The Regulation and Activation of Ciliary Neurotrophic Factor
Signaling Proteins in Adipocytes*
Sanjin
Zvonic
,
Peter
Cornelius§,
William C.
Stewart¶,
Randall L.
Mynatt
, and
Jacqueline M.
Stephens
**
From the
Department of Biological Sciences, Louisiana
State University, Baton Rouge, Louisiana 70803, § Pfizer
Central Research, the Department of Cardiovascular & Metabolic
Diseases, Groton, Connecticut 06340, the ¶ Department of
Biology, Middle Tennessee State University,
Murfreesboro, Tennessee 37132, and
Pennington Biomedical
Research Center, Baton Rouge, Louisiana 70808
Received for publication, June 12, 2002, and in revised form, October 10, 2002
 |
ABSTRACT |
Ciliary neurotrophic
factor (CNTF) is primarily known for its roles as a lesion factor
released by the ruptured glial cells that prevent neuronal
degeneration. However, CNTF has also been shown to cause weight loss in
a variety of rodent models of obesity/type II diabetes, whereas a
modified form also causes weight loss in humans. CNTF administration
can correct or improve hyperinsulinemia, hyperphagia, and
hyperlipidemia associated with these models of obesity. In order to
investigate the effects of CNTF on fat cells, we examined the
expression of CNTF receptor complex proteins (LIFR, gp130, and
CNTFR
) during adipocyte differentiation and the effects of CNTF on
STAT, Akt, and MAPK activation. We also examined the ability of CNTF to
regulate the expression of adipocyte transcription factors and other
adipogenic proteins. Our studies clearly demonstrate that the
expression of two of the three CNTF receptor complex components,
CNTFR
and LIFR, decreases during adipocyte differentiation. In
contrast, gp130 expression is relatively unaffected by differentiation. In addition, preadipocytes are more sensitive to CNTF treatment than
adipocytes, as judged by both STAT 3 and Akt activation. Despite
decreased levels of CNTFR
expression in fully differentiated 3T3-L1
adipocytes, CNTF treatment of these cells resulted in a time-dependent activation of STAT 3. Chronic treatment of
adipocytes resulted in a substantial decrease in fatty-acid synthase
and a notable decline in SREBP-1 levels but had no effect on the
expression of peroxisome proliferator-activated receptor
, acrp30,
adipocyte-expressed STAT proteins, or C/EBP
. However, CNTF resulted
in a significant increase in IRS-1 expression. CNTFR
receptor
expression was substantially induced in the fat pads of four rodent
models of obesity/type II diabetes as compared with lean littermates.
Moreover, we demonstrated that CNTF can activate STAT 3 in adipose
tissue and skeletal muscle in vivo. In summary, CNTF
affects adipocyte gene expression, and the specific receptor for this
cytokine is induced in rodent models of obesity/type II diabetes.
 |
INTRODUCTION |
Ciliary neurotrophic factor
(CNTF)1 was originally
characterized as a trophic factor that supports the survival of
embryonic chick ciliary ganglion neurons in vitro (1, 2).
However, subsequent cloning and sequencing of CNTF revealed that it is unrelated to neurotrophins but is a member of the gp130 cytokine family
along with interleukin-6, interleukin-11, LIF, OSM, leptin and CT-1
(3-5). The actions of CNTF are mediated, in part, by a CNTF-specific
receptor (CNTFR
) that has homology to the interleukin-6R
(6).
Upon translation, the C terminus of CNTFR
is cleaved. Mature
CNTFR
has no transmembrane or cytosolic domains and is found on the
outer surface of the cell membrane where it is attached by a
glycosylphosphatidylinositol linkage sensitive to
phosphatidylinositol-phospholipase C treatment (7). Initially, CNTFR
was described as being distributed predominantly within neural tissues
(7) but has since been reported in skeletal muscle, adrenal gland,
sciatic nerve, skin, kidney, and testes (8). CNTFR
can be cleaved
from the cell surface and exist and act in a soluble form. The soluble
CNTFR
has been detected in the serum and the cerebrospinal fluid and has been shown to initiate signaling in cells not responsive to CNTF
alone (6, 9). Mice lacking CNTF develop normally and appear to have no
visible defects well into adulthood, when they develop minor loss of
motor neurons (10). However, mice lacking CNTFR
tend to have severe
motor neuron defects and die perinatally because they fail to initiate
feeding behaviors (11). CNTF signaling is initiated when CNTF binds
CNTFR
, either in its soluble or membrane-bound form (12). Once a
CNTF·CNTFR
complex is formed, two of these heterodimers
come together and recruit a gp130 transducer protein, followed by a
subsequent recruitment of LIFR protein. The resulting receptor complex
is a hexamer of CNTF, CNTFR
, gp130, and LIFR in a 2:2:1:1 ratio,
respectively (13). Within this complex, CNTF and CNTFR
make direct
contacts with all the complex components (7, 12). CNTF·CNTFR
is
considered to be a low affinity binding complex until further bound to
gp130 and LIFR (14). Aside from this hexameric, high affinity binding
complex, CNTF can bind its receptors and can induce signaling in the
absence of CNTFR
, solely by binding to a gp130:LIFR dimeric receptor (15, 16).
Although CNTF was first identified as a trophic factor in the ciliary
ganglion, it was later found to act on other motor neuron populations
(17). Hence, it was evaluated as a therapeutic tool in patients
suffering from motor neuron diseases (18). Interestingly, during these
trials, CNTF administration resulted in unexpected weight loss (19).
