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INTRODUCTION |
Diacylglycerol kinases
(DGKs)1 are involved in the
modulation of subcellular levels of the second messengers,
diacylglycerol and phosphatidic acid, as well as in the synthesis of
triacylglycerols (1). Based on structure, eukaryotic DGKs are
classified into five subgroups. These DGKs share a conserved catalytic
domain and cysteine-rich regions. However, each group has unique
domains that bind with calcium, phosphatidylinositols, and proteins. By Northern and Western blot, DGKs have been found in a wide variety of
tissues, where different DGKs coexist in the same cells or tissue. Some
DGK isoforms, including DGK
, are expressed in high levels in brain,
muscle, and white blood cells. They are associated with the cell
membrane and are also present in the cytosol, nucleus, and other
specific subcellular organelles (2-6). Protein kinase C and
receptor tyrosine kinase regulate both the enzymatic activity and
subcellular location of the DGKs (4, 7-10).
This report focuses on DGK
, which belongs to a subgroup of DGKs that
has unknown physiological function (10-14). DGK
is characterized by
its four C-terminal ankyrin repeats and a unique region homologous to
MARCKS phosphorylation site domain. DGK
exists in both the cytosol
and nucleus under the regulation of specific types of protein kinase C
that phosphorylate the MARCKS site. In the nucleus, DGK
modulates
nuclear levels of diacylglycerol and increases the cell cycle duration,
probably through effects on gene expression (10, 13). Its role in
regulating gene activity is also indicated by the findings that its
expression is temporally and spatially regulated during embryonic
development and correlates with the development of sensory neurons and
regions undergoing apoptosis (13). Furthermore, DGK
may also
participate as a key enzyme in the biosynthesis of complex lipids. This
is suggested by the fact that DGK
is widely and abundantly expressed
throughout the body (14) and that it can promiscuously use various
kinds of long chain diacylglycerols as substrates, whether or not they are second messengers (11).
Dietary fat is an important factor that contributes to the development
of obesity. In rodents, it has been demonstrated that the concentration
of fat in the diet, but not protein or carbohydrate, is strongly,
positively correlated with the amount of body fat mass and that free
access to a high-fat diet causes obesity and hyperinsulinemia (15-17).
These effects of dietary fat may be mediated, at least in part, by
changes in the expression of genes in the brain that are involved in
energy balance (18-20). Dietary fat also affects plasma levels of
leptin, a hormone that exerts a key function in regulating food intake
and body weight (21). Leptin controls energy balance through its long
form receptor (Ob-Rb) on neurons in the hypothalamus (22). It is
believed to function through a Jak/STAT signal transduction pathway
(23) to promote fat oxidation (24), satiety (25), and homeostasis of
lipids (26). The mutation of this hormone or its receptor causes morbid
obesity in rodents and humans (27-30). Moreover, serum leptin levels
are strongly, positively correlated with body fat mass (31, 32).
In this study, we sought to identify genes that are functionally linked
to both dietary fat and Ob-Rb in the hypothalamus. We demonstrate
several lines of evidence indicating an interaction between DGK
and
the cytoplasmic portion of Ob-Rb in vitro and in
vivo. Further analyses demonstrate that DGK
is expressed in neurons of the hypothalamus and that a high-fat diet stimulates DGK
expression in hypothalamus. Moreover, hypothalamic DGK
expression is
found to be reduced in obese animals and strongly, inversely related to
both body fat mass and serum leptin level. Based on these results, we
propose that the enzymatic activity of DGK
may be activated in
response to a high-fat diet and that DGK
may participate in the
control of body fat accumulation.
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EXPERIMENTAL PROCEDURES |
Animals, Tissues, and Physiological Studies--
Male Harlan
Sprague-Dawley rats and inbred mouse strains, AKR/J, SWR/J, C57BL/6j,
and C57BL/6j ob-/ob- (Charles River Laboratories), and
C57BL/3j and C57BL/3j db-/db- (Dr. Cai Li, Texas
Southwestern Medical University, Dallas, TX), were individually housed
and maintained on either a low-fat diet (10% fat, 25% protein, 65% carbohydrate, 3.75 Kcals/g), moderate-fat diet (30% fat, 25% protein, 45% carbohydrate, 3.98 Kcals/g), or high-fat diet (60% fat, 25% protein, 15% carbohydrate, 5.10 Kcals/g). Procedures for diet preparation, measurement of food intake and body weight, and dissection of body fat pads (inguinal, epididymal, intraperitoneal, or mesenteric) and hypothalamus, are described elsewhere (18). Serum level of leptin
was determined by radioimmunoassay (Linco Research). All other rat
tissues were obtained from Harlan Bioproducts.
