Download Diacylglycerol kinase zeta in hypothalamus interacts with long form leptin receptor. Relation to dietary fat and body weight regulation

THE JOURNAL OF BIOLOGICAL CHEMISTRY
© 2001 by The American Society for Biochemistry and Molecular Biology, Inc.
Vol. 276, No. 8, Issue of February 23, pp. 5900 –5907, 2001
Printed in U.S.A.
Diacylglycerol Kinase ␨ in Hypothalamus Interacts with Long Form
Leptin Receptor
RELATION TO DIETARY FAT AND BODY WEIGHT REGULATION*
Received for publication, August 11, 2000, and in revised form, October 13, 2000
Published, JBC Papers in Press, November 14, 2000. DOI 10.1074/jbc.M007311200
Zhitong Liu, Guo-Qing Chang, and Sarah F. Leibowitz‡
From The Rockefeller University, New York, New York 10021
Leptin and its long form receptor, Ob-Rb, in hypothalamic nuclei play a key role in regulating energy balance. The mutation of Ob-Rb into one of its natural
variants, Ob-Ra, results in severe obesity in rodents. We
demonstrate here that diacylglycerol kinase ␨ (DGK␨)
interacts, via its ankyrin repeats, with the cytoplasmic
portion of Ob-Rb in yeast two-hybrid systems, in protein
precipitation experiments in vitro and in vivo. It does
not interact, however, with the short form, Ob-Ra, which
mediates the entry of leptin into the brain. Furthermore, we show by in situ hybridization that DGK␨ is
expressed in neurons of hypothalamic nuclei known to
synthesize Ob-Rb and to participate in energy homeostasis. The mutant ob-/ob- and db-/db- mice exhibit increased hypothalamic DGK␨ mRNA level compared with
their wild-type controls, suggesting a role for the leptin/
OB-Rb system in regulating DGK␨ expression. Further
experiments show that hypothalamic DGK␨ mRNA level
is stimulated by the consumption of a high-fat diet. In
addition, DGK␨ mRNA is statistically significantly lower
in rats and inbred mice that become obese on a high-fat
diet compared with their lean counterparts. In fact, it is
strongly, negatively correlated with both body fat and
circulating levels of leptin. Taken together, our evidence suggests that DGK␨ constitutes a downstream
component of the leptin signaling pathway and that reduced hypothalamic DGK␨ mRNA, and possibly activity,
is associated with obesity.
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
* The work was supported by National Institutes of Health Grant
MH43422 and the Price Foundation. The costs of publication of this
article were defrayed in part by the payment of page charges. This
article must therefore be hereby marked “advertisement” in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
‡ To whom correspondence should be addressed: The Rockefeller University, Box 278, 1230 York Ave., New York, NY 10021. Tel.: 212-3278378; Fax: 212-327-8447; E-mail: [email protected].
1
The abbreviations used are: DGK, diacylglycerol kinase; Jak, Janus
kinase; STAT, signal transducers and activators of transcription; RTPCR, reverse transcriptase-polymerase chain reaction; Trx, thioredoxin; RDA, representational difference analysis; GST, glutathione
S-transferase.
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
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This paper is available on line at http://www.jbc.org
DGK␨ Interacts with Ob-Rb
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.
EXPERIMENTAL PROCEDURES
Animals, Tissues, and Physiological Studies—Male Harlan SpragueDawley 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 ObRbt, pET-32a(⫹)-Ob-Rbc was digested by KpnI, followed by T4 DNA
polymerase and ligation. The bacteria expressing Trx-Ob-Rbc and TrxOb-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 glutathioneSepharose 4B column.
Protein Precipitation—The purified proteins were combined with
either 50 ␮l of 50% Ni-NTA Superflow-agarose resin or 50 ␮l 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 ␮g/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䡠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 ␮g/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 ␮g
of goat anti-Ob-Rb antibody (Santa Cruz Biotechnology) plus protein
G-agarose beads (Upstate biotechnology) or with 100 ␮g 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
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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 ␮l of 50 mM Tris-HCl, pH 8.9, 15 mM (NH4)2SO4, 1.5 mM
MgCl2, 1 ␮M of each primer, 0.2 mM dNTP, 5 units of Taq polymerase
(Promega), and 1/20 volume of 1 ␮g of medial hypothalamus RNA
reverse transcription reaction (20 ␮l) 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 SpragueDawley 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.
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 highfat 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).
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), mod-
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DGK␨ Interacts with Ob-Rb
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 ␮g of cDNA fragments made from the
hypothalamus of rats (n ⫽ 10) on a low-fat (10%) diet was used to
subtract 0.1 ␮g of cDNA fragments from high-fat (60%) diet rats (n ⫽
10), followed by PCR. The product obtained from each PCR (1 ␮g) was
resolved in 1% agarose gel. The position of DGK␨ is indicated (arrowhead) based on the length of the cDNA fragment.
erate-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).