Additional studies showed that, like leptin, CNTF can activate the same
signaling molecules and that CNTFR
is co-localized with ObR in the
hypothalamic nuclei involved in the regulation of feeding (20). CNTF
can also cross the blood-brain barrier in a manner similar to leptin
(21). CNTF treatment of leptin-deficient ob/ob mice was found to reduce
adiposity, hyperphagia, and hyperinsulinemia associated with this
genotype. Leptin administration had the same effect in these animals.
However, unlike leptin, CNTF also corrected obesity-related phenotypes
in leptin-resistant, ObR-deficient, db/db mice and in mice with
diet-induced obesity that are partially resistant to leptin (22). CNTF
and its synthetic analog, Axokine, have also been found to suppress NPY
gene expression (23) and pCREB in the feeding-relevant brain sites
(22). The weight loss caused by CNTF administration is due to the
preferential loss of fat (24). It is believed to occur by resetting the
hypothalamic weight set point, such that cessation of CNTF treatment
does not result in overeating and rebound weight gain (22). Unlike
cachectic cytokines, the appetite diminution during CNTF treatment does not appear to be due to stress, inflammatory responses, nausea, or
conditioned taste aversion but is possibly due to the modification of
NPYergic signaling (22, 25).
In this study, we examined the regulation and activation of STATs and
proteins by CNTF in adipocytes. The objective of this project was to
determine whether CNTF, a cytokine known to result in weight loss,
could have effects on peripheral tissues such as white adipose tissue.
Our results clearly demonstrate that two of the three CNTF receptors
are down-regulated during the adipogenesis of 3T3-L1 cells. However,
CNTF administration results in the activation of STAT 3 in both
cultured 3T3-L1 adipocytes and in rodent adipose tissue. Also this
study provides the first evidence that CNTFR
is expressed in adipose
tissue and that the expression of this receptor is regulated in four
rodent models of obesity/type II diabetes. We also observed that CNTF
treatment did not effect the expression of key adipogenic transcription factors such as PPAR
and C/EBP
but did result in a decrease of
fatty-acid synthase (FAS) expression. Also, unlike other cachectic cytokines such as tumor necrosis factor-
, chronic CNTF treatment did
not result in the development of insulin resistance in cultured adipocytes. Moreover, acute CNTF administration resulted in increased GLUT4 expression, whereas chronic CNTF treatment resulted in a substantial increase in IRS-1 expression. Also, acute CNTF treatment resulted in an increase in insulin-induced IRS-1 and Akt activation. In
summary, the results of this study demonstrate that both cultured and
native adipocytes, as well as skeletal muscle, are responsive to CNTF
and that this cytokine may act as an insulin-sensitizer in cultured
adipocytes. These studies support our hypothesis that the ability of
CNTF to result in weight loss is not solely mediated by the central
nervous system.
 |
EXPERIMENTAL PROCEDURES |
Materials--
Dulbecco's modified Eagle's media (DMEM) was
purchased from Invitrogen. Bovine and fetal bovine serum (FBS) were
obtained from Sigma and Invitrogen, respectively. Rat CNTF was
purchased from Calbiochem. The non-phospho-STAT antibodies were
monoclonal IgGs purchased from Transduction Laboratories or polyclonal
IgGs from Santa Cruz Biotechnology. A highly phospho-specific
polyclonal antibody for STAT 3 (Tyr705) was
purchased from BD Biosciences. LIFR and gp130 were rabbit polyclonals
from Santa Cruz Biotechnology. PPAR
was a mouse monoclonal from
Santa Cruz Biotechnology. SREBP-1 and ERK1/ERK2 were rabbit polyclonals
from Santa Cruz Biotechnology. Active ERK antibody was a rabbit
polyclonal from Promega. C/EBP
was a rabbit polyclonal from Dr.
Ormond MacDougald (Ann Arbor, MI), and GLUT 4 was a rabbit polyclonal
from Dr. Paul Pilch (Boston). CNTFR
was a mouse monoclonal purchased
from BD Biosciences. IRS-1 polyclonal was a polyclonal obtained from
Upstate Biotechology, Inc., and the phospho-specific IRS-1 polyclonal
was from BIOSOURCE International. PGNaseF was obtained from New England Biolabs.
Cell Culture--
Murine 3T3-L1 preadipocytes were plated and
grown to 2 days post-confluence in DMEM with 10% bovine serum. Medium
was changed every 48 h. Cells were induced to differentiate by
changing the medium to DMEM containing 10% fetal bovine serum, 0.5 mM 3-isobutyl-1-methylxanthine, 1 µM
dexamethasone, and 1.7 µM insulin. After 48 h this
medium was replaced with DMEM supplemented with 10% FBS, and cells
were maintained in this medium until utilized for experimentation.
Preparation of Whole Cell Extracts--
Monolayers of 3T3-L1
preadipocytes or adipocytes were rinsed with phosphate-buffered saline
and then harvested in a non-denaturing buffer containing 150 mM NaCl, 10 mM Tris, pH 7.4, 1 mM
EGTA, 1 mM EDTA, 1% Triton X-100, 0.5% Nonidet P-40, 1 µM phenylmethylsulfonyl fluoride, 1 µM
pepstatin, 50 trypsin inhibitory milliunits of aprotinin, 10 µM leupeptin, and 2 mM sodium vanadate.
