Insulin Inhibits Growth Hormone Signaling via the Growth Hormone
Receptor/JAK2/STAT5B Pathway*
Shaonin
Ji
,
Ran
Guan§¶,
Stuart J.
Frank§¶
, and
Joseph L.
Messina
**
From the
Department of Pathology, Division of
Molecular and Cellular Pathology, the § Department of
Medicine, Division of Endocrinology and Metabolism, and the
¶ Department of Cell Biology, University of Alabama at Birmingham,
and
Birmingham Veterans Administration Medical Center,
Birmingham, Alabama 35294
 |
ABSTRACT |
Insulin is important for maintaining the
responsiveness of the liver to growth hormone (GH). Insulin deficiency
results in a decrease in liver GH receptor (GHR) expression, which can
be reversed by insulin administration. In osteoblasts, continuous insulin treatment decreases the fraction of cellular GHR localized to
the plasma membrane. Thus, it is not clear whether hyperinsulinemia results in an enhancement or inhibition of GH action. We asked whether
continuous insulin stimulation, similar to what occurs in
hyperinsulinemic states, results in GH resistance. Our present studies
suggest that insulin treatment of hepatoma cells results in a
time-dependent inhibition of acute GH-induced
phosphorylation of STAT5B. Whereas total protein levels of JAK2 were
not reduced after insulin pretreatment for 16 h, GH-induced JAK2
phosphorylation was inhibited. There was a concomitant decrease in GH
binding and a reduction in immunoreactive GHR levels following
pretreatment with insulin for 8-24 h. In summary, continuous insulin
treatment in rat H4 hepatoma cells reduces GH binding, immunoreactive
GHR, GH-induced phosphorylation of JAK2, and GH-induced tyrosine
phosphorylation of STAT5B. These findings suggest that hepatic GH
resistance may develop when a patient exhibits chronic
hyperinsulinemia, a condition often observed in patients with obesity
and in the early stage of Type 2 diabetes.
 |
INTRODUCTION |
Growth hormone (GH)1 is
one of the prime regulators of body composition (1). Along with other
hormones and growth factors it increases muscle mass and decreases
subcutaneous and visceral fat (2, 3). Abdominal adiposity is prevalent
in human diseases of impaired GH function, including Laron syndrome, a
GH-resistant syndrome due to mutation of the GH receptor (GHR), and
Prader-Willi syndrome in which there is diminished circulating GH (4,
5). Abdominal obesity is also associated with human peripheral insulin resistance, hyperinsulinemia, and Type 2 diabetes (2, 6). Common to all
of these conditions is an increase in the ratio of insulin to GH (1, 2,
6).
The GHR belongs to the superfamily of cytokine receptors and in humans
and rabbits the full-length GHR is translated from a single mRNA
(7). Circulating GHBP results from proteolytic cleavage of the plasma
membrane-associated GHR (7). However, a recent study suggests that
primate GHBP may also arise from an alternatively spliced mRNA, a
mechanism first indicated in rodents (7, 8). Binding of GH to its
receptor results in dimerization of the receptor followed by tyrosine
phosphorylation of GHR itself and tyrosine phosphorylation and
activation of Janus activating kinase 2 (JAK2) (9, 10). Activation of
JAK2 by GH, and by other cytokines and growth factors, leads to
phosphorylation and activation of one or more signal
transducers and activators of
transcription (STAT) (10-12). The JAK-STAT pathway, is a
major pathway for GH regulation of gene transcription. Although GH
promotes activation of STAT1, STAT3, STAT5A, and STAT5B, gene
disruption experiments indicate that STAT5B is necessary for GH
regulation of sexually dimorphic hepatic genes (10, 13).
In vivo insulin appears to be necessary for normal liver GH
responsiveness, probably by maintaining liver GHR levels (14-17). In
Type 1 diabetic patients and streptozotocin-treated rodents, insulin
deficiency is correlated with hepatic GH resistance which, in most
studies, is associated with reduced levels of circulating GHBP in
patients or decreased liver GHR in rodents (14-24). In streptozotocin-treated rats, circulating insulin-like growth factor 1 (IGF-1), whose mRNA expression is regulated by GH, is reduced as is
GH binding capacity. Insulin treatment restores IGF-1 levels and in
some, but not all experiments, restores GH binding (14, 17, 25-27). In
Type 1 diabetic patients intraperitoneal insulin administration
restores GHBP levels better than subcutaneous insulin treatment (19,
20, 23). This suggests that peripheral (subcutaneous) insulin
administration may result in portal insulin concentrations insufficient, compared with intraperitoneal insulin infusion, to
properly regulate hepatic GHR expression and therefore circulating GHBP. Patients with Type 2 diabetes and peripheral insulin resistance also exhibit reduced circulating IGF-1 levels, possibly due to a
decrease in GH responsiveness, but it has not yet been studied whether
liver GHR or circulating GHBP levels are reduced accordingly (18,
23).
In vitro studies are also inconsistent concerning whether
insulin can increase GH binding and GHR mRNA. For example, in a study with primary cultures of rat hepatocytes, insulin treatment increases GH binding 4-fold with no significant effect on GHR mRNA
expression (28). In a study measuring the subcellular localization of
GHR in osteoblasts, continuous insulin treatment decreases the fraction
of cellular GHR presented at the plasma membrane via inhibition of
surface translocation of GHR with no effect on the total cellular
content of GHR (29). However, changes in GH-induced signaling pathways
have not been investigated in these studies measuring insulin-induced
changes in GHR and GH binding.
There is impaired GH action after 60 years of age in humans, possibly
resulting from decreases in circulating GHBP levels, and therefore most
likely hepatic GHR levels (30). Also, there may be defects in GH
stimulation of the JAK-STAT signaling pathway in aging humans, as there
are in aging mice (31). Therefore, investigation of factors that affect
GH responsiveness may help in the understanding of aging-related
changes in GH action.
In the present study, insulin pretreatment for 8-24 h was found to
reduce the acute effect of GH on STAT5B phosphorylation in rat H4
hepatoma cells. The GH-induced tyrosine phosphorylation of JAK2,
immunoreactive GHR, and binding of 125I-hGH were also
reduced following insulin pretreatment. Inhibition of GH-induced STAT5B
phosphorylation, immunoreactive GHR, and GH binding were all reduced by
insulin pretreatment with similar kinetics. Our study indicates an
extensive reduction in hepatoma cell GH responsiveness following
conditions that may mimic the chronic hyperinsulinemia observed in some
obese patients and patients in the early stages of Type 2 diabetes.
 |
EXPERIMENTAL PROCEDURES |
Materials--
Bovine GH (bGH; lot number APF11182B) and ovine
prolactin (oPRL, NIDDK-oPRL-21; lot number AFP-10692C) were kindly
provided by Dr. A. F. Parlow, Pituitary Hormones and Antisera
Center, Harbor-UCLA Medical Center (Torrance, CA) and the NIDDK,
National Institutes of Health National Hormone & Pituitary Program. The
125I-human GH (125I-hGH) was purchased from
NENTM Life Science Products. Porcine sodium-insulin was a
gift from Dr. Ron Chance (Eli Lilly, Co., Indianapolis, IN) and
unlabeled hGH was also kindly provided by Eli Lilly, Co. Fetal bovine
serum, calf serum, and horse serum were purchased from Life
Technologies, Inc. (Grand Island, NY). Protein G-Sepharose was obtained
from Pharmacia Biotech Inc. (Uppsala, Sweden) and ECL detection
reagents were obtained from Amersham Corp. Other materials were
purchased from Sigma and Fisher (Pittsburgh, PA) unless otherwise noted.
