From the Third Department of Medicine, Shiga
University of Medical Science, Otsu, Shiga 520-2192, Japan, the
¶ Department of Medicine, University of Tennessee and Research
Service, Veterans Affairs Medical Center, Memphis, Tennessee 38104, the ** Department of Physiology, Tulane University and
Department of Medicine, Louisiana State University,
New Orleans, Louisiana 70112
Received for publication, October 18, 2000, and in revised form, December 11, 2000
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ABSTRACT |
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Insulin signaling is regulated by tyrosine
phosphorylation of the signaling molecules, such as the insulin
receptor and insulin receptor substrates (IRSs). Therefore, the balance
between protein-tyrosine kinases and protein-tyrosine phosphatase
activities is thought to be important in the modulation of insulin
signaling in insulin-resistant states. We thus employed the
adenovirus-mediated gene transfer technique, and we analyzed the effect
of overexpression of a wild-type protein-tyrosine phosphatase-1B
(PTP1B) on insulin signaling in both L6 myocytes and Fao cells. In both
cells, PTP1B overexpression blocked insulin-stimulated tyrosine
phosphorylation of the insulin receptor and IRS-1 by more than 70% and
resulted in a significant inhibition of the association between IRS-1
and the p85 subunit of phosphatidylinositol 3-kinase and Akt
phosphorylation as well as mitogen-activated protein kinase
phosphorylation. Moreover, insulin-stimulated glycogen synthesis was
also inhibited by PTP1B overexpression in both cells. These effects
were specific for insulin signaling, because platelet-derived growth
factor (PDGF)-stimulated PDGF receptor tyrosine phosphorylation and Akt
phosphorylation were not inhibited by PTP1B overexpression. The present
findings demonstrate that PTP1B negatively regulates insulin signaling in L6 and Fao cells, suggesting that PTP1B plays an important role in
insulin resistance in muscle and liver.
After insulin binds to its own receptor, the insulin receptor is
phosphorylated on its tyrosine residues, and tyrosine kinase activity
is activated. The activated insulin receptor binds to insulin receptor
substrates (IRSs)1 via the
YMXM motif, and IRSs are also phosphorylated on tyrosine residues (1). The tyrosine-phosphorylated IRSs activate their downstream signaling molecules, such as phosphatidylinositol (PI) 3-kinase and p21ras. Since tyrosine phosphorylation is
essential for insulin signaling, the balance of activities between
protein-tyrosine kinases and protein-tyrosine phosphatases (PTPase)
appears to be very important for the effects of insulin.
Several lines of evidence demonstrate that PTPase activity is increased
in insulin-resistant states such as obesity and type 2 diabetes
mellitus. It has been reported that obese human subjects have increased
PTPase activity in skeletal muscle and adipose tissue (2, 3), and a
10% body weight reduction causes a decrease in overall adipose tissue
PTPase activity with enhanced insulin sensitivity (4). Furthermore,
insulin infusion in vivo produces a rapid 25% suppression
of soluble PTPase activity in muscles of insulin-sensitive subjects,
but this response is severely impaired in subjects who are
insulin-resistant (5). Moreover, increased PTPase activity is also
observed in the liver (6) and skeletal muscle (7) of diabetic rats.
Taken together, abnormalities of PTPase activity are thought to be
important to understand the molecular mechanism of insulin resistance.
Previous studies showed that the tandem domain transmembrane enzymes,
leukocyte antigen-related and leukocyte common antigen-related phosphatase/RPTP In the present study, we employed the adenovirus-mediating gene
transfer technique, and we analyzed the effect of PTP1B overexpression on insulin signaling in model cells of insulin target tissues, such as
L6 myocytes and Fao cells. In both cells, PTP1B overexpression markedly
inhibited insulin-stimulated tyrosine phosphorylation of the insulin
receptor and IRS-1 and resulted in a significant inhibition of the
association between IRS-1 and the p85 subunit of PI 3-kinase and Akt
phosphorylation as well as MAP kinase phosphorylation. Moreover,
glycogen synthesis was also inhibited under both basal and
insulin-stimulated conditions. The present findings demonstrate that
PTP1B negatively regulates insulin signaling in muscle and liver cells.