Samples were extracted for 30 min on ice and centrifuged at 15,000 rpm at 4 °C for 15 min. Supernatants containing whole cell extracts were
analyzed for protein content using a BCA kit (Pierce) according to the
manufacturer's instructions.
Preparation of Nuclear/Cytosolic Extracts--
Cell monolayers
were rinsed with phosphate-buffered saline and then harvested in a
nuclear homogenization buffer (NHB) containing 20 mM Tris,
pH 7.4, 10 mM NaCl, and 3 mM MgCl2.
Nonidet P-40 was added to a final concentration of 0.15%, and cells
were homogenized with 16 strokes in a Dounce homogenizer. The
homogenates were centrifuged at 1500 rpm for 5 min. Supernatants were
saved as cytosolic extract, and the nuclear pellets were resuspended in 0.5 volume of NHB and were centrifuged as before. The pellet of intact
nuclei was resuspended again in 0.5 of the original volume of NHB and
centrifuged again. A small portion of the nuclei was used for trypan
blue staining to examine the integrity of the nuclei. The majority of
the pellet (intact nuclei) was resuspended in an extraction buffer
containing 20 mM HEPES, pH 7.9, 420 mM NaCl,
1.5 mM MgCl2, 0.2 mM EDTA, and 25%
glycerol. Nuclei were extracted for 30 min on ice and then placed at
room temperature for 10 min. Two hundred units of DNase I was added to
each sample, and tubes were inverted and incubated an additional 10 min
at room temperature. Finally, the sample was subjected to
centrifugation at 15,000 rpm at 4 °C for 30 min. Supernatants
containing nuclear extracts were analyzed for protein content.
Gel Electrophoresis and Immunoblotting--
Proteins were
separated in 5, 7.5, 10, or 12% polyacrylamide (acrylamide from
National Diagnostics) gels containing SDS according to Laemmli (26) and
transferred to nitrocellulose (Bio-Rad) in 25 mM Tris, 192 mM glycine, and 20% methanol. Following transfer, the
membrane was blocked in 4% milk for 1 h at room temperature. Results were visualized with horseradish peroxidase-conjugated secondary antibodies (Sigma) and enhanced chemiluminescence (Pierce). Following chemiluminescence, some results were quantified by scanning film, and densitometry analysis was performed with associated analytical software (Biomax, Eastman Kodak Co.).
Determination of 2-Deoxyglucose--
The assay of
2-[3H]deoxyglucose was performed as described previously
(27). Prior to the assay, fully differentiated 3T3-L1 adipocytes were
serum-deprived for 4 h. Uptake measurements were performed in
triplicate under conditions where hexose uptake was linear, and the
results were corrected for nonspecific uptake, and absorption was
determined by 2-[3H]deoxyglucose uptake in the presence
of 5 µM cytochalasin B (Sigma). Nonspecific uptake and
absorption were always less than 10% of the total uptake.
Animals and Adipose Tissue Isolation--
Seven-week-old ob/+
and ob/ob mice were purchased from The Jackson Laboratories.
Eight-week-old fa/+ and fa/fa rats were purchased from Harlan. C57Bl/6J
mice were obtained from The Jackson Laboratories at 3-5 weeks of age,
and upon receipt were placed on a high fat/high sucrose diet (Research
Diets 12331, Surwit diet) or a low fat/high sucrose diet (Research
Diets 12329). In each experiment, at least five animals were used for
each condition. Twelve-week-old transgenic mice expressing agouti under
the control of the
-actin promoter were obtained from a colony at
the Pennington Biomedical Research Center. Rodents were euthanized by
cervical dislocation, and adipose tissue was quickly removed, weighed,
and frozen in liquid nitrogen. Frozen fat pads were homogenized in a
buffer containing 150 mM NaCl, 10 mM Tris, pH
7.4, 1 mM EGTA, 1 mM EDTA, 1% Triton X-100, 0.5% Nonidet P-40, 1 µM phenylmethylsulfonyl fluoride, 1 µM pepstatin, 50 trypsin inhibitory milliunits of
aprotinin, and 10 µM leupeptin, and 2 mM
sodium vanadate. Homogenates were centrifuged for 10 min at 5,000 rpm
to remove any debris and insoluble material and then analyzed for
protein content. All animal studies were carried out with protocols
that were reviewed and approved by the institutional animal care
and use committee. Adipocytes were isolated from the epididymal fat
pads of male C57Bl/6J mice (35g) by collagenase digestion.
 |
RESULTS |
The sensitivity of 3T3-L1 cells to cytokine treatment was examined
by treating undifferentiated preadipocytes and fully differentiated 3T3-L1 adipocytes with an acute treatment of 0.8 nM CNTF or
0.8 nM LIF. As shown in Fig.
1, immunoblotting of whole cell extracts demonstrated that both preadipocytes and adipocytes express STAT 3. Treatment of both preadipocytes and adipocytes with LIF or CNTF
resulted in the rapid activation of STAT 3, as evident by increased
tyrosine phosphorylation. However, treatment of preadipocytes resulted
in a greater stimulation of STAT 3 activation, relative to adipocytes,
despite equivalent expression of STAT 3 protein. In addition, CNTF and
LIF treatment caused a robust activation of Akt in preadipocytes,
whereas the same treatment of adipocytes did not result in a detectable
activation of Akt. The expression of two CNTF receptor complex
proteins, LIFR and gp130, was also examined. LIFR was expressed at a
substantially higher level in preadipocytes than in adipocytes, whereas
the expression of gp130 protein was not differentially expressed in
these two cell types.