Antibodies--
Anti-STAT5 monoclonal antibody (raised against
amino acid 451-649 of sheep STAT5A) was purchased from Transduction
Laboratories (Lexington, KY). Mouse anti-STAT5A (raised against the
unique C terminus of murine STAT5A) and mouse anti-STAT5B (raised
against the unique C terminus of murine STAT5B) monoclonal antibodies and rabbit anti-phosphotyrosine-STAT5 polyclonal antibodies (raised against the phospho-peptide around C-terminal Y694 of murine STAT5A that is conserved in both STAT5A and STAT5B of human, sheep, and rat)
were obtained from Zymed Laboratories Inc. (San
Francisco, CA). Rabbit anti-JAK2 peptide antiserum, directed at
residues 758-776 of murine JAK2 (called anti-JAK2UBI), and 4G10 mouse
monoclonal anti-phosphotyrosine antibody were purchased from Upstate
Biotechnology, Inc. (Lake Placid, NY). A second anti-JAK2 serum
(anti-JAK2AL33) was raised in rabbits against a glutathione
S-transferase fusion protein incorporating residues
746-1129 of the murine JAK2, as described (32, 33). Two anti-GHR
antisera were raised in rabbits against peptides of the cytoplasmic
domain of the GHR: anti-GHRcyt3728 against residues 317-620 of the
human GHR cytoplasmic domain and anti-GHRcytAL37 against residues
271-620 fused to glutathione S-transferase as described
(33, 34). Secondary antibodies including the peroxidase-linked sheep
anti-mouse serum and the peroxidase-linked donkey anti-rabbit serum
were obtained from Amersham Life Science.
Cell Culture--
Rat H4-II-E (H4) hepatoma cells were cultured
in Swim's medium supplemented with 10% serum mixture (5% horse
serum, 3% newborn calf serum, and 2% fetal calf serum) and 2 µg/ml
gentamycin sulfate (35). At about 50% confluence, cells were removed
from serum and maintained in serum-free media for 24 h prior to
protein extraction.
Protein Extraction--
Hormone treatment (detailed in the text)
was terminated by rinsing the cells once with TBS (10 mM
Tris-HCl, pH 7.4, 150 mM NaCl) and cells were collected in
100 °C SDS-lysis buffer (1% SDS, 10 mM Tris-HCl, pH
7.4, 1 mM phenylmethylsulfonyl fluoride, 50 mM
sodium fluoride, 0.5 mM Na3VO4) and
boiled for 5 min. One volume of 4 × Laemmli sample buffer (8%
SDS, 250 mM Tris-HCl, pH 6.8, 40% glycerol, 4%
-mercaptoethanol, 0.02% bromphenol blue) was added to 3 volumes of
whole cell lysates, the solution was boiled for an additional 5 min and
stored at
80 °C until subjected to polyacrylamide gel electrophoresis.
In an alternative method, cells were scraped into 4 °C TBS plus 1 mM phenylmethylsulfonyl fluoride, 50 mM sodium
fluoride, 0.5 mM Na3VO4 and cell
pellets were collected by centrifugation (800 × g for
2 min at 4 °C). Cell pellets were solubilized with mild agitation
for 1 h at 4 °C in Triton-lysis buffer (1% Triton X-100, 50 mM Tris-HCl, pH 8.0, 150 mM NaCl, 10%
glycerol, 1.5 mM MgCl2, 1 mM EDTA,
10 mM Na4O7P2, 1 mM phenylmethylsulfonyl fluoride, 50 mM sodium
fluoride, 1 mM Na3VO4, 10 mM benzamidine, 2 µg/ml aprotinin). After centrifugation
at 15,000 × g for 15 min at 4 °C, the supernatants were
immediately subjected to either immunoprecipitation or were boiled for
5 min in sample loading buffer, and stored at
80 °C until electrophoresis.
Immunoprecipitation, Electrophoresis, and
Immunoblotting--
For immunoprecipitation in nondenaturing
conditions, cells were lysed in Triton-lysis buffer. Protein
G-Sepharose was used to adsorb immune complexes and after extensive
washing with Triton-lysis buffer precipitated proteins were eluted by
boiling in sample loading buffer for 5 min. The proteins were then
subjected to 5-9% gradient SDS-polyacrylamide gel electrophoresis.
Western transfer of proteins were performed as described previously,
except for the use of Protran membrane from Schleicher & Schuell (BA 85) (34). Immunoblotting was performed with the antibodies at the
following dilutions: anti-STAT5 (1:500), anti-STAT5A (1:5000), anti-STAT5B (1:5000), anti-PY-STAT5 (1:5000), 4G10 (1:2500),
anti-GHRcyt3728 and anti-GHRcytAL37 (1:2000), or anti-JAK2
(1:2000) with horseradish peroxidase-conjugated anti-mouse or
anti-rabbit secondary antibodies (1:2000). Incubation with rabbit
primary antisera was at 4 °C overnight, while incubation with mouse
monoclonal antibodies was at room temperature for 1 h. Washing
times after primary and secondary antibodies were at room temperature
for 10 and 45 min, respectively, unless specified. Washing times after
anti-GHRcyt3728 and anti-rabbit IgG were at room temperature for 90 and
20 min, respectively. All antibodies are used in 0.7% Tween 20 in TBS,
pH 8.0, with 0.4% milk, 2% bovine serum albumin, and 0.04% azide
added to the primary antibodies except for 4G10, in which milk was
excluded. Detection of bound antibodies by ECL and stripping and
reprobing of blots were accomplished according to the manufacturer's suggestions.
125I-hGH-binding Assay--
H4 cells were grown to
50% confluence in 60-mm plates and treated as indicated in the text
before partial withdrawal of media to reduced volume to 1 ml. Cells
were then incubated with a constant amount of 125I-hGH
within a given experiment (between 6 × 105 and 9 × 105 cpm per plate in the separate experiments) with or
without 2 µg/ml unlabeled hGH, bGH, or oPRL at room temperature for
2 h with gentle agitation (36, 37). Following incubation, binding was measured by washing the cells twice with 2 ml of 4 °C binding buffer (25 mM Tris-HCl, pH 7.4, 120 mM NaCl, 5 mM KCl, 1.2 mM MgCl2, 0.1% bovine
serum albumin), harvesting the cells in a buffer of 1% SDS, 0.1 N NaOH and counting in an
-counter (Packard Instrument Co., Meriden, CT).
Densitometric and Statistical Analysis--
ECL images of
immunoblots were scanned and quantified with a Scion-image analysis
program (release beta 2) from Scion Corp. (Frederick, MD). All data was
analyzed by ANOVA using the InStat statistical program (version 3) by
GraphPad Software, Inc. (San Diego, CA).
 |
RESULTS |
STAT5A and STAT5B Were Expressed in H4 Cells at Comparable
Levels--
We first asked whether the STAT5 proteins were expressed
at appreciable amounts in rat H4 hepatoma cells. Using a pan-STAT5 antibody, the immunoreactive STAT5 bands from whole cell extracts from
untreated H4 cells migrated as at least 3 bands. From previously published studies, and as confirmed here with isoform-specific antibodies, the upper band that runs as p95 or p96, depending upon the
study (and referred to as p95/p96) was specific for STAT5A (Fig.
1A, lanes 1 and 3).
The lower p92/p94 doublet was STAT5B with the doublet arising because a
fraction of the STAT5B is serine-phosphorylated, even in quiescent
cells, and thus displays retarded migration (Fig. 1A, lanes
1 and 2, and Refs. 38 and 39). As observed in Fig.