Materials--
Human insulin was provided by Lilly. Anti-PTP1B
antibody, anti-IRS-1 antibody, and anti-p85 N-SH2 antibody were
purchased from Upstate Biotechnology Inc. (Lake Placid, NY).
Anti-phospho-Akt antibody and anti-phospho-MAP kinase antibody were
from New England Biolabs (Beverly, MA). Horseradish
peroxidase-conjugated phosphotyrosine antibody (RC20H) and insulin
receptor antibody were from Transduction Laboratories (Lexington, KY).
Horseradish peroxidase-linked anti-rabbit and anti-mouse antibodies,
anti-ERK2 antibody, anti-Akt1 antibody, and anti-PDGF receptor antibody
were from Santa Cruz Biotechnology (Santa Cruz, CA). Dulbecco's
modified Eagle's (DME) medium and fetal calf serum (FCS) were obtained
from Life Technologies, Inc. All radioisotopes were obtained from
PerkinElmer Life Sciences. XAR-5 film was obtained from Eastman Kodak
Co. All other reagents and chemicals were purchased from Sigma.
Cell Culture--
L6 cells, which were provided by Dr. A. Klip
(The Hospital for Sick Children, Toronto, Canada), were grown and
maintained in minimum Eagle's medium- Preparation of Recombinant Adenovirus--
The recombinant
adenovirus containing PTP1B cDNA was generated as
described previously (13).2
PTP1B wild-type cDNA was subcloned into pACCMVpLpASR(+)
plasmid (14). This plasmid contains 1.3 map units of adenovirus
5 (Ad5) left end, cytomegalovirus early promoter, PUC19 polylinker
site, and SV40 poly(A) signal sequences, followed by map units 9-18 of
the Ad5 genome. The resulting recombinant plasmid was then cotransfected into 293 packaging cells with pJM17 plasmid (15), which
carriers Ad5 genomic DNA and propagated as described previously (16).
Mature recombinant Ad5 encoding PTP1B wild type was thus generated after in vivo homologous recombination between
these two plasmids. Since 293 cells were originally derived from
adenovirus transformation, the missing E1 gene
function of pJM17 was provided in trans. The resulting
recombinant virus containing the PTP1B was denoted as
Ad5-PTP1B and was replication-defective (at least in cells lacking the
E1 region of the adenovirus) but fully infectious.
Cell Treatment--
L6 myocytes and Fao cells were infected at a
multiplicity of infection (m.o.i.) of 10-50 plaque formation
units/cell for 1 or 2 h with stocks of either a control
recombinant adenovirus (Ad5-ctrl) containing the cytomegalovirus
promoter, pUC 18 polylinker, a fragment of the SV40 genome, or the
recombinant adenovirus containing PTP1B (Ad5-PTP1B).
Transduced cells were incubated for 56 h at 37 °C in 5%
CO2 and appropriate medium with 2% heat-inactivated serum,
followed by incubation in the starvation media required for the assay.
The efficiency of adenovirus-mediated gene transfer was ~90% as
measured by immunocytochemistry.
Western Blotting--
Ad5-ctrl- or Ad5-PTP1B-infected cells were
starved for 16 h in DME regular glucose medium with 0.05% FCS.
The cells were stimulated with 100 ng/ml insulin for 5-10 min at
37 °C and lysed in a solubilizing buffer containing 20 mM Tris, 1 mM EDTA, 140 mM NaCl,
1% Nonidet P-40, 50 units/ml aprotinin, 1 mM
Na3VO4, 1 mM phenylmethylsulfonyl fluoride, 50 mM NaF, pH 7.5, for 30 min at 4 °C. The
cell lysates were centrifuged to remove insoluble materials. For
Western blot analysis, whole cell lysates (20 µg of protein per lane)
were denatured by boiling in Laemmli sample buffer containing 100 mM dithiothreitol and resolved by SDS-PAGE. Gels were
transferred to nitrocellulose by electroblotting in Towbin buffer
containing 20% methanol. For immunoblotting, membranes were blocked
and probed with specified antibodies. Blots were then incubated with
horseradish peroxidase-linked second antibody followed by
chemiluminescence detection, according to the manufacturer's
instructions (Amersham Pharmacia Biotech).