Identification and Cloning of DGK
--
Hypothalamus from 10 rats on a high-fat (60%) or low-fat (10%) diet were dissected and
pooled for the purification of mRNA, which was then used for
representational difference analysis as described (33). The
cDNA fragment of rat Ob-Rbc, obtained by RT-PCR with primers
5'-TCACACCAGAGAATGAAAAAG-3' and 5'-CACAGTTAAGTCACACATCTTA-3', was used
to screen a rat brain two-hybrid library
(CLONTECH). By RT-PCR, the cDNA fragment of rat
DGK
was obtained with primers 5'-TTTTCATATGGAGCCGCGGGACCCCAG-3' and
5'-TTTTGTCGACTACACAGCTGTCTCCTGGTCC-3'. DGK
with all four
ankyrin repeats deleted (DGK
a) was obtained with primers
5'-TTTTGAATTCATGGAGCCGCGGGACCCCAG-3' and
5'-TTTTGTCGACAGTGCGGCATCCCCCTGCAG-3'. The ankyrin repeats
of DGK
(DGK
a) was obtained with primers 5'-TTTTGAATTCGCACTGCCCCAAGGTGAAG-3' and
5'-TTTTGTCGACTACACAGCTGTCTCCTGGTCC-3'.
Protein Expression and Purification--
The cDNA fragment
of rat Ob-Rac was obtained by RT-PCR with primers
5'-TCACACCAGAGAATGAAAAAG-3' and 5'-AAGAGTGTCCGCTCTCTTTTG-3'. The
cDNA fragments for Ob-Rac and Ob-Rbc were subcloned into plasmid pET-32a(+) (Novagen) for expression as thioredoxin (Trx) fusion proteins in bacterial strain BL21(DE3)pLysS (Novagen). To generate Ob-Rbt, pET-32a(+)-Ob-Rbc was digested by KpnI, followed by
T4 DNA polymerase and ligation. The bacteria expressing Trx-Ob-Rbc and
Trx-Ob-Rbt were solubilized in buffer TUNN (10 mM Tris, 8 M urea, 100 mM NaH2PO4,
0.5% Nonidet P-40) plus 5 mM imidazole, pH 7.9. The
proteins were purified with a Ni-NTA Superflow column (Qiagen) by
washing sequentially with TUNN plus 20 mM imidazole, pH
7.9, and TUNN plus 20 mM imidazole, pH 6.3. The proteins
were eluted with TUNN plus 20 mM imidazole, pH 5.6, and
renatured by dialysis against 3 × 2 liters of phosphate-buffered
saline, pH 7.4, 2 mM dithiothreitol, 10% glycerol at
4 °C for 36 h. DGK
, DGK
a, and DGK
a were subcloned
in-frame into pGEX-5X-1 (Amersham Pharmacia Biotech), expressed in
bacterial strain BL21, and purified as GST-DGK
a with a
glutathione-Sepharose 4B column.
Protein Precipitation--
The purified proteins were combined
with either 50 µl of 50% Ni-NTA Superflow-agarose resin or 50 µl
of 50% glutathione-Sepharose 4B in 1 ml of 150 mM NaCl, 50 mM Tris-HCl, pH 7.4, 1% Nonidet P-40, 1 mM
EGTA and phenylmethylsulfonyl fluoride, and 1 µg/ml each of leupeptin
and pepstatin A. The mixture was shaken at 37 °C for 1 h,
pelleted at 400 × g in a microcentrifuge, and washed 4 times with 1 ml of the above buffer. Proteins were separated in a 12%
polyacrylamide gel and transferred onto an Immobilon-P membrane
(Millipore). Trx, Trx-Ob-Rac, Trx-Ob-Rbc, and Trx-Ob-Rbt were assayed
by a mouse monoclonal antibody against His·Tag (Oncogene Research
Products). GST and GST-DGK
a were assayed by a goat polyclonal antibody against GST (Amersham Pharmacia Biotech). Alkaline phosphatase conjugate secondary antibodies were from Sigma and detected with NBT/BCIP. For in vivo immunoprecipitation, 100 rat
hypothalamus were homogenized in 10 ml of phosphate-buffered saline, pH
7.4, plus 1 mM EGTA and 1 µg/ml each of leupeptin and
pepstatin A, followed by centrifugation at 24,000 × g
at 4 °C for 1 h. This extract (7.8 mg/ml) was then divided into
two parts and combined with 100 µg of goat anti-Ob-Rb antibody (Santa
Cruz Biotechnology) plus protein G-agarose beads (Upstate
biotechnology) or with 100 µg of normal goat IgG (Oncogene Research
Products) plus protein G-agarose beads, and shaken at 4 °C
overnight. The beads were washed 4 times, each with 12 ml of
phosphate-buffered saline, pH 7.4, by 400 × g at
4 °C for 5 min. After the final wash, the beads were transferred
into a column and eluted with 0.3 ml of 65 °C water. DGK
was
assayed by a polyclonal rabbit anti-DGK
antibody (11) in Western blot.