1A, lane 1, using the pan-STAT5 antibody, there was less STAT5A than STAT5B (approximately 70% as much STAT5A as STAT5B by
densitometry) in H4 cell extracts.

View larger version (38K):
[in this window]
[in a new window]
|
Fig. 1.
Effects of insulin treatment on STAT5,
STAT5A, and STAT5B levels. H4 cells were serum starved for 48 h and left untreated or treated with insulin (100 nM) for
the time indicated. A, B, and C, representative
experiments are presented with cells harvested, whole cell lysates
prepared, and subjected to Western blot analysis as described in the
text with antibodies specific for STAT5, STAT5A, and STAT5B as
indicated either above the lane in A and to the
left in B and C. D,
densitometric analysis of autoradiographs from four similar experiments
were performed to quantify changes in STAT5 and STAT5B and are
expressed as the mean ± S.E. of the mean. The levels of STAT5 or
STAT5B in control (untreated) cells was arbitrarily set to 100% within
each experiment and the levels following various times of insulin
pretreatment are expressed as a percentage of expression measured in
control cells. All insulin time points are not statistically
significant compared with the zero time by ANOVA.
|
|
Effect of Insulin on STAT5 Levels--
We then asked whether
insulin altered the cellular levels of the STAT5 isoforms. Using the
pan-STAT5 antibody, there was no appreciable change in cellular STAT5
levels following insulin treatment of H4 cells for 4 and 12 h
(Fig. 1B, lanes 1-3). Using the STAT5B-specific antibody, a
slight reduction of STAT5B was sometimes observed following insulin
treatment for 12, 20, and 24 h (Fig. 1C, lanes 1-5).
When multiple experiments were quantified by densitometry (Fig.
1D), total cellular levels of STAT5 were unchanged by
insulin treatment and STAT5B levels were reduced to approximately 80%
of that measured in untreated, control H4 cells after insulin treatment
for 12 or more hours. However, this small reduction of STAT5B was not
statistically significant.
Continuous Insulin Treatment Did Not Alter STAT5
Phosphorylation--
Previous studies demonstrated that
unphosphorylated STAT5B migrates faster on polyacrylamide gels and the
migration of tyrosine and/or serine-phosphorylated STAT5B is retarded
(38, 39). In the present studies using rat H4 hepatoma whole cell
extracts, and using an antibody specific only to STAT5B, in the absence of GH treatment STAT5B migrated as a doublet of p92/p94 (Fig. 2, top row, lanes 1 and
5) which were previously shown to be specific for
unphosphorylated STAT5B (p92) and STAT5B phosphorylated only on serine
residues (p94) (38, 39). Using the STAT5B-specific antibody, that does
not cross-react with STAT5A, bGH treatment (20 min) of rat H4 hepatoma
cells induced tyrosine phosphorylation of STAT5B (PY-STAT5B), as
suggested by the retarded mobility of STAT5B (Fig. 2, top row,
lanes 2 and 6 versus lanes 1 and 5,
respectively). This 95-96-kDa band (p95/p96) is thought to be specific
for STAT5B phosphorylated on both serine and tyrosine residues
(PY-STAT5B (38, 39)). Thus, when phosphorylated on both tyrosine and serine, STAT5B migrates at approximately the same size as STAT5A (Fig.
1A, lane 3). Insulin pretreatment of H4 cells for 8, 20, and
24 h did not alter the mobility, and therefore the
phosphorylation, of STAT5B (Fig. 2, top row, lanes 3, 4, and
7).

View larger version (32K):
[in this window]
[in a new window]
|
Fig. 2.
Insulin did not effect STAT5B migration and
tyrosine phosphorylation of STAT5. Western blots were performed as
described in the legend for Fig. 1 and in the text except that some H4
cells were treated with bovine growth hormone (bGH; 500 ng/ml) for 20 min and the antibodies used (as indicated to the left)
included that for STAT5B and phosphorylated tyrosine STAT5. A
representative experiment is presented.
|
|
Tyrosine phosphorylation of STAT5 was then measured by using a
phosphotyrosine-specific antibody and, consistent with the mobility
shift using the STAT5B antibody, bGH-induced the phosphorylation of
STAT5 on tyrosine (PY-STAT5; Fig. 2, bottom row, lanes 2 and 6 versus lanes 1 and 5, respectively). Also
consistent with the mobility shift using the STAT5B antibody, insulin
for 8, 20, and 24 h did not alter the tyrosine phosphorylation of
STAT5B (PY-STAT5; Fig. 2, bottom row, lanes 3, 4, and
7). Thus, continuous insulin treatment at the times studied
had little effect on the cellular content or phosphorylation state of STAT5B.
Insulin Inhibited GH-induced STAT5B Phosphorylation and STAT5
Tyrosine Phosphorylation--
Experiments were then designed to
examine whether insulin affected GH-stimulated phosphorylation of
STAT5B. The ability of bGH for 20 min to induce PY-STAT5B was again
observed by the retarded mobility of the STAT5B using the STAT5B
isoform-specific antibody (Fig. 3,
A and B, lanes 1 versus 2). After insulin
pretreatment of H4 cells for 8, 20, or 24 h, there was a reduction
of the bGH stimulation of STAT5B retardation. This is most likely due
to a smaller fraction of STAT5B being phosphorylated (a smaller
fraction migrates at the higher p95/96 PY-STAT5B band) upon bGH
addition following insulin pretreatment compared with untreated cells
(compare Fig. 3, A, lanes 4 versus 2; and B, lanes 4 versus 2 and 6 versus 2). There was no significant
inhibition of bGH-stimulated STAT5B phosphorylation by 4 h of
insulin pretreatment, but this inhibition became highly significant
between 8 and 24 h of insulin pretreatment (p < 0.001 by ANOVA), with maximum inhibition to approximately 25-30% of
control at all times tested between 12 and 24 h (Fig. 3C).

View larger version (28K):
[in this window]
[in a new window]
|
Fig. 3.
Insulin reduced GH-induced STAT5B
phosphorylation. A and B, Western blots were
performed as described in the legend for Fig. 1 and in the text except
that some groups of H4 cells were pretreated with insulin (100 nM) for the indicated durations prior to addition of bGH
(500 ng/ml) for 20 min. Representative Western blots are presented.
C, densitometric analysis of autoradiographs from four
similar experiments (except n = 3 at insulin 20 h)
were performed to quantify the proportion of
tyrosine/serine-phosphorylated STAT5B following bGH addition and the
indicated times of insulin pretreatment. The data is expressed as the
mean ± S.E. The ratio of densitometric measurements of the p95/96
band of STAT5B following bGH (500 ng/ml) for 20 min to that of all
STAT5B bands was arbitrarily set to 100% in samples from H4 cells
without insulin pretreatment (control cells) within each experiment.
The corresponding ratio in whole cell extracts from H4 cells treated
with insulin (100 nM) for the indicated times prior to bGH
addition for 20 min is expressed as a percentage of expression measured
in control cells. The 4-h insulin time point is not statistically
significant compared with the zero time, whereas the 8-, 12-, 16-, 20-, and 24-h insulin time points are significant (p < 0.001) by ANOVA.
|
|
Using an antibody specific for tyrosine-phosphorylated STAT5, no
pretreatment or pretreatment with insulin for 4 h resulted in
similar levels of bGH-induced PY-STAT5 (Fig.
4A, top row, lanes 2 and
4). However, following insulin pretreatment for 16, 20, and
24 h, bGH-induced PY-STAT5 was reduced (Fig. 4B, top row, lanes 2 versus 4; and 6 versus 8 and 10).
