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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online February 26, 2001 as doi:10.1096/fj.00-0530fje. |
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The Rockefeller University, New York, New York 10021, USA
2Correspondence: The Rockefeller University, Box 278, 1230 York Ave., New York, NY 10021, USA. E-mail: leibow{at}rockvax.rockefeller.edu
SPECIFIC AIM
To identify and clone genes that are expressed in the hypothalamus and involved in the development of obesity through the regulation of food intake and body fat accrual.
PRINCIPAL FINDINGS
1. Identification of Apo D
We used representational difference analysis (RDA) to identify
genes that exhibit increased expression in the hypothalamus of rats
maintained on a high-fat diet, which is known to cause obesity and
stimulate hypothalamic expression of peptides involved in energy
balance. We then investigated whether any of these RDA clones encode
proteins that interact with the long-form receptor of leptin, which
controls food intake and body weight and is stimulated by a high-fat
diet. We performed this experiment by screening a yeast two-hybrid
library and searching the resultant clones for DNA sequences identical
to those generated by RDA.
In the RDA experiment, the cDNA fragments prepared from the medial hypothalamus of rats (Sprague-Dawley, male, n=10/group) maintained for 3 wk on a low-fat diet (10% fat, 3.75 Kcal/g) were subtracted from those of the rats on a high-fat diet (60%, 5.10 Kcal/g) diet, and the resultant cDNA fragments were amplified by PCR. After three rounds of subtraction and amplification, distinct DNA bands were obtained in agarose gel. The subsequent cloning and sequencing of these DNA fragments (53 clones) revealed a clone containing a 0.5 kb cDNA fragment of Apo D. In a GAL4 yeast two-hybrid system, the cytoplasmic domain immediately following the transmembrane region of rat Ob-Rb, hereafter referred as Ob-Rbc (for carboxyl terminal), was used as the bait to screen a rat brain cDNA library (2x106 yeast colonies). Sequencing of 57 positive clones from an X-GAL filter assay revealed a clone that contained a 0.9 kb amino-terminal truncated cDNA fragment of Apo D.
2. Dietary fat stimulates Apo D expression in the hypothalamus
To confirm that dietary fat stimulates the hypothalamic mRNA level
of Apo D, we measured Apo D mRNA in the medial hypothalamus by
quantitative RT-PCR in an additional set of rats
(n=5–6/group) maintained for 3 wk on either a low-fat
(10%), moderate-fat (30%), or high-fat (60%) diet. The results
demonstrate that the relative (to actin) Apo D mRNA level increases
significantly as dietary fat rises from 10% to 30% (+19%,
P<0.03) and even further in rats on a 60% fat diet (+25%,
P<0.001). This increase in dietary fat concentration and
Apo D mRNA was accompanied by a significant rise in circulating levels
of leptin. Body fat pad weights (retroperitoneal, inguinal, mesenteric,
and epididymal), as well as body weight and total daily intake, were
also elevated in the high-fat diet rats.
3. Apo D interacts with Ob-Rb
To confirm the binding between Apo D and Ob-Rbc, we tested their
interaction in a different LexA yeast two-hybrid system. In these
experiments, only the yeast harboring Apo D and Ob-Rbc fusion proteins
grew, and the resultant colonies turned blue within 3 days on the test
medium, demonstrating that Apo D interacts specifically with Ob-Rbc.
We confirmed this interaction with purified proteins by protein-to-protein interaction experiments in vitro. Apo D and Ob-Rbc were expressed in bacteria as a GST fusion protein and a thioredoxin (Trx) fusion protein, respectively, and purified. It was found that 20 µg of Trx·Ob-Rbc coprecipitated with 1 µg of GST · Apo D, but not with 1 µg of GST when GST and GST · Apo D were precipitated with glutathione agarose beads. Reciprocally, 1 µg of GST · Apo D coprecipitated with 20 µg of Trx · Ob-Rbc, but not with 20 µg of Trx, when Trx and Trx · Ob-Rbc were precipitated with Ni-NTA SuperflowTM resin.