Quantitative RT-PCR and Quantification--
PCR was set up in a
total volume of 20 µl of 50 mM Tris-HCl, pH 8.9, 15 mM (NH4)2SO4, 1.5 mM MgCl2, 1 µM of each primer,
0.2 mM dNTP, 5 units of Taq polymerase
(Promega), and 1/20 volume of 1 µg of medial hypothalamus RNA reverse
transcription reaction (20 µl) as template. Primers for actin
(Invitrogen, Carlsbad, CA) were included for simultaneous amplification
with either DGK
or Ob-Rbc. PCR was conducted in 18 cycles in a
Thermal cycler 480 (PerkinElmer Life Sciences). PCR products were
separated in a 5% polyacrylamide gel, stained by ethidium bromide, and
digitally quantified by an imaging densitometer GS-700 (Bio-Rad). The
results were averaged from four independent experiments.
In Situ Hybridization--
Antisense and sense riboprobes were
transcribed in vitro from linearized DNA of plasmid
pGEM-Teasy containing a cDNA fragment of DGK
by using SP6 or T7
RNA polymerase in the presence of biotin-UTP (PerkinElmer Life
Sciences). The probe for DGK
(736 base pairs) corresponded to
2054-2790 base pairs of the coding sequence at the 3' end of the
cDNA. In situ hybridization was performed on frozen
brain sections of adult male Harlan Sprague-Dawley rats on a high-fat
diet as described (18), and the signal was enhanced with tyramide
(PerkinElmer Life Sciences) and detected by nitro blue
tetrazoliium/5-bromo-4-chloro-3-indolyl phosphate.
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RESULTS |
Identification of DGK
--
In an attempt to clone genes that
regulate food ingestion and body fat accrual, we used representational
difference analysis (RDA) (33) to identify genes that exhibit increased
expression in the hypothalamus of rats maintained on a high-fat diet,
which is known to enhance hypothalamic expression of peptides involved in energy balance (18, 20, 34). This method resulted in a large number
of candidate clones. To choose the most promising clones from these
candidates, we then explored if any of these encode proteins that
interact with the long form receptor of leptin, which controls food
intake and body weight and is also stimulated by a high-fat diet (35,
36). We used the yeast two-hybrid technique (37) to screen a rat brain
cDNA library for proteins that interact with the cytoplasmic domain
of Ob-Rb, and searched the resultant clones for DNA sequences that are
identical to those generated by RDA.
In the RDA experiment, cDNA fragments made from the hypothalamus of
adult, male Harlan Sprague-Dawley rats (n = 10/group) maintained for 3 weeks on a low-fat diet (10% fat, 3.75 Kcals/g) were
subtracted from those of rats on a high-fat diet (60% fat, 5.1 Kcals/g), and the quantity of the resultant fragments was amplified by
PCR (Fig. 1). After three rounds of
subtractive hybridization and amplification, the resultant distinct DNA
bands were cloned, obtaining 53 clones. Sequencing of these RDA
products revealed a clone encoding part of the ankyrin repeats of
DGK
. In a GAL4 yeast two-hybrid system, the cytoplasmic domain
immediately following the transmembrane region of rat Ob-Rb (Ob-Rbc)
was used as the bait to screen a rat brain two-hybrid cDNA library.
An initial screening of ~2 × 106 yeast colonies
yielded 436 clones, of which 57 clones tested positive by
5-bromo-4-chloro-3-indolyl
-D-galactopyranoside (X-gal) filter assay. Sequencing analysis revealed that two of these clones contain a 0.8-kilobase cDNA fragment that encodes a partial
sequence of the ankyrin repeats of DGK
(Fig.
2). Comparison with the published sequence of rat DGK
(14) reveals that this partial sequence encodes
the third and fourth ankyrin repeats of DGK
, as well as the last 12 amino acids of the second repeat (Fig. 2).

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Fig. 1.
Identification of
DGK . Three rounds of subtractive
hybridization (1st, 2nd, and 3rd) were used to
obtain small cDNA fragments representing up-regulated genes
expressed in the hypothalamus of rats maintained on a high-fat diet. 40 µg of cDNA fragments made from the hypothalamus of rats
(n = 10) on a low-fat (10%) diet was used to subtract
0.1 µg of cDNA fragments from high-fat (60%) diet rats
(n = 10), followed by PCR. The product obtained from
each PCR (1 µg) was resolved in 1% agarose gel. The position of
DGK is indicated (arrowhead) based on the length of the
cDNA fragment.
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Fig. 2.