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Fig. 1.
The effects of acute CNTF or LIF treatment on
3T3-L1 preadipocytes and adipocytes. Whole cell extracts were
prepared from confluent undifferentiated preadipocytes and from fully
differentiated 3T3-L1 adipocytes following a 15-min treatment with CNTF
(0.8 nM) or LIF (0.8 nM). Whole cell extracts
were prepared, and 75 µg of each extract was separated by SDS-PAGE,
transferred to nitrocellulose, and subjected to Western blot analysis.
The detection system was horseradish peroxidase-conjugated secondary
antibodies and enhanced chemiluminescence. This is a representative
experiment independently performed three times.
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The expression of CNTF receptor complex proteins was also examined
during a time course of adipocyte differentiation. As shown in Fig.
2, the expression of CNTFR
protein
decreases notably after 15 min of induction of differentiation, and
this lower level of expression is maintained for 48 h. However,
there were no detectable levels of CNTFR
72 h after the
initiation of differentiation in whole cell extracts. Yet we did
observe the presence of CNTFR
in the media at 72, 96, and 120 h
at lower levels (data not shown). As indicated in Fig. 1, the
expression of LIFR decreased during adipogenesis, and there was a
slight modulation of gp130 expression. The expression of STAT 5A is
known to be induced during adipocyte differentiation and is shown as a
positive control for adipogenesis.

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Fig. 2.
The expression of CNTF receptor complex
proteins during adipocyte differentiation. Whole cell extracts
were prepared from 3T3-L1 cells at various times following the
induction of differentiation. Cells were induced to differentiate at 2 days post-confluence with the addition of a differentiation mixture
containing 10% FBS, 0.5 mM 3-isobutyl-1-methylxanthine,
1.0 µM dexamethasone, and 1.7 µM insulin.
After 48 h this medium was replaced with DMEM supplemented with
10% FBS, and cells were maintained in this medium until utilized for
experimentation. One hundred µg of each extract was separated by
SDS-PAGE, transferred to nitrocellulose, and subjected to Western blot
analysis. Samples were processed, and results were visualized as
described in Fig. 1 legend. This is a representative experiment
independently performed three times.
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|
Because the expression of two CNTF receptor complex proteins was
reduced during adipogenesis, we wanted to determine whether these
proteins were expressed in adipose tissue and to compare the expression
levels to other tissues. Whole cell extracts were isolated from the
various tissues indicated in Fig. 3.
Western blot analysis revealed lung, stomach, epididymal fat, spleen, heart, brain, testes, and skeletal muscle as tissues expressing both
CNTFR
and LIFR. All of these tissues had comparable receptor expression levels, except for the brain, which had significantly higher
levels of CNTFR
expression. Also the molecular weight of CNTFR
in
stomach and brain was greater than in other tissues. In agreement with
our earlier observations (Fig. 2), the expression of CNTFR
was
abundant in preadipocytes and undetectable in 3T3-L1 adipocytes. We
also observed that the expression of CNTFR
was up-regulated in the
epididymal fat pad of an obese Zucker rat as compared with a lean
littermate.

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Fig. 3.
Tissue distribution of CNTF receptor complex
components in rodents. Tissue extracts were prepared from
8-week-old Sprague-Dawley rats. Seventy-five µg of each extract was
separated by SDS-PAGE, transferred to nitrocellulose, and subjected to
Western blot analysis. Samples were processed, and results were
visualized as described in Fig. 1 legend. Whole cell extracts from
3T3-L1 preadipocytes and adipocytes as well as tissue extracts from the
epididymal fat pads from lean and obese Zucker rats were also examined.
This is a representative experiment independently performed two
times.
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To determine whether the altered mobility of CNTFR
was due to
glycosylation, tissue extracts were incubated with PNGaseF. As shown in
Fig. 4, treatment with PNGaseF resulted
in the deglycosylation of CNTFR
and LIFR. In particular, the
CNTFR
bands of larger molecular weights (brain and stomach)
co-migrated with the CNTFR
from other tissues following digestion,
indicating that the size difference between CNTFR
in these tissues
was due to different glycosylation patterns. Also all LIFR bands
migrated at the same molecular weight following PNGaseF treatment.

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Fig. 4.
Glycosylation of CNTF receptor complex
components in rat tissues. Tissue extracts were prepared from
8-week-old Sprague-Dawley rats. Eighty µg of each extract was
incubated with 4 µl of PNGaseF (5,000 units/µl) as directed by the
manufacturer's instruction and then separated by SDS-PAGE, transferred
to nitrocellulose, and subjected to Western blot analysis. Samples were
processed, and results were visualized as described in Fig. 1 legend.
This is a representative experiment independently performed two
times.
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Although our results demonstrate that fully differentiated 3T3-L1
adipocytes do not express CNTFR
, it has been demonstrated previously
(15, 16) that CNTF can signal via gp130 and LIFR in the absence of
CNTFR
. Therefore, we examined the ability of CNTF to activate STATs
in a time-dependent manner in 3T3-L1 adipocytes. Serum-deprived fully differentiated 3T3-L1 adipocytes were exposed to
CNTF and examined over an 8-h period. Cells were harvested at the times
indicated at the top of Fig. 5 and
fractionated into cytosolic and nuclear extracts. As shown in Fig.