JAK2 protein levels were unaltered at any period of insulin
pretreatment when the same Western blots were re-probed with a
JAK2-specific antibody (Fig. 4, A, bottom row, lanes 1-4;
and B, bottom row, lanes 1-10). When multiple experiments
were quantified by densitometry, there was a clear and highly
significant reduction (p < 0.001 by ANOVA) in
bGH-induced PY-STAT5 by insulin pretreatment using the
PY-STAT5-specific antibody at the 8-24-h insulin time points (Fig.
4C). The extent and kinetics of reduction of the bGH
stimulation of STAT5 phosphorylation following insulin pretreatment was
almost identical when using the PY-STAT5-specific (Fig. 4C)
and STAT5B-specific antibody (Fig. 3C). Again, there was no
change in JAK2 protein levels at any period of insulin pretreatment
when the same Western blots were re-probed with a JAK2-specific
antibody (Fig. 4D). From the use of 2 different antibodies
measuring the change in mobility of STAT5B and tyrosine-phosphorylated
STAT5 directly, we conclude that the effects of insulin are to reduce
the ability of GH to stimulate the phosphorylation of STAT5B.

View larger version (21K):
[in this window]
[in a new window]
|
Fig. 4.
Insulin reduced GH-induced STAT5
phosphorylation without changing JAK2 levels. A and
B, Western blots were performed as described in the legend
for Fig. 1 and in the text except the PY-STAT5 and anti-JAK2UBI or
anti-JAK2AL33 antibodies were used as indicated. Insulin (100 nM) and bGH (500 ng/ml) treatments were for the times
indicated. Representative Western blots are presented. C,
densitometric analysis of autoradiographs from four similar experiments
(except n = 3 at insulin 20 and 24 h) were
performed to quantify the changes in PY-STAT5 following various times
of insulin pretreatment. The data is expressed as the mean ± S.E.
The levels of PY-STAT5 immunoreactivity in control (untreated) cells
was arbitrarily set to 100% within each experiment and the levels
following various times of insulin pretreatment are expressed as a
percentage of that in control cells. The 4-h insulin time point is not
statistically significant compared with the zero time, whereas the 8-, 12-, 16-, 20-, and 24-h insulin time points are significant
(p < 0.001) by ANOVA. D, densitometric
analysis of autoradiographs from three similar experiments were
performed, using the anti-JAK2UBI and anti-JAK2AL33 antibodies to
quantify the changes in JAK2 following various times of insulin
pretreatment. The data is expressed as the mean ± S.E. The levels
of JAK2 immunoreactivity in control (untreated) cells was arbitrarily
set to 100% within each experiment and the levels following various
times of insulin pretreatment are expressed as a percentage of
expression measured in control cells. All insulin time points are not
statistically significant compared with the zero time by ANOVA.
|
|
Insulin Reduced GH-induced JAK2 Tyrosine
Phosphorylation--
Multiple possibilities exist as to the mechanism
by which insulin pretreatment could inhibit STAT5B phosphorylation.
Since STAT5B is phosphorylated by JAK2, the simplest possibility is that there is a reduction in JAK2 levels or activity. As indicated in
Fig. 4, A, B and D, there were no measurable
changes in the cellular levels of JAK2 at any time following insulin
addition. Thus, the next logical question was whether insulin
pretreatment altered GH-induced JAK2 activity. Since phosphorylation of
JAK2 on tyrosine is required for its activation and ability to tyrosine phosphorylate STAT5 (9), we proceeded to determine the effect of
insulin pretreatment on JAK2 tyrosine phosphorylation. Consistent with
the data described above with whole cell lysates, there was no effect
of acute bGH or 16 h of insulin pretreatment on the levels of
immunoprecipitable JAK2 protein (Fig. 5,
top row). Following 10 min of bGH, a fraction of the
immunoprecipitated JAK2 has been phosphorylated on tyrosine residues as
measured by a mouse monoclonal antibody raised against
phosphorylated tyrosine residues (Fig. 5, bottom row, lanes 3 versus 1). Insulin pretreatment for 16 h significantly
inhibited the ability of bGH to induce tyrosine phosphorylation of
immunoprecipitated JAK2 in the absence of any changes in the cellular
levels of immunoprecipitable JAK2 (Fig. 5, bottom row, lanes 4 versus 3). Thus, chronic insulin pretreatment inhibits both
GH-stimulated JAK2 and STAT5B phosphorylation.

View larger version (51K):
[in this window]
[in a new window]
|
Fig. 5.
Insulin for 16 h reduced JAK2 tyrosine
phosphorylation. H4 cells were treated with insulin (100 nM; 16 h) and bGH (500 ng/ml; 10 min) as indicated and
harvested to obtain detergent extracts for immunoprecipitation as
described in the text. Top row, JAK2 proteins were
immunoprecipitated with one JAK2 antiserum, anti-JAK2AL33, and then
immunoblotted with JAK2 antiserum from Upstate Biotechnology, Inc.
(anti-JAK2UBI). Bottom row, the same blot was stripped and
re-probed with anti-phosphotyrosine antibody 4G10 (PY antibody).
|
|
Insulin Reduced 125I-hGH Binding to H4 Cells--
The
next question was to determine whether the effect of insulin was even
further upstream in the GH signaling pathway, at the GHR itself. The
published results on insulin regulation of hepatic GH binding are
contradictory (14, 24, 25, 28) and no studies have been performed using
H4 or related rat hepatoma cell lines. Radiolabeled hGH was used to
measure GH binding and in our studies, the total binding of
125I-hGH binding to H4 cells was approximately 3.0% of the
total counts/min of 125I-hGH added (Fig.
6A). Since hGH is capable of
binding to rat somatogenic and lactogenic sites, both unlabeled hGH and
bGH were used in competition experiments to determine the level of
radiolabeled hGH binding to somatogenic binding sites. Excess unlabeled
hGH or bGH competed for 125I-hGH binding to H4 cells to a
similar degree, with a slightly better competition by hGH, resulting in
specific binding of approximately 1.6-1.7% (Fig. 6A). This
suggests that there was little 125I-hGH binding to
lactogenic binding sites in H4 cells. This was expected since H4 cells
are derived from a hepatocellular carcinoma of a male rat (40). Male
rats normally express low levels of hepatic lactogenic binding sites
(14). To further test this, the ability of unlabeled oPRL to compete
for 125I-hGH binding was measured and found to reduce
125I-hGH binding to only a small degree suggesting few
lactogenic binding sites (Fig. 6A). Thus, the majority of
125I-hGH binding is to somatogenic sites and the use of
125I-hGH binding is an accurate estimate of binding to H4
cell GHR.

View larger version (31K):
[in this window]
[in a new window]
|
Fig. 6.
Insulin for 16 h reduced specific
125I-hGH binding to H4 cells. The binding assays were
performed as described in the text to examine 125I-hGH
binding to H4 cells using 40,000-60,000 cpm of 125I-hGH
per well in the separate experiments. In A,
125I-hGH binding to H4 cells not pretreated with insulin
and either no addition (first bar; ) or 2 mg/ml unlabeled
hGH, bGH, or oPRL were added as competitors, as indicated. In
B, H4 cells were not pretreated (first and
second bars) or were treated with insulin (100 nM) for 16 h as indicated (third and
fourth bars). Unlabeled hGH was added to obtain nonspecific
binding in the second and fourth groups as indicated. Specific binding
is computed as the difference between no unlabeled hGH and when
unlabeled hGH is added. The data are the mean ± S.E. of
triplicate observations.
|
|
In a study to examine the effect of insulin pretreatment on GH binding
to H4 cells, specific binding of 125I-hGH, when using hGH
as competitor, was determined to be approximately 2.1% of the total
radioactivity added (Fig. 6B). When H4 cells were pretreated
with insulin, total binding of 125I-hGH was diminished from
2.8 to 1.2%. Upon competition with an excess of unlabeled hGH,
nonspecific binding was found to be equal to that observed prior to
insulin pretreatment. When specific binding was calculated, insulin
pretreatment reduced specific 125I-hGH binding from 2.1 to
0.55% of the total counts added (Fig. 6B). Thus, insulin
pretreatment for 16 h reduced specific hGH binding, the vast
majority of which is to somatogenic binding sites, by over 70%, to
approximately 26% of the specific GH binding obtained prior to insulin pretreatment.