The partial sequence of
DGK ankyrin repeats obtained by screening a
two-hybrid cDNA library, compared with the sequence of the intact
ankyrin repeats of rat DGK . The four
ankyrin repeats of DGK are underlined, with each of the
repeats containing 33 amino acids. The partial sequence starts at 2530 base pairs and encodes the last 12 amino acids of the second repeat and
the complete third and fourth repeats.
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Hypothalamic Expression of DGK
in Relation to Dietary
Fat--
The above RDA experiment indicates that dietary fat
stimulates hypothalamic DGK
expression. We confirmed this, by
quantitative RT-PCR, in an additional set of rats (n = 5-6/group) fed for 3 weeks on either a low-fat (10% fat),
moderate-fat (30%) or high-fat (60%) diet. The results demonstrate
that the DGK
mRNA level (relative to actin) increases (+20%,
p < 0.02) as dietary fat rises from 10 to 30%, and it
increases even further (+36%, p < 0.001) in rats on a 60% fat diet (Table I). This
increase in dietary fat concentration and DGK
mRNA is
accompanied by a significant rise in circulating levels of leptin
(Table I). Body fat pad weights (retroperitoneal, inguinal, mesenteric,
and epididymal), as well as body weight and total daily intake, are
also elevated in the high-fat diet rats (Table I).
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Table I
Dietary fat stimulates hypothalamic DGK mRNA in rats
Hypothalamus was excised from each individual rat or mouse and was used
to purify total mRNA and synthesize the first strand cDNA. The
cDNA fragments of DGK and actin were amplified by PCR for 20 cycles, which was determined to be within the exponential range. The
PCR products were resolved on agarose gels, and their quantities were
determined by a Bio-Rad GS-700 densitometer. The relative DGK
mRNA level was obtained by comparing optical densities of DGK
fragments with those of corresponding actin fragments and by averaging
these data from four independent experiments. Circulating leptin level
was determined in each rat or mouse. Body fat scores reflect weight of
4 dissected fat pads, and total intake (over 24 h) reflects an
average of frequent measures taken over the 3-week test period.
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DGK
Interacts with Ob-Rb via Its Ankyrin Repeats--
The
identification of DGK
by the yeast two-hybrid technique indicates
that DGK
interacts with Ob-Rbc. Since only two DGK
clones were
obtained from the rat brain cDNA library, the binding of DGK
to
Ob-Rbc may be weak. To confirm this, we performed
-galactosidase activity assays in the GAL4 yeast two-hybrid system to measure the
strength of this interaction. While the negative control generated 0.2 units of
-galactosidase activity, 5 units of activity were found in
the interaction between DGK
and Ob-Rbc. This contrasts with 108 units of
-galactosidase activity in a positive control interaction
between p53 and T antigen. This low
-galactosidase activity confirms
that the interaction between Ob-Rbc and DGK
is weak and explains the
low yield of DGK
clones in the library screening.
The interaction between DGK
and Ob-Rbc was confirmed in a different
LexA yeast two-hybrid system (38). The growth of yeast on a control
medium (Fig. 3, left)
indicates the presence of vectors expressing DGK
and Ob-Rbc fusion
proteins in the yeast cells, and the blue colony color indicates the
interaction between DGK
and Ob-Rbc. When these yeasts were plated
onto a test medium that selects for the interaction between the
expressed fusion proteins, only the yeasts expressing both DGK
and
Ob-Rbc grew and turned blue within 3 days (Fig. 3, right).
This experiment, again, demonstrates the interaction of DGK
with
Ob-Rbc.

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Fig. 3.
Protein interaction in the LexA yeast
two-hybrid system. Left panel, plating of the
yeast on the control medium. The growth of the yeast indicates the
presence of vectors expressing the indicated fusion proteins in the
yeast cells. The blue colony color indicates the interaction
between the recombinant proteins. Right panel, plating of
the yeast on the test medium. Only the yeast containing interacting
proteins grows on this medium and produces colony color change.