5A, CNTF administration to 3T3-L1 adipocytes resulted in the
nuclear translocation of STAT 3. STAT 3 was present in the nucleus
after a 10- or 30-min treatment with CNTF, and the amount of STAT 3 nuclear protein was decreased after a 1-h treatment. After 2 h,
there was little STAT 3 present in the nucleus. CNTF treatment did not
result in the activation/nuclear translocation of STAT 1 or STAT 5B,
indicating the specificity of the response. Also CNTF did not effect
the distribution of STAT 5A. Unlike other adipocyte-expressed STATs, some STAT 5A is always present in the adipocyte nucleus (28).

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Fig. 5.
Time- and dose-dependent effects
of CNTF administration on the phosphorylation and nuclear translocation
of STAT proteins in 3T3-L1 cells. A, cytosolic and
nuclear extracts were prepared from fully differentiated 3T3-L1
adipocytes following a treatment with 0.8 nM CNTF for the
times indicated at the top of the figure. B,
cytosolic and nuclear extracts were prepared from fully differentiated
3T3-L1 adipocytes following a 10-min treatment with CNTF at the doses
indicated. C, whole cell extracts were prepared from both
preadipocytes and from fully differentiated 3T3-L1 adipocytes following
a 10-min treatment with CNTF at the doses shown in the figure. Seventy-
five µg of each extract was separated by SDS-PAGE, transferred to
nitrocellulose, and subjected to Western blot analysis. Samples were
processed, and the results were visualized as described in Fig. 1
legend. This is a representative experiment independently performed
three times.
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The dose-dependent effects of CNTF on 3T3-L1 adipocytes
were also examined by treating adipocytes for 10 min with varying concentrations of CNTF. As shown in Fig. 5B, CNTF had no
effect on STATs 1 or 5A but resulted in the tyrosine phosphorylation and nuclear translocation of STAT 3. In addition, CNTF treatment resulted in a dose-dependent activation of MAPK (ERKs 1 and
2). To assess the dose effects of CNTF on STAT 3 activation, we
compared STAT 3 activation in preadipocytes and adipocytes. As shown in Fig. 5C, CNTF results in a dose-dependent effect
on STAT 3 activation in preadipocytes but not in adipocytes.
To characterize further the effects of CNTF, we treated fully
differentiated 3T3-L1 adipocytes for a 12-h period, and we isolated whole cell extracts at the times indicated at the top of
Fig. 6. As indicated previously, acute
CNTF treatment resulted in a time-dependent activation of
STAT 3 and MAPK but was unable to activate Akt. A positive control for
Akt activation (10 min of treatment of 3T3-L1 adipocytes with 50 nM insulin) is shown in the bottom panel of Fig.
6. Acute CNTF treatment did not affect the expression levels of STATs
1, 3, or 5A. There were also no observable differences in the
expression of SREBP-1 protein, as indicated by the levels of the
cleaved 67-kDa form of the protein.

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Fig. 6.
The effects of acute CNTF administration on
the expression of adipocyte proteins. Whole cell extracts were
prepared from fully differentiated 3T3-L1 adipocytes treated with 0.8 nM CNTF for the times shown. Seventy-five µg of each
extract was separated by SDS-PAGE, transferred to nitrocellulose, and
subjected to Western blot analysis. Samples were processed and results
were visualized as described in Fig. 1 legend. This is a representative
experiment independently performed three times.
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Next, we examined the effects of chronic CNTF administration on the
expression of adipocyte transcription factors and other adipocyte
proteins. Fully differentiated 3T3-L1 adipocytes were exposed to CNTF
over a 96-h period. A fresh bolus of CNTF was added to the cells every
24 h. Whole cell extracts were isolated at the times indicated at
the top of Fig. 7 and
subjected to Western blot analysis. Chronic administration of CNTF did
not alter the expression of adipocyte expressed STATs, PPAR
, or
C/EBP
. There were no notable differences in the levels of gp130 and
LIFR expression, and chronic CNTF treatment was insufficient to induce
the expression of CNTFR
. A positive control of confluent
preadipocytes is shown for CNTFR
expression. We also observed that
CNTF treatment did not alter the expression of acrp 30 in 3T3-L1
adipocytes. Moreover, CNTF had no effect on the expression or secretion
of leptin from 3T3-L1 adipocytes (data not shown). Interestingly, CNTF
treatment resulted in a decrease of FAS expression and a substantial
increase in the expression of IRS-1. Also, the levels of the 67-kDa
SREBP-1 protein were slightly decreased by CNTF treatment after 72 h.

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Fig. 7.
The effects of chronic CNTF administration on
the expression of adipocyte proteins. Whole cell extracts were
prepared from fully differentiated 3T3-L1 adipocytes treated with 0.8 nM CNTF for the times indicated at the top of
the figure. Seventy-five µg of each extract was separated by
SDS-PAGE, transferred to nitrocellulose, and subjected to Western blot
analysis. Samples were processed, and results were visualized as
described in Fig. 1 legend. This is a representative experiment
independently performed three times.
