulated by circulating levels of its soluble receptor
and possibly other binding proteins. This model predicts that a higher
total leptin concentration in plasma may not reflect the body's
sensitivity to leptin, because bound leptin is not active.
|
|
 |
DISCUSSION |
In a previous study, we demonstrated that the soluble leptin
receptor circulates in plasma and is capable of binding to leptin (18).
Other groups also found that soluble leptin receptor level is elevated
by up to 40-fold at late stages of mouse gestation (19), suggesting
that expression of the soluble leptin receptor is regulated and that it
may modulate leptin's biological activity in vivo.
In the current study, we extended these observations further and
demonstrate that a feedback regulation of the expression of both leptin
and its soluble receptor occurs in vivo. In the absence of
leptin signaling, both proteins are increased by more than 20-fold in
plasma. Although plasma leptin level is closely correlated with
adiposity in most individuals (26), our data demonstrate that its
soluble receptor also plays key roles in determining the amount of
leptin in circulation. This may be an important mechanism of regulating
leptin's availability and bioactivity, because no post-translational
modification of leptin occurs in vivo (27). The simultaneous
upsurge of both leptin and its soluble receptor during late stages of
mouse pregnancy could be explained by the stabilization of leptin by
its soluble receptor (19). Similarly, elevated leptin levels in
fa/fa pups compared with lean controls (fa/+ and
+/+) may also be the combined effect of increased leptin expression and
delayed clearance (28). The mechanism governing the expression of
soluble leptin receptor remains to be determined.
The main site of expression of the soluble leptin receptor in
vivo is not known. Previously, we failed to detect a signal for
OB-Re when Northern blot analysis was performed (15). Available data
have demonstrated that OB-Re is expressed by the placenta in mice. Its
expression starts at day 14 of pregnancy, peaking just before
parturition to about 40-fold the level found in nonpregnant mice (19,
29). In rats and humans, the pregnancy-associated rise of circulating
leptin and its soluble receptor is relatively modest, achieving only a
2-fold increase versus a more than 40-fold increase in mice
(30). Alternatively, soluble leptin receptor may also be produced by
proteolytic cleavage of membrane-associated receptor.
Why is leptin expression in the adipose tissue of db/db mice
or ZDF rats increased? One possibility is the feedback inhibition of
leptin via its receptor in wild type animals. It is known that OB-R is
expressed on the surface of adipocytes (6, 10). At a certain threshold
concentration, leptin may directly signal the adipocyte to stop its own
production utilizing the receptor on the surface of these adipocytes.
Alternatively, the central nervous system may also respond to leptin
signaling by releasing neurotransmitters or neuropeptides to control
the synthesis and release of leptin into circulation. A loss of these
pathways due to leptin signaling deficiency in db/db mice or
ZDF rats may contribute to the increased production of leptin.
Our findings provided a plausible explanation of several unexplained
observations regarding the level of circulating leptin. For example,
there is a lack of correlation between the leptin mRNA level and
protein level in some obese subjects. The leptin mRNA level is
lower in obese subjects than expected based on differences of
circulating leptin (31). This could be explained by the presence of
increased soluble leptin receptor in these individuals. A support for
this hypothesis came from a recent clinical study of patients with the
same mutations of the leptin receptor that truncate OB-R 5' of the
transmembrane region. The truncated product resembles the soluble
leptin receptor. As a result, circulating levels of leptin in these
patients are very high (32), analogous to the elevation of leptin by
its soluble receptor that we reported here. Similarly, the circulating
level of leptin in C57BL/Ks db/db mice was unchanged after
15 days of food restriction, whereas its level became undetectable in
lean mice 6 days after food restriction (24). The persistence of
circulating leptin in food-restricted db/db mice may also be
regulated by its soluble leptin receptor. Alternatively, other leptin
binding protein in serum may also play a role (33, 34).
We did not observe a major change of food intake or body weight in
Zucker lean rats overexpressing the soluble leptin receptor. Although
OB-Re overexpression raises circulating leptin by manyfold, it is
obviously inactive when bound to its soluble receptor. The slightly
enhanced leptin effect on food intake and body weight in
ob/ob mice may be due to a higher level of free leptin in
the presence of OB-Re overexpression. When a large pool of
leptin-receptor complex is present in circulation, bound leptin is
constantly released, resulting in a net increase of free leptin in
plasma. Conversely, when new leptin release is low, such as during food restriction, free leptin may not decrease as rapidly if a pool of
leptin-receptor complex preexists in plasma. Studies are underway to
determine whether bound leptin affects leptin signal transduction through its long form receptor in vitro.
The new mechanism in regulation of circulating leptin concentration
reported here has added an additional level of complexity to the
modulation of leptin's biological activity and availability. In the
absence of leptin signaling, increased output of its soluble receptor
results in an elevation of circulating leptin that is not from leptin
overexpression. The leptin that is bound by its soluble receptor
appeared to be inactive but may be made available for release into
circulation and activate leptin responses. It remains to be determined
what is the main site of expression of the soluble leptin receptor and
how its own stability is regulated. A fuller understanding of these
issues should shed more light on the mechanisms of action used by
leptin in the control of so many distinct yet important physiological
processes, ranging from appetite and body weight to fertility, immune
function, and bone formation.