4. Apo D does not interact with Ob-Ra
Since the mutation of Ob-Rb into the natural short form, Ob-Ra,
results in obesity in C57BL/ks db-/db-
mice, it was interesting to investigate whether Apo D also interacts
with Ob-Ra. We expressed the cytoplasmic domain of rat Ob-Ra (Ob-Rac)
as a Trx fusion protein (Trx · Ob-Rac) in bacteria and purified it.
In a protein-to-protein interaction experiment, 1 µg of GST · Apo
D did not coprecipitate with 20 µg of Trx · Ob-Rac when
Trx · Ob-Rac was precipitated by Ni-NTA
SuperflowTM resin. Apo D did not interact with
Ob-Rac in the LexA yeast two-hybrid system. Therefore, through
independent approaches, we have demonstrated that Apo D fails to
interact with Ob-Ra.
The above experiments indicate that the amino acid sequence responsible for the interaction with Apo D is present in Ob-Rbc but not Ob-Rac. To confirm this, we generated a truncated Ob-Rbc (Ob-Rbt) by removing a stretch of sequence at the NH2 terminus of Ob-Rbc. We found that 20 µg of Trx · Ob-Rbt coprecipitated with 1 µg of GST · Apo D but not with 1 µg of GST when GST and GST · Apo D were precipitated with glutathione agarose beads. Reciprocally, 1 µg of GST · Apo D coprecipitated with 20 µg of Trx · Ob-Rbt, but not with 20 µg Trx · Ob-Rac, when the Trx fusion proteins were precipitated by Ni-NTA SuperflowTM resin. These experiments indicate that Ob-Rbt is sufficient for the interaction between Apo D and Ob-Rbc. However, the amino acids or motifs on both Apo D and Ob-Rbt involved in this interaction remain to be determined.
We also demonstrated the interaction between Apo D and Ob-R in
vivo in immunoprecipitation experiments with proteins extracted
from pooled rat hypothalamus (Fig. 1
). A single band of 
30 kDa, representing the glycosylated Apo D, was
detected (Fig. 1
, lane 1). In contrast, a mock precipitation generated
no signal (Fig. 1
, lane 2).
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5. Apo D and Ob-Rb are coexpressed in hypothalamic neurons
By using a monoclonal anti-human Apo D antibody, we also observed
the existence of Apo D protein in neurons of hypothalamic nuclei in
immunohistochemical experiments. As shown in Fig. 2
(top panel), the immunoreactivity for Apo D is evident in the cytoplasm
of neurons, both parvocellular and magnocellular, of the
paraventricular nucleus and in small neurons of the arcuate nucleus.
This immunoreactivity is specific to Apo D protein, since no signal was
generated when the above antibody was preabsorbed by purified human Apo
D (not shown). Ob-R immunoreactivity is also present in neurons of the
paraventricular and arcuate nuclei, as revealed by using a polyclonal
anti-Ob-R antibody (Fig. 2
, middle panel). In this double-labeling
experiment, we found that the proteins of both Apo D and Ob-R are
clearly colocalized in the same neurons of these hypothalamic nuclei
(Fig. 2
, lower panel). This coexistence is also observed in other
areas, including the hypothalamic supraoptic nucleus, cortex, and
choroid plexus (not shown).
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6. Hypothalamic Apo D mRNA levels correlate positively with body
fat and circulating leptin
We used quantitative RT-PCR to determine relative Apo D mRNA
levels in the medial hypothalamus of rats exhibiting differential body
fat accrual on a high-fat diet. In this experiment, Sprague-Dawley rats
were fed ad libitum on a high-fat diet for 3 wk. Based on
the amount of body fat accumulated (measured in four dissected depots)
over this period, these subjects were then divided into two subgroups,
referred to as ‘lean’ (n=7) with 15–21 g body fat or
‘obese’ (n=8) with 26–34 g body fat. Whereas both groups
were similar in their total daily intake, the obese rats with 50% greater body fat had significantly higher Apo D mRNA levels in
the medial hypothalamus (1.57±0.05 vs. 1.38±0.04,
P<0.05). They also had considerably higher levels of
circulating leptin (21.5±3.1 vs. 9.8±2.0, P<0.05). Across
the whole group, Apo D mRNA was found to be strongly, positively
correlated with total body fat (r=+0.87, P<0.01), body
weight (r=+0.82, P<0.01), and the level of leptin (r=+0.76,
P<0.01), which in turn was positively related to adiposity
(r=+0.87, P<0.01). Similar results were obtained in inbred
mouse strains (SWR/J and AKR/J) with a differential propensity toward
obesity.