Insulin Pretreatment Reduced Immunoreactive GHR in Whole Cell
Lysates of H4 Cells in a Time- and Dose-dependent
Manner--
To further extend our studies of the effects of insulin
pretreatment on the GHR, immunoreactive GHR was studied using 2 antibodies raised against the intracellular domain of the GHR. In the
first experiments, GHR was immunoprecipitated as described in the
experiments for JAK2 (see Fig. 5), except using the anti-GHRcytAL37
antibody for both immunoprecipitation and immunoblotting.
Immunoreactive GHR migrated as a broad band with
Mr 110-125 due to its complex and variable
state of glycosylation (41). Insulin pretreatment for 16 h
significantly reduced the amount of immunoprecipitable GHR (Fig.
7, lane 2 versus lane 1).

View larger version (25K):
[in this window]
[in a new window]
|
Fig. 7.
Insulin for 16 h reduced the amount of
immunoprecipitable GHR. H4 cells were treated with insulin (100 nM; 16 h) as indicated and harvested to obtain
detergent extracts for immunoprecipitation as described in the text.
GHR were immunoprecipitated with anti-GHRcytAL37 antiserum and then
immunoblotted with the same antiserum. In a parallel lane size markers
were run, and the location of one size marker (108 kDa) is shown.
|
|
We also measured by immunoblotting the effects of insulin on the
abundance of immunoreactive GHR in rat H4 hepatoma whole cell lysates.
Using the anti-GHRcytAL37 antibody, immunoreactive GHR was largely
unchanged following insulin for 4 h (Fig.
8A, top row, lanes 1 and
2) but by 8 h, and even more so by 12 h, there
were significant decreases in immunoreactive GHR as measured by the
anti-GHRcytAL37 antibody (Fig. 8A, top row, lanes 3 and 4). There were no consistent changes in total JAK2 protein
levels in the same blot that was stripped and re-probed with a
anti-JAK2UBI antibody (Fig. 8A, bottom row, lanes 1-4).
There were also large reductions in the amount of immunoreactive GHR
following insulin pretreatment for 16, 20, and 24 h when using a
second polyclonal antibody, anti-GHRcyt3728, to measure changes in GHR
expression (Fig. 8B, top row, lanes 2, 3, and 4
compared with lane 1). Again, there were no significant
changes in JAK2 levels in the same blot that was stripped and re-probed
with the second JAK2 antibody, anti-JAK2AL33 (Fig. 8B, bottom
row, lanes 1-4). When several experiments were quantified by
scanning densitometry of the autoradiographs, it was clear that there
was little or no change in GHR abundance following insulin treatment
for 4 h when measured by with either anti-GHR serum
(anti-GHRcyt3728 or anti-GHRcytAL37; Fig. 8C). However, by
8 h of insulin pretreatment, immunoblotting with either of the two
antisera revealed a significant reduction in immunoreactive GHR, with
maximum decreases to only 20% of control values by 12-16 h and was
still significantly reduced at 20 and 24 h (p < 0.05 to p < 0.001; see figure legends for exact
p values at each time point; Fig. 8C).

View larger version (30K):
[in this window]
[in a new window]
|
Fig. 8.
Time-dependent effect of insulin
on GHR levels. A and B, Western blots were
performed as described in the legend for Fig. 1 and in the text. Some
H4 cells were left untreated or were treated with insulin (100 nM) for the times indicated and the antibodies used (as
indicated to the left) included 2 for GHR (anti-GHRcyt3728
or anti-GHRcytAL37) and 2 for JAK2 (anti-JAK2UBI and anti-JAK2AL33).
Representative Western blots are presented. C, densitometric
analysis of autoradiographs from three or four similar experiments were
performed, using two antisera of GHR (anti-GHRcyt3728 or
anti-GHRcytAL37) following different times of insulin pretreatment (100 nM). The data is expressed as the mean ± S.E. The
levels of GHR immunoreactivity in control (untreated) cells was
arbitrarily set to 100% within each experiment for each of the two
antibodies and the levels following various times of insulin
pretreatment are expressed as a percentage of expression measured in
control cells. For the anti-GHRcytAL37 antibody, the 4-h insulin time
point is not statistically significant compared with the zero time
whereas the 8-, 20-, and 24-h insulin time points are significant
(p < 0.01) and the 12- and 16-h insulin time points
are significant (p < 0.001) by ANOVA. For the
anti-GHRcyt3728 antibody, the 4-h insulin time point is not
statistically significant compared with the zero time, whereas the 8- and 20-h insulin time points are significant (p < 0.05), the 12- and 16-h insulin time points are significant
(p < 0.001), and the 24-h insulin time point is
significant (p < 0.01) by ANOVA.
|
|
When various concentrations of insulin were added to H4 cells for
16 h, a dose-dependent decrease in immunoreactive GHR
was observed. There was little effect of 0.1 nM insulin for
16 h compared to untreated cells and a moderate but significant
decrease in immunoreactive GHR following 16 h of 1 nM
insulin (Fig. 9, lanes 1-3).
Insulin was only added once in these studies, so this may be an
underestimation of the sensitivity of the response, since insulin may
have been degraded over the extended pretreatment period. In previous
studies, 80-98% of insulin (0.02-5 nM) added to H4 cells
is still present 6 h later when measured by radioimmunoassay (42).
However, the present experiments were performed at 16 h following
insulin addition which may have resulted in further degradation.
Clearly, insulin at concentrations or 10 and 100 nM reduced
GHR to a similar extent, comparable to that shown in Fig. 8C
(Fig. 9, lanes 4 and 5). Thus, insulin
pretreatment results in 70-80% reductions in GH binding,
immunoreactive GHR, JAK2 phosphorylation, and STAT5B
phosphorylation.

View larger version (20K):
[in this window]
[in a new window]
|
Fig. 9.
Dose-dependent effect of 16-h
insulin on GHR levels. Western blots were performed as described
in the legend for Fig. 7 and in the text using the anti-GHRcyt3728
antibody to GHR and no insulin treatment (lane 1) or insulin
for 16 h at the indicated concentrations (0.1-100 nM;
lanes 2-5). A representative Western blot is
presented.
|
|
 |
DISCUSSION |
Although usually thought of as a "counter-regulatory" hormone,
with many actions opposing those of insulin, GH can have acute insulin-like effects, especially in the setting of GH deficiency (1).
In contrast, GH excess induces insulin resistance (43) but little is
known about the ability of insulin to promote GH resistance. A
significant fraction of the adult population exhibit peripheral insulin
resistance and hyperinsulinemia. This is true both of Type 2 diabetic
patients and a large number of individuals, many of which are obese,
but do not exhibit overt diabetes (6). In the present study, prolonged
treatment of hepatoma cells with high insulin concentrations, similar
to those in the hepatic portal circulation in patients with
hyperinsulinemia (see below), resulted in reduced GH binding and a
severe diminution of GH-induced STAT5B phosphorylation.