Glycogen Synthase Activity--
Glycogen synthase activity was
determined as described previously (17). Differentiated L6 myocytes
were infected with Ad5-PTP1B or Ad5-ctrl at 50 m.o.i. for 1 h
and grown in medium containing heat-inactivated serum (2%) for 72 h. The cells were serum- and glucose-starved in DME with no
glucose, 0.1% BSA, 2 mM pyruvate medium for 3 h and
then stimulated with or without 200 ng/ml insulin for 30 min in 5 mM glucose containing medium. Cells were washed with
ice-cold phosphate-buffered saline three times, scraped in the buffer
containing 50 mM Tris-HCl, 10 mM EDTA, 100 mM KF, pH 7.4, and sonicated. After centrifugation, the
protein concentration was measured. 10 µg of protein was used to
determine the ability to stimulate incorporation of
[14C]UDP-glucose into glycogen in the presence or absence
of glucose 6-phosphate.
Glycogen Synthesis--
Glycogen synthesis was measured as
described previously (18) with some modification. Fao cells were
infected with Ad5-PTP1B or Ad5-ctrl at 10 m.o.i. for 2 h and
grown in medium containing 2% heat-inactivated serum for 56 h.
The cells were serum-starved for 16 h, and the medium was then
replaced with DME medium containing 1% BSA. The cells were incubated
with [14C]glucose (0.4 µCi/well) and 100 ng/ml insulin
for 2 h in a CO2 incubator, washed with
phosphate-buffered saline 3 times, and lysed with 2 N NaOH
at 55 °C. The synthesized [14C]glycogen was
precipitated with cold glycogen in 66% ethanol and washed, and the
radioactivity was measured.
Statistics--
The values are expressed as mean ± S.E.,
unless otherwise stated. Scheffe's multiple comparison test was used
to determine the significance of any differences among more than three
groups. p < 0.05 was considered significant.
Expression of PTP1B in L6 Myocytes and Fao
Cells--
Differentiated L6 myocytes and Fao cells were infected with
recombinant adenovirus expressing wild-type PTP1B as described under
"Experimental Procedures". Following 72 h of incubation, the
cells were lysed and analyzed by SDS-PAGE followed by Western blotting
with anti-PTP1B antibody (Fig. 1). Small
amounts of endogenous PTP1B were observed in both cell lines. PTP1B was
expressed in a dose-dependent manner in each cell line.
Total phosphatase activity also increased dose-dependently
in PTP1B overexpressing cells as reported previously2 (13)
(data not shown).
Effects of PTP1B Expression on Tyrosine Phosphorylation of Insulin
Receptor and IRS-1, and Association between IRS-1 and the p85 Subunit
of PI 3-Kinase--
The infected cells were stimulated with 100 ng/ml
insulin for 5 min and lysed. The cell lysates (200-500 µg of
protein) were immunoprecipitated with anti-insulin receptor antibody or
anti-IRS-1 antibody. The immunocomplexes were analyzed by Western
blotting with anti-phosphotyrosine antibody. Tyrosine phosphorylation
of the insulin receptor and IRS-1 were decreased more than 70% in the
Ad5-PTP1B infected cells in both cell lines (Fig.
2, A, C, F, G, I, and
L). The membranes were stripped and reblotted with anti-p85
antibody. The association between IRS-1 and the p85 subunit of PI
3-kinase also decreased in PTP1B overexpressing cells (Fig. 2,
D and J) and paralleled the reduction of IRS-1
tyrosine phosphorylation (Fig. 2, C and I).
Effect of PTP1B Expression on Akt Phosphorylation--
Next, we
studied the downstream signaling of PI 3-kinase. Either PTP1B
expressing L6 myocytes or Fao cells were stimulated with insulin for 10 min, lysed, and analyzed by Western blotting with phospho-specific Akt
antibody (Fig. 3). Overexpression of PTP1B almost completely inhibited insulin-induced Akt phosphorylation in both cell lines.
Phosphorylation of p70S6 kinase, which is another downstream molecule
of PI 3-kinase, was also decreased by PTP1B overexpression (data not shown).