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.
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 inter-
action 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 ␮g of
Trx-Ob-Rbc was found to co-precipitate with 1 ␮g of GSTDGK␨, but not with 1 ␮g 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 ␮g of Trx-Ob-Rbc co-precipitated
with 1 ␮g of GST-DGK␨a, but not with 1 ␮g of GST, when GST
and GST-DGK␨a were precipitated with glutathione-agarose
beads (Fig. 4c, lanes 3 and 4). Reciprocally, 1 ␮g of GST-DGK␨a
co-precipitated with 20 ␮g of Trx-Ob-Rbc, but not with 20 ␮g 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 ␮g 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.
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 ␮g of GST-DGK␨a did not
coprecipitate with 20 ␮g 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
␮g of GST-DGK␨a co-precipitated with 20 ␮g 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 ␮g of Trx-Ob-Rbt co-precipitated with 1 ␮g
of GST-DGK␨a, but not with 1 ␮g 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 antiOb-R antibody was used to bring down Ob-R and its associated
DGK␨ Interacts with Ob-Rb
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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.
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.
a
b
Diet (% fat)
n
DGK␨ mRNA
relative to actin
ng/ml
Low-fat (10%)
Moderate-fat (30%)
High-fat (60%)
5
6
5
0.64 ⫾ 0.02
0.77 ⫾ 0.02a
0.87 ⫾ 0.03a,b
2.6 ⫾ 0.4
4.5 ⫾ 0.9a
6.7 ⫾ 0.7a,b
Leptin
Body fat
Body weight
Total intake
387 ⫾ 6
402 ⫾ 6a
414 ⫾ 6a,b
89.3 ⫾ 2.2
91.0 ⫾ 1.7
101.6 ⫾ 3.7a,b
gm
14.0 ⫾ 0.7
15.4 ⫾ 0.8
20.5 ⫾ 1.2a,b
Kcals/day
p ⬍ 0.05 compared to low-fat diet.
p ⬍ 0.05 compared to moderate-fat diet.
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.
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.
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 predom-
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DGK␨ Interacts with Ob-Rb
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䡠Tag, 6 ⫻ histidine, and S䡠Tag was fused into
the N terminus of Ob-Rbc, Ob-Rac, and Ob-Rbt when these proteins were expressed in bacteria. Box 1 and Box 2 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 ␮g of GST-DGK␨ and GST-DGK␨⌬a and assay of Trx-Ob-Rbc. Lanes 3 and 4, precipitation of 1 ␮g 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.
inantly in intestine, liver, spleen, and ovary (Fig. 6a).
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-/dbmice, 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␨
DGK␨ Interacts with Ob-Rb
5905
FIG. 5. Interaction of DGK␨ with Ob-R in vivo. Proteins were
precipitated by a goat polyclonal anti-Ob-R antibody plus protein Gagarose 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).
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).
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).
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 1) 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 2) 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
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 glyceraldehyde3-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.
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
DGK␨ Interacts with Ob-Rb
5906
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.
Animals
a
DGK␨ mRNA
Leptin
relative to actin
ng/ml
n
Body fat
Body weight
gm
Total intake
Kcals/day
Lean rats
Obese rats
7
8
1.11 ⫾ 0.02
0.95 ⫾ 0.01a
9.8 ⫾ 2.0
21.5 ⫾ 3.1a
19.1 ⫾ 1.4
28.3 ⫾ 2.4a
418 ⫾ 4
480 ⫾ 7a
119 ⫾ 14
115 ⫾ 8
Lean SWR/J
Obese AKR/J
6
6
0.84 ⫾ 0.01
0.73 ⫾ 0.01a
2.1 ⫾ 0.2
2.3 ⫾ 0.3
0.95 ⫾ 0.18
1.63 ⫾ 0.12a
22.6 ⫾ 0.5
31.0 ⫾ 0.8a
15.6 ⫾ 0.8
15.9 ⫾ 0.9
p ⬍ 0.05 compared to lean animals.
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 wildtype 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 diacyl-
glycerol 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.
Acknowledgments—We are grateful to Dr. Guo-Ching Chang (The
Rockefeller University, New York) who conducted the in situ hybridization for this work, Dr. Stephen Prescott (University of Utah, Salt Lake
city, UT) for the polyclonal rabbit anti-DGK␨ antibody, and Drs. Zhen
Pang (St. Jude Hospital, Memphis, TN), Cai Li (South Western Medical
University, Dallas, TX), and Stephen Prescott for critical reading and
help during the preparation of this manuscript.
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