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Although we did not observe substantial levels of CNTFR
in cultured
3T3-L1 adipocytes (Figs. 2, 3, and 5), we were able to detect the
expression of CNTFR
in rodent adipose tissue from an obese Zucker
rat, and the levels of this receptor appeared to be up-regulated in
conditions of obesity (Fig. 3). Hence, we examined the expression of
CNTF receptors in adipose tissue of additional rodent models of
obesity/type II diabetes. Whole cell extracts were prepared from
epididymal fat pads of five ob/ob and five ob/+ lean littermates. As
shown in Fig. 8A, we observed very little expression of CNTFR
in the adipose tissue of lean mice,
but we observed a substantial increase in the expression of this
receptor in three of the five obese insulin-resistant ob/ob
littermates. In the other two ob/ob mice, there was a modest increase
in CNTFR
expression. In addition, we observed increased LIFR
expression in all five ob/ob mice compared with lean littermates, but
we did not observe any substantial changes in gp130 expression. We also
examined the expression of these proteins in the epididymal fat pads of
fa/+ and fa/fa rats. As shown in Fig. 8B, the expression of
CNTFR
was also substantially up-regulated in this rodent model of
obesity/type II diabetes. However, we did not observe an increase in
LIFR levels in the fa/fa rats as compared with their lean littermates, although there was a modest increase in gp130 levels in adipose tissue
from fa/fa rats. We also examined the expression of these receptors in
transgenic mice that over express agouti under the control of the
-actin promoter, a condition that causes obesity and type II
diabetes (29). We observed a substantial increase in CNTFR
levels in
the epididymal fat pads of three obese transgenic mice (Tg/+) compared
with wild-type lean (+/+) mice. There was also a modest decrease in
LIFR and gp130 in the fat pads of mice with agouti-induced obesity.
Finally, we examined the expression of CNTF receptors after low fat or
high fat feeding in C57B1/6J mice. Seven mice from each condition were
analyzed for CNTF receptor expression. The results in Fig.
8D only include three animals per condition. However, this
pattern of regulation was observed for all seven animals examined for
each condition (data not shown). In C57B1/6J mice, we observed an
increase in CNTFR
levels with high fat feeding after 12 weeks. A
similar pattern was also observed after 7 weeks (data not shown).
Overall, there was no modulation of LIFR or gp130 with high fat feeding
in the C57Bl/6J mice.

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Fig. 8.
The expression of CNTF receptor components in
the adipose tissue of lean and obese rodents. Whole cell extracts
were isolated from the epididymal fat pads of 7-week-old ob/+ (lean)
and ob/ob (obese) littermate mice (A), 8-week-old fa/+
(lean) and fa/fa (obese) littermate rats (B), 12-week-old
lean mice or obese agouti (Tg/+) littermates (C), and
17-week-old C57B1/6J mice fed a low or high fat diet for 12 weeks
(D). In each panel, 75 µg of each extract was separated by
SDS-PAGE, transferred to nitrocellulose, and subjected to Western blot
analysis. Samples were processed and results were visualized as
described in Fig. 1 legend.
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|
We have shown that cultured adipocytes do not express CNTFR
, but
rodent adipose tissues express detectable levels of the receptor.
Therefore, we examined the ability of CNTF to activate STAT 3 in
vivo. C57Bl/6J mice were given an intraperitoneal injection of
CNTF (33.3 µg/kg) or vehicle (saline) control. Fifteen minutes after
the injection, the mice were sacrificed, and epididymal adipose tissue,
brains, and skeletal muscle were immediately removed and frozen in
liquid nitrogen. Whole cell extracts were prepared from these tissues
and analyzed for STAT 3 phosphorylation by Western blot analysis. As
shown in Fig. 9, acute CNTF treatment resulted in the activation of STAT 3 in epididymal adipose tissue. We
were unable to detect STAT 3 phosphorylation in the adipose tissue of
five saline-injected mice, but four of the five CNTF injected mice had
readily detectable levels of phosphorylated STAT 3. The increase in
STAT 3 phosphorylation was not due to increased STAT 3 expression.
Also, the expression of LIFR was not changed, and the levels of
CNTFR
were variable in the 10 mice. The results in Fig.
9B demonstrate constitutive STAT 3 phosphorylation in brain,
which was unresponsive to exogenous CNTF. Moreover, we observed an
increase in STAT 3 phosphorylation in the skeletal muscle of
CNTF-treated animals, as compared with saline controls. As indicated
previously (Fig. 2), the levels of CNTFR
in the brain are
substantially greater than the levels in skeletal muscle.

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Fig. 9.
In vivo effect of acute CNTF
administration in rodents. Six-week-old C57Bl/6J mice were given
an intraperitoneal injection of CNTF (33.3 µg/kg) or vehicle (saline)
control. Fifteen minutes after the injection, the mice were sacrificed,
and epididymal fat pads, brains, and skeletal muscle were immediately
removed and frozen in liquid nitrogen. Tissue extracts were analyzed
from epididymal fat pads (A) and brain and skeletal muscle
(B). In each panel, 75 µg of each extract was separated by
SDS-PAGE, transferred to nitrocellulose, and subjected to Western blot
analysis. Samples were processed, and results were visualized as
described in Fig. 1 legend. This is a representative experiment
independently performed two times.
|
|
Our results demonstrate that CNTFR
receptor expression was decreased
during the adipogenesis of 3T3-L1 cells but expressed in the fat pads
of rodents. Therefore, we fractionated epididymal fat pads from
C57Bl/6J mice to determine whether the CNTFR
receptor was expressed
in the stromovascular fraction or in the adipocytes. As shown in Fig.