There are two closely related STAT5 isoforms, STAT5A and STAT5B (44).
They clearly mediate different functions and the loss of either cannot
be compensated by the other isoform in vivo, as indicated by
studies with knockout mouse models (13, 45). Acute insulin treatment
stimulates STAT5B tyrosine phosphorylation in Chinese hamster ovary
cells overexpressing either the insulin receptor or STAT5B (46, 47).
This change in phosphorylation is not observed in native Chinese
hamster ovary cells or hepatoma cells expressing the normal complement
of insulin receptors (46). In the present work, it is shown that
continuous insulin treatment does not significantly alter STAT5B
protein concentrations, STAT5B phosphorylation, or STAT5 tyrosine
phosphorylation in rat H4 hepatoma cells.
Patients with peripheral insulin resistance and hyperinsulinemia often
exhibit abdominal adiposity, a morphology similar to that found in
patients or animal models with GH deficiency or a disruption of GH
signaling, such as STAT5B knockout mice (4-6, 13, 48). This lead us to
hypothesize that hyperinsulinemia may result in GH resistance. This
hypothesis is consistent with our presented results indicating that
continuous treatment with high concentrations of insulin reduced the
ability of GH to stimulate phosphorylation of STAT5B in hepatoma cells.
As determined with two different antibodies measuring a GH-induced
mobility shift in STAT5B due to phosphorylation and directly measuring
tyrosine phosphorylation of STAT5, GH-induced STAT5B phosphorylation
was severely reduced by insulin pretreatment for 8-24 h. In addition, the GH-induced tyrosine phosphorylation of immunoprecipitated JAK2 was
almost completely inhibited by insulin pretreatment with little change
in JAK2 protein levels.
Since a reduction in GH binding could contribute to the reductions in
STAT5B and JAK2 phosphorylation, GH binding was measured using
125I-hGH. Ovine PRL was ineffective in competing for
125I-hGH, so we conclude that there were few, if any,
lactogenic binding sites on rat H4 hepatoma cells, a cell line derived
from an hepatocarcinoma of a male rat (40), and the specific
125I-hGH binding was to somatogenic sites. Due to the low
levels of GH-specific binding in H4 cells, we choose to measure binding for 2 h at room temperature. The measured specific binding is likely due to both cell surface-binding sites and a small amount of
internalized 125I-hGH·GHR complexes. The percentage that
is degraded or released into the media is thought to be minimal due to
the room temperature incubation and relatively short time of incubation
(49, 50). The low amount of total and specific GH binding in this cell
line is not surprising since GHR mRNA levels in H4 cells are 40%
or less of those in rat liver (51). To our knowledge these represent the first experiments to demonstrate GH binding in H4 cells.
Insulin pretreatment for 16 h reduced specific
125I-hGH specific binding to somatogenic binding sites to
26% of the specific binding prior to pretreatment, a decrease in GH
binding comparable to the reduction of GH-induced STAT5B
phosphorylation. Previous studies are inconsistent on whether insulin
increases GH binding and GHR mRNA or not (14-28). This may be due
to multiple, and sometimes separate, controlling factors for GHR
expression and for GH binding. For example, while in vivo
data suggests that insulin treatment of insulin-deficient individuals
might increase GHBP (and therefore most likely GHR expression), high
physiological levels of GHBP may feedback to decrease GH-receptor
mRNA levels and low levels of GHBP may increase GH-receptor
mRNA expression (18, 22, 52). Additionally, continuous insulin
treatment of osteoblasts decreases the fraction of cellular GHR
presented at the plasma membrane via inhibition of surface
translocation of GHR with no effect on the total cellular content of
GHR (29). Thus, there may multiple feedback mechanisms controlling GHR
expression and the subcellular localization of GHR. Most in
vivo studies do not maintain high insulin concentrations for
extended periods and therefore do not study the effects of insulin
under conditions similar to patients with insulin resistance and
hyperinsulinemia. The effect of insulin on GHR expression may be
dependent on the concentration of insulin, the duration of insulin
treatment and may be tissue and cell-type type specific (27).
Decreases in GH binding could be due to insulin-dependent
changes in GHR synthesis or degradation, or
insulin-dependent reduction of cell surface GHR without
changes in total GHR expression. GHR normally migrates as a broad band
with Mr 110-125 due to its complex and variable
glycosylation (41, 53). Using 2 different antibodies that recognize the
cytoplasmic domain of the GHR, there was no significant change in
immunoreactive GHR during the first 4 h of insulin treatment.
However, immunoreactive GHR was reduced by insulin pretreatment for
8-24 h, with a maximum reduction to less than one-fourth of the
untreated cells with insulin for 16 h. Densitometric analysis of
the broad GH bands indicated a similar reduction of GHR using either
antiserum. Insulin treatment (16 h) at 10 and 100 nM
reduced GHR to a similar extent, while 1 nM insulin
resulted in an intermediate reduction and 0.1 nM insulin did not alter the amount of immunoreactive GHR, respectively. Thus,
chronic hyperinsulinemia resulted in a similar reduction in GH binding,
whole cell immunoreactive GHR, and the ability of GH to induce
JAK2 and STAT5B phosphorylation.
The finding of reduced GHR by both binding studies and Western analysis
suggests that insulin might reduce expression of GHR, thereby
decreasing the number of available somatogenic binding sites and
decreasing the levels of immunoreactive GHR. A second, more remote
possibility, is that insulin pretreatment results in a
post-translational alteration of the GHR resulting in decreased GH
binding. This modification would then also have to result in a reduced
immuno-detectability by Western blot analysis, possibly due to a
conformational change in the cytoplasmic region of the GHR, to which
the GHR antibodies were raised. Since two separate polyclonal
antibodies were used, it is unlikely that a covalent modification would
result in such similar decreases in immunoreactivity of the GHR and in
GH binding. However, we cannot exclude this possibility.
In a number of species, IGF-1 may have some actions similar to those of
insulin. Since GH is one of the main regulators of IGF-1 expression,
elevated IGF-1 may also act to reduce GHR and GH responsiveness as part
of a negative feedback system. However, in the present studies the
effects of insulin are through the insulin receptor since H4 hepatoma
cells, like the H35 cells from which H4 cells were derived and rat
liver, contain few if any IGF-1 receptors (54, 55).
The similarity between the timing and percentage decrease in GHR levels
and STAT5B phosphorylation following insulin pretreatment suggests that
the decrease in GHR is the cause of the reduction of GH-induced STAT5B
phosphorylation. The remaining GHR are still functional since a small
amount of GH-induced STAT5B phosphorylation is observed even following
extended insulin pretreatment. The reduction of GHR levels and
GH-induced STAT5B phosphorylation required more than 4 h of
elevated insulin and the insulin concentration needed to be 1 nM or greater. In normal human subjects basal insulin concentrations in the hepatic portal circulation are approximately 0.2 nM, severalfold higher than peripheral insulin
concentrations (56). Peak post-prandial insulin concentrations reach
0.5-1.0 nM in the peripheral circulation and approximately
3 nM in the portal circulation (56, 57). However, in obese
individuals with peripheral insulin resistance, peak post-prandial
systemic insulin concentrations reach 3-7 nM and this
increase in insulin concentration is more prolonged than in normal
subjects (58). Portal circulation insulin concentrations were not
measured in these obese individuals, but if the ratio of peripheral to
portal insulin remains consistent, portal circulation concentrations of
insulin would be expected in the range of 20-50 nM.
Therefore, normal postprandial concentrations of insulin may be
insufficient to result in substantial or prolonged GH resistance.