Effect of PTP1B Expression on Glycogen
Synthesis--
Differentiated L6 myocytes were infected with PTP1B and
control adenoviruses at 50 m.o.i. for 1 h. Following 72 h of incubation, the cells were serum- and glucose-starved for 3 h
and stimulated with 200 ng/ml of insulin for 30 min. Overexpression of
PTP1B almost completely inhibited insulin-stimulated glycogen synthase activity (control 2.51 ± 0.29% versus PTP1B 1.55 ± 0.19%, p < 0.02, Fig.
4).
Next, we examined [14C]glucose incorporation into
glycogen in Fao cells. The adenovirus-infected Fao cells were incubated
with insulin and [14C]glucose for 2 h, and glycogen
synthesis was measured as described under "Experimental
Procedures." As shown in Fig. 5,
overexpression of PTP1B inhibited glycogen synthesis at both basal
(control 126.4 ± 0.99% versus PTP1B 88.5 ± 7.24%, p < 0.01) and insulin-stimulated conditions
(control 190.9 ± 6.93 versus PTP1B 121.9 ± 7.80, p < 0.001).
Effect of PTP1B Expression on MAP Kinase Phosphorylation--
The
PTP1B expressing cells were stimulated with insulin for 10 min, lysed,
and subjected to SDS-PAGE followed by Western blotting with
anti-phospho-specific MAP kinase antibody (Fig.
6). PTP1B expression totally inhibited
insulin-stimulated MAP kinase phosphorylation in both cell lines.
PTP1B Overexpression Did Not Affect PDGF-stimulated Receptor and
Akt Phosphorylation--
Finally, we examined the effects of PTP1B on
platelet-derived growth factor (PDGF) signaling. Either Ad5-ctrl- or
Ad5-PTP1B-infected L6 myocytes were stimulated with 30 ng/ml PDGF for 5 min, lysed, and then analyzed by SDS-PAGE followed by Western blotting
with anti-phosphotyrosine antibody or anti-phospho-specific Akt
antibody (Fig. 7). PTP1B expression did
not affect either PDGF-induced PDGF receptor tyrosine phosphorylation
or Akt phosphorylation. Thus, the observation concerning PTP1B
overexpression appears to be specific for insulin signaling.
We previously reported that exposing Rat 1 fibroblasts expressing
human insulin receptors to high glucose conditions impaired the
insulin-stimulated tyrosine phosphorylation of the insulin receptor and
IRS-1 due to the increased expression and activity of PTP1B (10). More
direct evidence has shown that overexpression of PTP1B reduced the
level of GLUT4 on the cell surface in primary cultured rat adipose
cells (11). Recently, it was reported (12) that the PTP1B knockout mice
showed increased insulin sensitivity and resistance to high fat
diet-induced obesity with enhanced insulin-induced tyrosine
phosphorylation of the insulin receptor and IRS-1 in muscle and liver.
However, in those mice, insulin-induced tyrosine phosphorylation of the
insulin receptor and IRS-1 in adipose tissue was not affected. Another
study also showed that increased insulin sensitivity in PTP1B-deficient
mice was tissue-specific, as insulin-stimulated glucose uptake was
elevated in skeletal muscle, whereas adipose tissue was unaffected
(19). Furthermore, it has been reported that overexpression of PTP1B
decreased insulin-induced tyrosine phosphorylation of the insulin
receptor and IRS-1 by about 50% but did not affect the glucose
transport in 3T3-L1 adipocytes (20). Therefore, the role of PTP1B for
insulin effects appears to be tissue-specific.
In the current studies, we overexpressed PTP1B wild-type in L6 myocytes
and Fao cells using adenovirus-mediated gene transfer to analyze the
effect of PTP1B on insulin signaling. PTP1B overexpression markedly
inhibited insulin-stimulated MAP kinase phosphorylation, Akt
phosphorylation, and glycogen synthesis in both L6 myocytes and Fao
cells with marked inhibition of tyrosine phosphorylation of the insulin
receptor and IRS-1. These inhibitory effects were specific for insulin
signaling, because PTP1B overexpression did not affect PDGF-stimulated
phosphorylation of PDGF receptor and Akt (Fig. 7).