7. Apo D mRNA levels are reduced in medial hypothalamus of
ob-/ob- and db-/db- mice
We further investigated whether Apo D remains positively
associated with body fat mass in mouse strains that are obese due
to a mutated leptin or Ob-Rb gene. We first examined Apo D expression
in C57BL/3j db-/db- mice using quantitative RT-PCR. Due to
their mutational loss of the cytoplasmic portion of Ob-Rb, these mice
presumably do not support an interaction between Apo D and the
mutant Ob-Rb. The level of medial hypothalamic Apo D mRNA in C57BL/3j
db-/db- mice was found to be considerably reduced relative
to that of the wild-type mice (0.69±0.01 vs. 0.88±0.01,
P<0.05). To investigate the influence of the loss of
leptin itself on Apo D expression, we compared hypothalamic Apo D mRNA
in obese C57BL/6j ob-/ob- mice to that of the lean wild-type
C57BL/6j mice. With a mutant leptin but intact Ob-Rb in C57BL/6j
ob-/ob- mice, Ob-Rb could not be stimulated, even though it
may still interact with Apo D. Similar to the result in
db-/db- mice, the Apo D mRNA level was 30% lower in the C57BL/6j ob-/ob- mice compared to their lean
wild-type littermates (0.71±0.01 vs. 1.06±0.01,
P<0.05). These findings, indicating that a functional
leptin/Ob-Rb signaling process is required for the up-regulation of Apo
D expression, support a possible role for hypothalamic Apo D in the
control of body fat
accrual.
CONCLUSIONS AND SIGNIFICANCE
We provide strong evidence suggesting that Apo D interacts with Ob-Rb, but not Ob-Ra, in hypothalamic neurons in vivo. We have also found that fat intake significantly stimulates hypothalamic expression of Apo D, which in turn is strongly, positively correlated with body fat pad weights and circulating levels of leptin. However, the mutant ob-/ob- and db-/db- mice have considerably reduced levels of hypothalamic Apo D mRNA compared to their lean wild-type littermates. These findings indicate that the leptin/Ob-Rb signaling pathway modulates Apo D expression in hypothalamic neurons. We propose that the reduced expression of hypothalamic Apo D in ob-/ob- and db-/db- mice, possibly resulting in a deficiency of Apo D protein, contributes to the development of the obesity, particularly on a high-fat diet.
Apo D belongs to the lipocalin protein family, the members of
which bind and transport small hydrophobic ligands. However, the
specific ligand to which Apo D binds has not been unequivocally
identified. Published evidence suggests that Apo D may bind to multiple
ligands, and each ligand is specific to the tissue or cell type where
Apo D is expressed. We thus speculate that Apo D may be activated
through its interaction with a leptin-stimulated Ob-Rb and may bind a
specific ligand in hypothalamic neurons, where it exerts signaling
functions. This ligand may be produced after leptin stimulation and
serve as a paracrine signal within particular hypothalamic nuclei or a
hormone that enters the circulation. Apo D may thus play a contributing
role in the regulation of body fat accrual. This proposed function of
hypothalamic Apo D is consistent with a previous report that a
Taq I Apo D polymorphism is linked to obesity and
hyperinsulinemia, as well as to noninsulin-dependent diabetes mellitus,
a condition commonly associated with obesity in animals and
humans.FIGURE 3
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FOOTNOTES
1 To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.00-0530fje ; to cite this
article, use FASEB J. (February 22, 2001) 10.1096/fj.00-0530fje 
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