However, in patients with peripheral insulin resistance, where there
are elevated basal insulin levels and extended postprandial peripheral insulin concentrations above 1 nM, peripheral GH resistance
may develop. In these same individuals, where insulin concentrations in
the hepatic portal circulation may rise to well above 10 nM for extended periods, there is an even greater possibility for the
development of hepatic GH resistance. Thus, prior to commencement of
exogenous GH administration, knowledge of a patient's insulin sensitivity and circulating insulin concentrations may be warranted.
 |
ACKNOWLEDGEMENTS |
We thank A. Keeton, M. Amsler, S.-O. Kim, and
J. Jiang for helpful and insightful discussions and suggestions. We are
grateful to A. F. Parlow, Pituitary Hormones and Antisera Center,
Harbor-UCLA Medical Center (Torrance, CA), and the NIDDK, National
Institutes of Health, National Hormone & Pituitary Program for the gift
of bGH, to Dr. Ron Chance (Eli Lilly, Co., Indianapolis, IN) for the
porcine sodium insulin and hGH, which was also kindly provided by Eli
Lilly Co.
 |
FOOTNOTES |
*
This work was supported by National Institutes of Health
Grant DK40456, grants from the American Diabetes Association and the
Alabama affiliate of the American Heart Association (to J. L. M.), National Institutes of Health Grant DK46395 and VA Merit Review
award (to S. J. F.).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 Pathology,
Div. of Molecular and Cellular Pathology, Volker Hall, G019, 1670 University Blvd., University of Alabama at Birmingham, Birmingham, AL
35294-0019. E-mail: messina{at}vh.path.uab.edu; Tel.: 205-934-4921; Fax:
205-934-1775.
 |
ABBREVIATIONS |
The abbreviations used are:
GH, growth hormone;
GHR, growth hormone receptor;
JAK, Janus activating kinase;
STAT, signal transducers and activators of transcription;
PRL, prolactin;
IGF, insulin growth factor;
GHBP, growth hormone binding protein;
h, human;
o, ovine;
b, bovine.
 |
REFERENCES |
-
Ho, K. K.,
O'Sullivan, A. J.,
and Hoffman, D. M.
(1996)
Endocr. J.
43 (suppl.),
63
-
Bjorntorp, P.
(1997)
Human Reprod.
12 Suppl. 1,
21-25[Medline]
[Order article via Infotrieve]
-
Cuneo, R. C.,
Judd, S.,
Wallace, J. D.,
Perry-Keene, D.,
Burger, H.,
Lim-Tio, S.,
Strauss, B.,
Stockigt, J.,
Topliss, D.,
Alford, F.,
Hew, L.,
Bode, H.,
Conway, A.,
Handelsman, D.,
Dunn, S.,
Boyages, S.,
Cheung, N. W.,
and Hurley, D.
(1998)
J. Clin. Endocrinol. Metab.
83,
107-116[Abstract/Free Full Text]
-
Laron, Z.
(1995)
J. Clin. Endocrinol. Metab.
80,
1526-1531[Abstract]
-
Angulo, M.,
Castro-Magana, M.,
Mazur, B.,
Canas, J. A.,
Vitollo, P. M.,
and Sarrantonio, M.
(1996)
J. Pediatr. Endocrinol. Metab.
9,
393-400[Medline]
[Order article via Infotrieve]
-
Bjorntorp, P.
(1997)
Nutrition
13,
795-803[CrossRef][Medline]
[Order article via Infotrieve]
-
Baumann, G.
(1995)
Exp. Clin. Endocrinol. Diabetes
103,
2-6[Medline]
[Order article via Infotrieve]
-
Martini, J. F.,
Pezet, A.,
Guezennec, C. Y.,
Edery, M.,
Postel-Vinay, M. C.,
and Kelly, P. A.
(1997)
J. Biol. Chem.
272,
18951-18958[Abstract/Free Full Text]
-
Argetsinger, L. S.,
Campbell, G. S.,
Yang, X.,
Witthuhn, B. A.,
Silvennoinen, O.,
Ihle, J. N.,
and Carter-Su, C.
(1993)
Cell
74,
237-244[Medline]
[Order article via Infotrieve]
-
Carter-Su, C.,
Schwartz, J.,
and Smit, L. S.
(1996)
Annu. Rev. Physiol.
58,
187-207[CrossRef][Medline]
[Order article via Infotrieve]
-
Witthuhn, B. A.,
Quelle, F. W.,
Silvennoinen, O.,
Yi, T.,
Tang, B.,
Miura, O.,
and Ihle, J. N.
(1993)
Cell
74,
227-236[Medline]
[Order article via Infotrieve]
-
Dusanter-Fourt, I.,
Muller, O.,
Ziemiecki, A.,
Mayeux, P.,
Drucker, B.,
Djiane, J.,
Wilks, A.,
Harpur, A. G.,
Fischer, S.,
and Gisselbrecht, S.
(1994)
EMBO J.
13,
2583-2591[Abstract]
-
Udy, G. B.,
Towers, R. P.,
Snell, R. G.,
Wilkins, R. J.,
Park, S. H.,
Ram, P. A.,
Waxman, D. J.,
and Davey, H. W.
(1997)
Proc. Natl. Acad. Sci. U. S. A.
94,
7239-7244[Abstract/Free Full Text]
-
Baxter, R. C.,
Bryson, J. M.,
and Turtle, J. R.
(1980)
Endocrinology
107,
1176-1181[Abstract]
-
Bereket, A.,
Lang, C. H.,
Blethen, S. L.,
Gelato, M. C.,
Fan, J.,
Frost, R. A.,
and Wilson, T. A.
(1995)
J. Clin. Endocrinol. Metab.
80,
1312-1317[Abstract]
-
Menon, R. K.,
Arslanian, S.,
May, B.,
Cutfield, W. S.,
and Sperling, M. A.
(1992)
J. Clin. Endocrinol. Metab.
74,
934-938[Abstract]
-
Menon, R. K.,
Stephan, D. A.,
Rao, R. H.,
Shen-Orr, Z.,
Downs, L. S., Jr.,
Roberts, C. T., Jr.,
Leroith, D.,
and Sperling, M. A.
(1994)
J. Endocrinol.
142,
453-462[Abstract]
-
Kratzsch, J.,
Kellner, K.,
Zilkens, T.,
Schmidt-Gayk, H.,
Selisko, T.,
and Scholz, G. H.
(1996)
Clin. Endocrinol. (Oxf.)
44,
673-678[CrossRef][Medline]
[Order article via Infotrieve]
-
Hanaire-Broutin, H.,
Sallerin-Caute, B.,
Poncet, M. F.,
Tauber, M.,
Bastide, R.,
Chale, J. J.,
Rosenfeld, R.,
and Tauber, J. P.
(1996)
Diabetologia
39,
1498-1504[CrossRef][Medline]
[Order article via Infotrieve]
-
Hanaire-Broutin, H.,
Sallerin-Caute, B.,
Poncet, M. F.,
Tauber, M.,
Bastide, R.,
Rosenfeld, R.,
and Tauber, J. P.
(1996)
Diabetes & Metab.
22,
245-250[Medline]
[Order article via Infotrieve]
-
Mercado, M.,
and Baumann, G.
(1995)
Arch. Med. Res.
26,
101-109[Medline]
[Order article via Infotrieve]
-
Clayton, K. L.,
Holly, J. M.,
Carlsson, L. M.,
Jones, J.,
Cheetham, T. D.,
Taylor, A. M.,
and Dunger, D. B.
(1994)
Clin. Endocrinol. (Oxf.)