10, our results clearly demonstrate
that CNTFR
is expressed highly in the adipocytes, and STAT 3 is
expressed at higher levels in the stromovascular portion. We
hypothesize that the loss of CNTFR
that occurs during
differentiation in vitro (Fig. 2) could be an artifact of
cell culture because this receptor is expressed in native adipocytes
(Fig. 10) and in media obtained from cultured 3T3-L1 adipocytes (data
not shown).

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Fig. 10.
In vivo expression of
CNTFR in epididymal fat pads. Epididymal
fat pads were extracted from 6-week-old lean C57Bl/6J mice and
fractionated into adipocyte and stromovascular fractions. Seventy-five
µg of each extract was separated by SDS-PAGE, transferred to
nitrocellulose, and subjected to Western blot analysis. Samples were
processed, and results were visualized as described in Fig. 1
legend.
|
|
Because CNTF administration of ob/ob, db/db, and diet-induced obesity
mice has been shown to improve insulin sensitivity in vivo,
we examined the ability of CNTF to regulate insulin-sensitive glucose
uptake in 3T3-L1 adipocytes. Fully differentiated 3T3-L1 adipocytes
were treated for 24 h with CNTF. As shown in Fig.
11A, CNTF treatment resulted
in a notable increase (25-50%) in GLUT 4 levels. However, additional
treatments of CNTF did not result in a further increase in GLUT 4 levels as chronic CNTF treatment did not substantially increase GLUT 4 mRNA or protein levels (data not shown). Therefore, we examined the
ability of CNTF to affect glucose uptake. Fully differentiated
adipocytes were treated for 72 h with CNTF. Every 24 h, cells
were treated with a fresh bolus of CNTF. Acute insulin treatment (50 nM, 7 min) resulted in a 5-fold increase in
insulin-stimulated glucose uptake and was relatively unaffected by
chronic CNTF treatment (Fig. 11B). In addition, CNTF had no
effect on basal glucose uptake.

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Fig. 11.
CNTF does not cause insulin resistance but
increases GLUT 4 expression. A, whole cell extracts
were prepared from fully differentiated 3T3-L1 adipocytes treated with
0.8 nM CNTF for the times shown. Seventy-five µg of each
extract was separated by SDS-PAGE, transferred to nitrocellulose, and
subjected to Western blot analysis. Samples were processed, and results
were visualized as described in Fig. 1 legend. B, fully
differentiated 3T3-L1 adipocytes were treated with CNTF for 72 h.
A fresh bolus of CNTF was added to the cells every 24 h.
Monolayers of adipocytes were used to examine glucose uptake as
indicated under "Experimental Procedures." This is a representative
experiment independently performed four times.
|
|
Because CNTF treatment resulted in an increase in IRS-1 expression
levels (Fig. 7), we examined the ability of this cytokine to induce
IRS-1 activation, as judged by tyrosine phosphorylation at residue 896. As shown in Fig. 12, acute insulin
treatment (15 min) results in the activation of IRS-1 and Akt, whereas
acute CNTF treatment does not. However, CNTF pretreatment (30 min)
prior to insulin stimulation resulted in an increased IRS-1 activation (>20%) and increased Akt phosphorylation (>25%). The efficacy of
the CNTF is demonstrated by the activation of STAT 3.

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Fig. 12.
The effects of acute CNTF treatment on IRS-1
and Akt activation in 3T3-L1 adipocytes. Whole cell extracts were
prepared from fully differentiated 3T3-L1 adipocytes treated with 0.8 nM CNTF or 50 nM insulin for the times
indicated in the figure. Seventy-five µg of each extract was
separated by SDS-PAGE, transferred to nitrocellulose, and subjected to
Western blot analysis. Samples were processed, and results were
visualized as described in Fig. 1 legend. This is a representative
experiment independently performed three times.
|
|
 |
DISCUSSION |
In the light of recent findings demonstrating that CNTF
administration results in weight loss and correction of many other obesity/type II diabetes-related symptoms (19, 22, 24, 30), we
hypothesized that the effects of this cytokine may not be limited to
the central nervous system and that CNTF may also have effects on
peripheral tissues such as adipose tissue. Our in vitro
studies using 3T3-L1 preadipocytes and adipocytes have shown that CNTF indeed has significant, yet different, effects on these two cell types.
In 3T3-L1 adipocytes we observed that CNTF was a potent activator of
the Jak/STAT pathway, in particular STAT 3, as well as an activator of
a MAPK signaling cascade that resulted in activation of ERKs 1 and 2. In preadipocytes CNTF elicited similar effects but also resulted in the
activation of Akt. Our studies revealed that two of the three CNTF
receptor components, LIFR and CNTFR
, were down-regulated during the
adipogenesis of 3T3-L1 cells. Our study clearly demonstrates that the
expression of CNTFR
is substantially decreased during the course of
adipocyte differentiation. Other studies (16) have shown that CNTFR
is down-regulated during astrocyte differentiation. A previous
investigation had also indicated a decrease in LIFR during adipogenesis
(31), but this is the first investigation to demonstrate a decrease in
CNTFR
during adipogenesis. We hypothesize that decreased expression
of CNTF receptors upon differentiation accounts for cultured adipocytes being less sensitive to CNTF treatment than preadipocytes, as judged by
STAT 3 or Akt activation.