Similar to data derived from animal studies, the inhibitory effects of
PTP1B on insulin signaling appears cell type-specific. In 3T3-L1
adipocytes, PTP1B overexpression inhibited tyrosine phosphorylation of
the insulin receptor and IRS-1 by about 50%, but Akt phosphorylation
and glucose uptake were intact (20). The inhibition of tyrosine
phosphorylation of the insulin receptor and IRS-1 were greater in L6
myocytes and Fao cells, so this may be one explanation for the
different effects of PTP1B among these cells. It has been reported that
PTP1B anchors to the endoplasmic reticulum and is activated after
release into the cytosol by truncation of its COOH terminus (21, 22).
Localization of overexpressed PTP1B may be different in each cell line.
Another explanation for these findings may be the different expression
levels of endogenous PTP1B. In 3T3-L1 adipocytes, PTP1B may not play an
important role because its expression level is relatively low, and
other phosphatases may be important. Thus, overexpression of PTP1B
might show smaller effects than in L6 and Fao cells. In L6 myocytes and
Fao cells, both insulin-stimulated MAP kinase phosphorylation and PI
3-kinase pathway were inhibited by PTP1B overexpression. However, only insulin-stimulated MAP kinase phosphorylation was significantly blocked
by PTP1B overexpression in 3T3-L1 adipocytes (20). This difference may
be able to explain the cell type specificity of PTP1B effects.
Adenovirus-mediated liver-specific PTP1B overexpression in rats did not
cause insulin resistance in the recent study by Wang et
al.2 The reason for this difference between in
vivo and in vitro study is unclear. One possible
explanation is the difference of the expression level. PTP1B
overexpression was increased by about 2-3-fold in the liver of the
rats in this study, whereas it was increased by 20-30 times in our cells.
PTP1B overexpression showed different effects among L6 myocytes, Fao
cells, and 3T3-L1 adipocytes. PTP1B caused insulin resistance in muscle
and liver cells but had small effects in adipocytes (20) in the culture
system, similar to the observation of the PTP1B knockout mice (12, 19).
It was reported that insulin signaling was impaired in muscle but was
not in liver of IRS-1 knockout mice by rescuing up-regulated IRS-2
expression (23), but the disruption to the irs-2 gene
in mice caused severe insulin resistance in the liver (24).
Furthermore, it was reported that IRS-3 plays an important role in
adipose tissue of IRS-1 knockout mice (25). These findings demonstrate
that specific molecules mediate insulin signaling in different tissues.
RPTP In conclusion, the present findings indicate that PTP1B negatively
regulates insulin signaling in muscle and liver cells. Investigating
the activity of PTP1B may be important in understanding the molecular
mechanism of insulin resistance in the type 2 diabetic patients.
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
, and the intracellular, single domain enzymes, protein-tyrosine phosphatase-1B (PTP1B) and SHP2 are candidate PTPases
for the regulation of the insulin signaling pathway. In particular,
PTP1B directly interacts with the activated insulin receptor (8) and
exhibits the highest specific activity toward IRS-1 (9). We have also
reported that exposing Rat 1 fibroblasts expressing human insulin
receptors to a high glucose condition impairs insulin-stimulated
tyrosine phosphorylation of the insulin receptor and IRS-1 due to the
increased PTP1B expression and activity (10). Furthermore,
overexpression of PTP1B by the electroporation method reduces the level
of GLUT4 on the cell surface in primary cultured rat adipose cells
(11). Moreover, it is reported that mice lacking the
ptp1b gene show increased insulin sensitivity and
resistance to high fat diet-induced obesity, which is supported by
enhanced insulin-induced tyrosine phosphorylation of the insulin receptor and IRS-1 in muscle and liver (12). Thus, PTP1B appears to
play an important role in the regulation of insulin signaling.
EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
containing 50 units/ml
penicillin, 50 µg/ml streptomycin, and 10% FCS in a 5%
CO2 environment. The cells were reseeded in the appropriate
culture dishes and, after reaching subconfluency, the medium was
changed to minimum Eagle's medium-
containing 2% FCS. The medium
was then changed every 2 days until the cells were fully
differentiated, typically after 5 days. Fao cells, which were from Dr.