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The identification of the ankyrin repeats of DGK
in our two-hybrid
library screening suggests that DGK
may use its ankyrin repeats to
interact with Ob-Rbc. To substantiate this observation, we then used an
in vitro protein to protein interaction experiment to
demonstrate that the ankyrin repeats by themselves are responsible for
the interaction. In this experiment, we expressed rat DGK
, a DGK
with all four ankyrin repeats deleted (DGK
a), and the four
ankyrin repeats of DGK
(DGK
a) in bacteria as GST fusion proteins
and purified them (Fig. 4, a
and b). We also expressed Ob-Rbc in bacteria as a Trx fusion
protein, solubilized from inclusion bodies by 8 M urea,
purified, and renatured in a phosphate buffer. In the protein to
protein interaction experiment in vitro, 20 µg of
Trx-Ob-Rbc was found to co-precipitate with 1 µg of GST-DGK
, but
not with 1 µg of GST-DGK
a, when GST-DGK
and GST-DGK
a were precipitated with glutathione-agarose beads (Fig. 4c, lanes 1 and 2). This result indicates that the ankyrin
repeats are responsible for the interaction. To confirm this, we
further found that 20 µg of Trx-Ob-Rbc co-precipitated with 1 µg of
GST-DGK
a, but not with 1 µg of GST, when GST and GST-DGK
a were
precipitated with glutathione-agarose beads (Fig. 4c, lanes
3 and 4). Reciprocally, 1 µg of GST-DGK
a
co-precipitated 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 (Fig. 4c, lanes 5 and
6). The partial ankyrin repeats identified by two-hybrid
library screening, which contain the last two repeats and the last 12 amino acids of the second repeats, was also found to interact with
Ob-Rbc in protein to protein interaction experiments in
vitro (not shown). In addition, we found that 20 µg of
Trx-Ob-Rbc was required for the interaction to be detected, which may
indicate that only a small fraction of Trx-Ob-Rbc was correctly
renatured and bound with DGK
a. To demonstrate that native Trx-Ob-Rbc
interacts with DGK
a, we used soluble Trx-Ob-Rbc concentrated from
the bacterial extract in the protein binding experiment and obtained
the same result (not shown). This evidence indicates that DGK
interacts with Ob-Rb via its ankyrin repeats.

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Fig. 4.
Interaction of ankyrin repeats of
DGK with the cytoplasmic portion of Ob-Rb.
a, expression of GST-DGK , GST-DGK a, and GST-DGK a
in bacteria. These fusion proteins were expressed with pGEX5 × 1 in Escherichia coli strain BL21 and partially purified by
glutathione-agarose beads. The proteins were separated in a 10%
SDS-polyacrylamide gel and silver stained. b, diagram of
GST-DGK , GST-DGK a, GST-DGK a, Ob-Rb, Ob-Rbc, Ob-Rac, and
Ob-Rbt. A leader peptide containing a Trx·Tag, 6 × histidine,
and S·Tag was fused into the N terminus of Ob-Rbc, Ob-Rac, and Ob-Rbt
when these proteins were expressed in bacteria. Box B1 and Box B2 are
identified motifs on the cytoplasmic domain of Ob-Rb that interact with
Jak kinase and STAT proteins, respectively. c, protein to
protein interactions in vitro. The proteins were combined
and shaken at 37 °C for 1 h in the presence of either
glutathione-agarose beads or Ni-NTA SuperflowTM resin,
followed by washing 4 times and separation in a 12% polyacrylamide
gel. The proteins were transferred onto a polyvinylidene difluoride
membrane and assayed in Western blot by either an anti-His antibody,
which is targeted to a His-Tag in Trx-Ob-Rbc, or an anti-GST antibody.
Lanes 1 and 2, precipitation of 1 µg of
GST-DGK and GST-DGK a and assay of Trx-Ob-Rbc. Lanes
3 and 4, precipitation of 1 µg of GST and GST-DGK a
and assay of Trx-Ob-Rbc. Lanes 5 and 6, precipitation of Trx and Trx-Ob-Rbc and assay of GST-DGK a.
Lanes 7 and 8, precipitation of Trx-Ob-Rac and
Trx-Ob-Rbt and assay of GST-DGK a. Lanes 9 and
10, precipitation of GST and GST-DGK a and assay of
Trx-Ob-Rbt.
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DGK
Does Not Interact with OBRa--
Since a mutation that
changes Ob-Rb to Ob-Ra results in morbid obesity (27, 29), it is
important to determine whether DGK
a also interacts with the
cytoplasmic domain of Ob-Ra, which exists naturally and is thought to
mediate the entry of leptin into the brain (39). Therefore, the
cDNA encoding the cytoplasmic domain of rat Ob-Ra (Ob-Rac) was
cloned by RT-PCR and expressed as a Trx fusion protein (Trx-Ob-Rac) in
bacteria and purified. In the protein precipitation experiment, 1 µg
of GST-DGK
a did not coprecipitate with 20 µg of Trx-Ob-Rac when
Trx-Ob-Rac was precipitated by Ni-NTA SuperflowTM resin
(Fig. 4c, lane 7). Additionally, in the LexA yeast
two-hybrid system, the yeast containing DGK
and Ob-Rac did not grow
on the test medium, nor did the colony color change in 3 days (Fig. 3). Thus, through independent approaches, we have demonstrated that DGK
does not interact with Ob-Ra.