Although LIFR and CNTFR
protein levels are reduced in cultured
adipocytes, as compared with preadipocytes, we observed that adipocytes
were still responsive to CNTF. It has been demonstrated previously (15,
16) that CNTF can induce signaling in the absence of CNTFR
, solely
by binding to a gp130:LIFR dimeric receptor. Acute treatment of CNTF
did not alter the expression levels of any STATs or any other adipocyte
transcription factors in 3T3-L1 adipocytes. Hence, we examined the
chronic effects of CNTF on 3T3-L1 adipocytes, and we observed that this
cytokine affected the expression of several adipocyte-enriched
proteins, including SREBP-1, FAS, GLUT4, and IRS-1. The reduction in
the levels of SREBP-1 and FAS is indicative of decreased biosynthesis
of fatty acids that may account for some portion of weight loss and
decreased fat mass observed in patients treated with CNTF (Axokine). In agreement with previous findings that this CNTF-induced weight loss was
not due to cachexia or inflammation (22, 25), we did not observe any
effect of CNTF on PPAR
or C/EBP
, two transcription factors known
to be down-regulated by inflammatory cytokines such as tumor necrosis
factor-
and interferon-
(32-34). Also, unlike the in
vitro effects of other cytokines (32, 35-37), CNTF treatment of
3T3-L1 adipocytes did not result in the onset of insulin resistance (Fig. 10). Moreover, chronic CNTF treatment of these cells actually resulted in an increase in both GLUT4 and IRS-1 protein levels. However, we did not observe any effects of CNTF on basal or
insulin-stimulated glucose uptake. Clearly, additional experiments are
required to determine whether CNTF can act as an insulin sensitizer.
Nonetheless, we have shown that CNTF appears to act synergistically
with insulin to increase the level of IRS-1 and Akt phosphorylation in
3T3-L1 adipocytes.
Our results strongly suggest that CNTF affects adipose tissue and
skeletal muscle in vivo because an acute intraperitoneal injection of CNTF resulted in STAT 3 activation in both tissues. We
also observed that CNTFR
is expressed not only in brain and skeletal
muscle but also in adipose tissue, spleen, heart, testes, lungs, and
stomach. The receptor expression levels, as well as the protein size,
vary among these tissues, but our deglycosylation studies clearly
demonstrate that they all express CNTFR
. One of the most important
findings we observed was that, in vivo, CNTFR
expression
was significantly increased in four different rodent models of
obesity/type II diabetes, including both genetic and diet-induced
obesity. Moreover, we have shown that CNTFR
is expressed at higher
levels in the adipocytes as compared with the stromovascular portion of
the fat pad (Fig. 10). Although we observed an increase in the
expression of the LIFR in the ob/ob mice, as compared with lean
littermates, the expression of this receptor was not altered in the
fa/fa rats or in C57Bl/6J mice with diet-induced obesity.
The results of our study suggest that CNTF and CNTFR
may play a role
in the regulation of adipocyte metabolism and, perhaps, the control of
adipose tissue mass. Our results have led us to hypothesize that CNTF
can act as an insulin sensitizer in adipocytes. Therefore, the
up-regulation of CNTFR
in adipose tissue of obese/type II diabetic
rodents could be an adaptive response attempting to increase insulin
sensitivity. Interestingly, some studies suggest that CNTFR
may not
only act as receptor for CNTF but also as a receptor for another
unknown CNTF-like factor. For example, mice lacking CNTF develop
normally and appear to have no visible defects well into adulthood,
when they develop minor loss of motor neurons (10). Yet mice lacking
CNTFR
tend to have severe motor neuron defects and die perinatally
because they fail to initiate feeding behaviors (11). Also the finding
that CNTF expression is undetectable in the feeding-relevant brain
sites, which express high levels of CNTFR
(30), further supports the
notion that CNTFR
may have additional ligands and/or functions.
In summary, we observed that native as wells as cultured adipocytes are
responsive to CNTF treatment. Interestingly, CNTFR
is not highly
expressed in cultured adipocytes but is readily detectable in rodent
adipose tissue and furthermore highly up-regulated in multiple rodent
models of obesity/type II diabetes. This is the first demonstration
that this receptor is expressed in adipose tissue and that it is highly
regulated in obesity/type II diabetes. Current studies are underway to
determine the role of CNTFR
in adipose tissue function and examine
the ability of CNTF to act as insulin sensitizer in fat and muscle.
 |
ACKNOWLEDGEMENT |
We thank EunSun Lee for technical assistance.
 |
FOOTNOTES |
*
This work was supported by Grant R01DK52968-02 from the
National Institutes of Health (to J. M. S.).The costs of publication of this
article were defrayed in part by the
payment of page charges. The 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: Dept. of Biological
Sciences, Louisiana State University, 202 Life Sciences Bldg., Baton
Rouge, LA 70803. Tel.: 225-578-1749; Fax: 225-578-2597; E-mail:
jsteph1@.lsu.edu.
Published, JBC Papers in Press, November 6, 2002, DOI 10.1074/jbc.M205871200
 |
ABBREVIATIONS |
The abbreviations used are:
CNTF, ciliary
neurotrophic factor;
CNTFR
, CNTF-specific receptor;
PPAR, peroxisome
proliferator-activated receptor;
MAPK, mitogen-activated protein
kinase;
STAT, signal transducers and activators of transcription;
ERK, extracellular signal-regulated kinase;
DMEM, Dulbecco's modified
Eagle's medium;
FBS, fetal bovine serum;
PNGaseF, peptide:N-glycosidase F;
FAS, fatty-acid synthase;
gp, glycoprotein.
 |
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