41,
517-524[Medline]
[Order article via Infotrieve]
-
Mercado, M.,
Molitch, M. E.,
and Baumann, G.
(1992)
Diabetes
41,
605-609[Abstract]
-
Baxter, R. C.,
and Turtle, J. R.
(1978)
Biochem. Biophys. Res. Commun.
84,
350-357[Medline]
[Order article via Infotrieve]
-
Maes, M.,
Ketelslegers, J. M.,
and Underwood, L. E.
(1983)
Diabetes
32,
1060-1069[Abstract]
-
Bornfeldt, K. E.,
Arnqvist, H. J.,
Enberg, B.,
Mathews, L. S.,
and Norstedt, G.
(1989)
J. Endocrinol.
122,
651-656[Abstract]
-
Chen, N. Y.,
Chen, W. Y.,
and Kopchick, J. J.
(1997)
Endocrinology
138,
1988-1994[Abstract/Free Full Text]
-
Tollet, P.,
Enberg, B.,
and Mode, A.
(1990)
Mol. Endocrinol.
4,
1934-1942[Abstract]
-
Leung, K. C.,
Waters, M. J.,
Markus, I.,
Baumbach, W. R.,
and Ho, K. K.
(1997)
Proc. Natl. Acad. Sci. U. S. A.
94,
11381-11386[Abstract/Free Full Text]
-
Maheshwari, H.,
Sharma, L.,
and Baumann, G.
(1996)
J. Clin. Endocrinol. Metab.
81,
995-997[Abstract]
-
Xu, X.,
Bennett, S. A.,
Ingram, R. L.,
and Sonntag, W. E.
(1995)
Endocrinology
136,
4551-4557[Abstract]
-
Frank, S. J.,
Yi, W.,
Zhao, Y.,
Goldsmith, J. F.,
Gilliland, G.,
Jiang, J.,
Sakai, I.,
and Kraft, A. S.
(1995)
J. Biol. Chem.
270,
14776-14785[Abstract/Free Full Text]
-
Jiang, J.,
Liang, L.,
Kim, S. O.,
Zhang, Y.,
Mandler, R.,
and Frank, S. J.
(1998)
Biochem. Biophys. Res. Commun.
253,
774-779[CrossRef][Medline]
[Order article via Infotrieve]
-
Frank, S. J.,
Gilliland, G.,
and Van Epps, C.
(1994)
Endocrinology
135,
148-156[Abstract]
-
Messina, J. L.
(1989)
Endocrinology
124,
754-761[Abstract]
-
Frank, S. J.,
Gilliland, G.,
Kraft, A. S.,
and Arnold, C. S.
(1994)
Endocrinology
135,
2228-2239[Abstract]
-
Messina, J. L.,
Eden, S.,
and Kostyo, J. L.
(1985)
Am. J. Physiol.
249,
E56-E62[Abstract/Free Full Text]
-
Ram, P. A.,
Park, S. H.,
Choi, H. K.,
and Waxman, D. J.
(1996)
J. Biol. Chem.
271,
5929-5940[Abstract/Free Full Text]
-
Beadling, C.,
Ng, J.,
Babbage, J. W.,
and Cantrell, D. A.
(1996)
EMBO J.
15,
1902-1913[Abstract]
-
Pitot, H. C.,
Peraino, C.,
Morse, P. A., Jr.,
and Potter, V. R.
(1963)
Natl. Cancer Inst. Monogr.
13,
229-245
-
Asakawa, K.,
Hedo, J. A.,
McElduff, A.,
Rouiller, D. G.,
Waters, M. J.,
and Gorden, P.
(1986)
Biochem. J.
238,
379-386[Medline]
[Order article via Infotrieve]
-
Messina, J. L.,
Hamlin, J.,
and Larner, J.
(1985)
J. Biol. Chem.
260,
16418-16423[Abstract/Free Full Text]
-
Luger, A.,
Prager, R.,
Gaube, S.,
Graf, H.,
Klauser, R.,
and Schernthaner, G.
(1990)
Exp. Clin. Endocrinol.
95,
339-343[Medline]
[Order article via Infotrieve]
-
Liu, X.,
Robinson, G. W.,
Gouilleux, F.,
Groner, B.,
and Hennighausen, L.
(1995)
Proc. Natl. Acad. Sci. U. S. A.
92,
8831-8835[Abstract]
-
Feldman, G. M.,
Rosenthal, L. A.,
Liu, X.,
Hayes, M. P.,
Wynshaw-Boris, A.,
Leonard, W. J.,
Hennighausen, L.,
and Finbloom, D. S.
(1997)
Blood
90,
1768-1776[Abstract/Free Full Text]
-
Chen, J.,
Sadowski, H. B.,
Kohanski, R. A.,
and Wang, L. H.
(1997)
Proc. Natl. Acad. Sci. U. S. A.
94,
2295-2300[Abstract/Free Full Text]
-
Sawka-Verhelle, D.,
Filloux, C.,
Tartare-Deckert, S.,
Mothe, I.,
and Van, O. E.
(1997)
Eur. J. Biochem.
250,
411-417[Abstract]
-
Zhou, Y.,
Xu, B. C.,
Maheshwari, H. G.,
He, L.,
Reed, M.,
Lozykowski, M.,
Okada, S.,
Cataldo, L.,
Coschigamo, K.,
Wagner, T. E.,
Baumann, G.,
and Kopchick, J. J.
(1997)
Proc. Natl. Acad. Sci. U. S. A.
94,
13215-13220[Abstract/Free Full Text]
-
Lobie, P. E.,
Mertani, H.,
Morel, G.,
Morales-Bustos, O.,
Norstedt, G.,
and Waters, M. J.
(1994)
J. Biol. Chem.
269,
21330-21339[Abstract/Free Full Text]
-
Gavin, J. R.,
Saltman, R. J.,
and Tollefsen, S. E.
(1982)
Endocrinology
110,
637-643[Abstract]
-
Ooi, G. T.,
Cohen, F. J.,
Tseng, L. Y.,
Rechler, M. M.,
and Boisclair, Y. R.
(1997)
Mol. Endocrinol.
11,
997-1007[Abstract/Free Full Text]
-
Mullis, P. E.,
Wagner, J. K.,
Eble, A.,
Nuoffer, J. M.,
and Postel-Vinay, M. C.
(1997)
Mol. Cell. Endocrinol.
131,
89-96[CrossRef][Medline]
[Order article via Infotrieve]
-
Frick, G. P.,
Tai, L. R.,
Baumbach, W. R.,
and Goodman, H. M.
(1998)
Endocrinology
139,
2824-2830[Abstract/Free Full Text]
-
Massague, J.,
Blinderman, L. A.,
and Czech, M. P.
(1982)
J. Biol. Chem.
257,
13958-13963[Free Full Text]
-
Krett, N. L.,
Heaton, J. H.,
and Gelehrter, T. D.
(1987)
Endocrinology
120,
401-408[Abstract]
-
Blackard, W. G.,
and Nelson, N. C.
(1970)
Diabetes
19,
302-306[Medline]
[Order article via Infotrieve]
-
Cerasi, E.,
Hallberg, D.,
and Luft, R.
(1970)
Horm. Metab. Res.
2,
302-303[Medline]
[Order article via Infotrieve]
-
Malherbe, C.,
Heller, F.,
Gasparo, M.,
Hertogh, R.,
and Hoet, J. J.
(1970)
J. Clin. Endocrinol. Metab.
30,
535-538[Medline]
[Order article via Infotrieve]
Copyright © 1999 by The American Society for Biochemistry and Molecular Biology, Inc.