Additional experiments were conducted to confirm that the amino acid
sequence responsible for the interaction of DGK
with Ob-Rb is
present in Ob-Rbc but not Ob-Rac. A truncated Ob-Rbc (Ob-Rbt) was
generated by removing a stretch of sequence at the N terminus of Ob-Rbc
(Fig. 4b). We found that 1 µg of GST-DGK
a co-precipitated with 20 µg of Trx-Ob-Rbt, but not with Trx-Ob-Rac, when the Trx fusion proteins were precipitated by Ni-NTA
SuperflowTM resin (Fig. 4c, lanes 7 and
8). Reciprocally, 20 µg of Trx-Ob-Rbt co-precipitated with
1 µg of GST-DGK
a, but not with 1 µg of GST, when GST and
GST-DGK
a were precipitated with glutathione-agarose beads (Fig.
4c, lanes 9 and 10). These results indicate that
Ob-Rbt is sufficient for the interaction between DGK
a and
Ob-Rbc.
To provide evidence for their interaction in vivo, we
conducted immunoprecipitation experiments in which a goat anti-Ob-R antibody was used to bring down Ob-R and its associated proteins from a
protein extract made from pooled rat hypothalamus. After washing 4 times, the precipitates were separated on a polyacrylamide gel and
assayed for the presence of DGK
in Western blot by an anti-DGK
antibody (11). We detected two immunoreactive bands of DGK
(117 and
120 kDa), which have been previously observed in mouse and transfected
cell lines (10, 11, 13), as well as a 130 kDa alternatively spliced
DGK
(12) (Fig. 5, lane 1). In contrast, a mock precipitation performed by using normal goat IgG
generated no immunoreactive signal (Fig. 5, lane 2). This experiment indicates that the interaction between DGK
and Ob-R may
occur in vivo.

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Fig. 5.
Interaction of DGK
with Ob-R in vivo. Proteins were
precipitated by a goat polyclonal anti-Ob-R antibody plus protein
G-agarose beads (lane 1) and by normal goat IgG plus protein
G-agarose beads as a control (lane 2). After washing 4 times, these proteins were separated in a 7.5% polyacrylamide gel and
assayed for DGK by a polyclonal anti-DGK antibody in Western
blot. The 117-, 120-, and 130-kDa DGK bands are indicated by
arrowheads. The two smaller bands represent degraded DGK .
No signal was detected in the control (lane 2).
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DGK
a Is Expressed in Areas Similar to Ob-Rb--
An additional
experiment using quantitative RT-PCR demonstrates that DGK
is
broadly expressed throughout the body, with levels from highest to
lowest detected in the spleen, thymus, ovary, hypothalamus, lung,
brain, intestine, liver, and pituitary (Fig. 6a). The Ob-Rb mRNA is
found to be dense in tissues where DGK
is detected, notably the
hypothalamus, brain, pituitary, and thymus. This contrasts with Ob-Ra,
which exhibits a very different distribution pattern, expressed
predominantly in intestine, liver, spleen, and ovary (Fig.
6a).

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Fig. 6.
Distribution of DGK .
a, distribution of mRNAs of DGK , Ob-Rb, and Ob-Ra in
various tissues. The mRNA was purified from the tissues and was
used to synthesize cDNA, which was then used as a template for
quantitative PCR. A control fragment of glyceraldehyde-3-phosphate
dehydrogenase (G3PDH) was amplified simultaneously with
DGK , Ob-Rb, or Ob-Ra. These PCR products were resolved in a 5%
polyacrylamide gel, transferred onto a nylon membrane, and hybridized
with their specific 32P-labeled PCR primers. b,
hypothalamic distribution of DGK in rats on a high-fat diet. A
biotin-labeled antisense cRNA probe was used for the in situ
hybridization. The hybridization was enhanced by tyramide signal
amplification. DGK mRNA was detected in cells of the
paraventricular (PVN, ×10), arcuate (ARC, ×10),
and ventromedial (VMH, ×4) nuclei of the hypothalamus.
In situ hybridization with control sense DGK probe
yielded no signal (PVN, 4×, lower right).
V, the third ventricle.
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To identify the specific locations of DGK
expression in brain and
hypothalamus, the DGK
mRNA was detected by in situ
hybridization. Biotin-labeled, DGK
-specific antisense RNA probes
were synthesized and used. The control sense RNA probes demonstrated
almost no signal (Fig. 6b). Whereas the DGK
mRNA
level is quite low in the hypothalami of rats on a low-fat diet, rats
on a high-fat diet have detectable DGK
mRNA throughout the
hypothalamus. DGK
mRNA is clearly evident in several medial
hypothalamic nuclei known to be involved in energy homeostasis (40).
These include the paraventricular, arcuate, and ventromedial nuclei
(Fig. 6b). It is notable that this expression pattern
detected for DGK
, while different from that of Ob-Ra (39), is
similar to that seen for Ob-Rb (41). This is consistent with the
possibility that DGK
and Ob-Rb are colocalized within the same
hypothalamic neurons.
Obese ob/ob and db/db Mice Have Higher Hypothalamic DGK
mRNA
Level--
The above evidence supports the hypothesis that
hypothalamic DGK
may be functionally associated with leptin/Ob-Rb in
regulating eating and body fat accrual. This association is further
demonstrated in our experiments conducted in mice with a mutant
leptin or Ob-Rb gene. We used quantitative RT-PCR
to measure the hypothalamic DGK
mRNA level in C57BL/6j
ob-/ob- mice, which have a dysfunctional leptin
gene. Compared with that of the lean wild-type C57BL/6j mice, the
C57BL/6j ob-/ob- mice were found to have an elevated mRNA level of DGK
, relative to actin, in the hypothalamus
(0.86 ± 0.01 versus 0.75 ± 0.01, p < 0.05). A similar result was obtained in the obese
C57BL/3j db-/db- mice, which have lost the cytoplasmic domain of Ob-Rb by mutation, compared with their lean wild-type controls (1.32 ± 0.02 versus 0.79 ± 0.01, p < 0.05). These experiments indicate that the signal
transduction process of the leptin/Ob-Rb system participates in the
regulation of hypothalamic DGK
expression.
Obese Rats and Mice Have Lower Hypothalamic DGK
mRNA
Level--
In rats and mice with an intact leptin/Ob-Rb system,
further evidence demonstrates that body fat accrual is, in fact, linked to reduced DGK
mRNA in the hypothalamus. Using quantitative
RT-PCR, we compared the hypothalamic mRNA level of DGK
in Harlan
Sprague-Dawley rats that either become obese or remain lean after 3 weeks on a high-fat diet. Whereas both subgroups are similar in their
total caloric intake (Table II), the
weight of the dissected body fat pads of the obese rats (26-34 g) is
~50% greater than that of the lean rats (15-21 g). This greater
body fat in the obese is associated with a statistically significant
reduction in hypothalamic DGK
mRNA levels, along with 100%
higher levels of circulating leptin (Table II). Moreover, across the
entire group, the level of hypothalamic DGK
mRNA is negatively
correlated with total body fat (r = -0.85, p < 0.01), as well as with leptin (r = -0.79, p < 0.01).
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Table II
Obese rats and mice have lower hypothalamic DGK mRNA
Hypothalamus was excised from each individual rat or mouse and was used
to purify total mRNA and synthesize the first strand cDNA. The
cDNA fragments of DGK and actin were amplified by PCR for 20 cycles, which was determined to be within the exponential range. The
PCR products were resolved on agarose gels, and their quantities were
determined by a Bio-Rad GS-700 densitometer. The relative DGK
mRNA level was obtained by comparing optical densities of DGK
fragments with those of corresponding actin fragments and by averaging
these data from four independent experiments. Circulating leptin level
was determined in each rat or mouse. Body fat scores reflect weight of
4 dissected fat pads, and total intake (over 24 h) reflects an
average of frequent measures taken over the 3-week test period.
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This inverse relationship between DGK
and body fat or leptin is
similarly detected in inbred mouse strains that have a differential propensity to accumulate body fat (42). In subjects maintained on a
high-fat diet, hypothalamic DGK
mRNA was measured, via
quantitative RT-PCR, in AKR/J mice, which are prone to obesity on this
diet, and was compared with that of SWR/J mice, which are resistant to
obesity despite their equal level of caloric intake (Table II). As in
the rats, the greater adiposity of the AKR/J strain is accompanied by a
statistically significant decrease in hypothalamic DGK
mRNA
compared with that of the SWR/J mice (Table II).
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DISCUSSION |
In these experiments, we have found that DGK
, via its ankyrin
repeats, interacts with the cytoplasmic portion of Ob-Rb (Ob-Rbc). Ankyrin repeats are known to be involved in a wide variety of protein
to protein interactions (43-45). It is thus not surprising that DGK
interacts with Ob-Rb via this domain. We have demonstrated this
interaction by reciprocal protein to protein interaction experiments
in vitro and additionally in the LexA yeast two-hybrid system. Ob-Rbc has several known protein binding motifs for interacting with Jak kinase and STAT proteins (27, 29). To obtain further information regarding the binding site on Ob-Rbc, we removed the Jak
binding motif (Box B1) at the N terminus of Ob-Rbc. We found that this
N-terminal truncated Ob-Rbc is sufficient for the interaction with
DGK
, indicating that the involved amino acid sequence (or motif) may
be in a 229-amino acid sequence at the C terminus of Ob-Rb. A
STAT-binding motif (Box B2) exists in this stretch of sequence, which
binds the SH2 domain on STAT. However, we have not found an obvious SH2
sequence homologue in the ankyrin repeats of DGK
. This may indicate
that DGK
interacts with other unidentified motifs in Ob-Rbc.
In support of this interaction in vivo, we have demonstrated
that DGK
can be co-precipitated with Ob-R by an anti-Ob-R
antibody from protein extract made from rat hypothalamus. Furthermore, by using in situ hybridization, we have shown that DGK
is
expressed in hypothalamic nuclei that are known to synthesize Ob-Rb
(41, 46) and are involved in feeding and body weight regulation (40). These hypothalamic areas include the paraventricular, arcuate, and
ventromedial. This overlap of expression in the hypothalamus provides
anatomical evidence for a direct interaction between DGK
and Ob-Rb
in vivo.
The expression of DGK
and Ob-Rb appears to overlap in other brain
areas as well. Similar to the areas reported for Ob-Rb (41, 46), DGK
mRNA is detected in the hippocampus, cerebral cortex, and
cerebellum, as well as in other areas of the brain (14). Moreover, by
using RT-PCR, we have found both DGK
and Ob-Rb expression in
pituitary and lung (Fig. 6a). Thus, an interaction between
DGK
and Ob-Rb in vivo may occur in multiple areas,
although the functional significance of the interaction in these areas remains to be determined.
This interaction places DGK
downstream of the signal transduction
pathway of leptin/Ob-Rb and supports a novel function for hypothalamic
DGK
in energy homeostasis. In agreement with this hypothesis, we
have found that the hypothalamic mRNA level of DGK
is
statistically significantly elevated in obese ob-/ob- and
db-/db- mice compared with their wild-type controls. Since these mice have a mutant leptin or Ob-Rb gene,
respectively, this experiment indicates that, in addition to regulating
the expression of other genes (21), the signaling activities of leptin
have impact on the hypothalamic expression of DGK
. Furthermore, we find that the consumption of a high-fat diet, which is known to affect
the expression of other genes (18, 20, 34) together with leptin
production (35, 36), potentiates hypothalamic mRNA level of DGK
.
In wild-type rats and inbred mice maintained on a high-fat diet, we
additionally detect lower levels of hypothalamic DGK
mRNA in
those subjects that become obese compared with the lean animals and
also a negative relationship between hypothalamic DGK
mRNA level
and body fat. This supports the idea that reduced activity of DGK
may accompany or contribute to the accrual of body fat.
Based on these results showing that DGK
mRNA is higher in the
wild-type lean rats and inbred mice, one may interpret the elevated
DGK
mRNA in the mutant, morbidly obese ob-/ob- and
db-/db- mice as indicating that these animals regard
themselves as "lean," as suggested previously (21), and
consequently oversynthesize DGK
mRNA. However, the specific
enzymatic activity of DGK
in the hypothalamus of these mutant mice
is unknown. In fact, there is suggestive evidence that Ob-Rb mutation
may cause a reduction of DGK activity in obese Zucker rats. These rats,
which have a mutant Ob-Rb (47), exhibit elevated diacylglycerol levels
and protein kinase C activity (48, 49), which are known to be direct
consequences of lower DGK activity (1). It is therefore possible that
ob-/ob- and db-/db- mice, similar to obese Zucker rats in having a dysfunctional leptin-signaling pathway, may also have
reduced DGK activity in the hypothalamus. Further experiments are
needed to test this and to determine the enzymatic activity, as well as
the expression, of the specific isoforms of DGK that may be affected by
leptin activity and by high-fat diet consumption. In our RDA
experiments with different rat and mouse models, we have only detected
DGK
and have not found the expression of other DGK types/isoforms to
be affected by fat consumption. We have also not found other
types/isoforms to interact with Ob-Rb in our screening of a two-hybrid
rat brain cDNA library. This evidence leads us to propose that the
activity of DGK
is specifically regulated by the interaction of its
ankyrin repeats with a leptin-stimulated Ob-Rb and that the mutation of
leptin or Ob-Rb in ob-/ob- or db-/db- mice
results in a decline of DGK
enzymatic activity, despite the elevated
hypothalamic mRNA level. The evidence that DGK
in porcine aortic
endothelial cells is primarily associated with the cell membrane (50)
may further support the possibility that this enzyme associates with
Ob-Rb on the membrane where leptin stimulation of Ob-Rb leads to the
activation and then dissociation of DGK
from the membrane.
Our evidence for the first time links the function of DGK
to the
activities of leptin in the hypothalamus. It supports the hypothesis
that hypothalamic DGK
is activated through its interaction with a
leptin-stimulated Ob-Rb. DGK
may participate in regulating body fat
mass by directly controlling diacylglycerol in the synthesis of complex
lipids and/or by controlling gene expression via modulating levels of
the second messengers, diacylglycerol and phosphatidic acid. Based on
our experimental results in rodent animals, we further propose that a
reduction in DGK activity in the hypothalamus, whether derived from low
mRNA level in spontaneously obese rats and inbred mice or from
failed stimulation by mutant leptin/Ob-Rb, is associated with obesity.