C. R. Kahn (Joslin Diabetes Center, Boston, MA), were grown and
maintained in DME medium containing 50 units/ml penicillin, 50 µg/ml
streptomycin, and 10% FCS in a 5% CO2 environment. Prior
to experimentation, the cells were trypsinized and reseeded in the
appropriate culture dishes. The Ad-E1A-transformed human embryonic
kidney cell line 293 was cultured in DME high glucose medium containing
50 units/ml penicillin, 50 µg/ml streptomycin, and 10% FCS in a 5%
CO2 environment.
RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Expression of PTP1B in L6 myocytes and Fao
cells. The differentiated L6 myocytes (A) and Fao cells
(B) were infected with Ad5-ctrl (ctrl) or
Ad5-PTP1B (PTP1B) at the indicated m.o.i.s for 1 or 2 h, respectively. After 72 h, the cells were lysed and analyzed by
SDS-PAGE followed by Western blotting with anti-PTP1B antibody. Each
Western blot is a representative of three independent
experiments.
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Fig. 2.
Effects of PTP1B overexpression on tyrosine
phosphorylation of insulin receptor and IRS-1 and insulin-induced
association of IRS-1 and the p85 subunit of PI 3-kinase. The
differentiated L6 myocytes (A-F) and Fao cells
(G-L) were infected with Ad5-ctrl (ctrl) or
Ad5-PTP1B (PTP1B) at 50 or 10 m.o.i. for 1 or 2 h,
respectively. After 56 h, the cells were starved for 16 h and
stimulated without or with 100 ng/ml of insulin for 5 min. Then the
cells were lysed and immunoprecipitated with anti-insulin receptor
antibody (A, B, G, and H) or anti-IRS-1 antibody
(C, D, E, I, J, and K). Immunocomplexes were
analyzed by Western blotting with phosphotyrosine antibody (RC20H)
(A, C, G, and I), anti-insulin receptor antibody
(B and H), anti-p85 antibody (D and
J), and anti-IRS-1 antibody (E and K).
Each Western blot is a representative of four independent experiments.
Tyrosine phosphorylation level was quantitated by NIH Image, and the
percentage of phosphorylation (count of tyrosine
phosphorylation/protein amount) was calculated. The graph
shows the means ± S.E. of the percentage of uninfected and
insulin-stimulated cells (F and L). * indicates
the difference from insulin-stimulated values in the cells with control
virus at p < 0.01.
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Fig. 3.
Effect of PTP1B overexpression on Akt
phosphorylation in L6 myocytes and Fao cells. The differentiated
L6 myocytes (A and B) and Fao cells (C
and D) were infected with Ad5-ctrl (ctrl) or
Ad5-PTP1B (PTP1B) at 50 or 10 m.o.i. for 1 or 2 h,
respectively. Following infection, the cells were serum-starved (16 h)
and incubated in the absence or presence of insulin (100 ng/ml) for 10 min. Total cell lysates (20 µg) were subjected to SDS-PAGE and
immunoblotted with phospho-specific Akt antibody (A and
C). The membrane was stripped and re-blotted with anti-Akt1
antibody (B and D). The Western blot is a
representative of four independent experiments.
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Fig. 4.
Effect of PTP1B overexpression on glycogen
synthase activity in L6 myocytes. The differentiated L6 myocytes
were infected with Ad5-ctrl (ctrl) or Ad5-PTP1B
(PTP1B) at 50 m.o.i. for 1 h. After 72 h, the
cells were serum- and glucose-starved in DME with no glucose,
0.1% BSA, 2 mM pyruvate medium for 3 h and then
stimulated with or without 200 ng/ml insulin for 30 min in medium
containing 5 mM glucose. Results are expressed as
means ± S.E. of percentage of glycogen synthase index (% GSI) for three independent experiments. % glycogen synthase index
was determined as (activity without Glc-6-P/activity with
Glc-6-P) × 100. * indicates the difference from
insulin-stimulated values in the cells with control virus at
p < 0.02.
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Fig. 5.
Effect of PTP1B overexpression on glycogen
synthesis in Fao cells. Fao cells were infected with Ad5-ctrl
(ctrl) or Ad5-PTP1B (PTP1B) at 10 m.o.i. for
2 h. After 56 h, the cells were serum-starved for 16 h
and then the medium was changed to DME with 1% BSA. The cells were
incubated with 100 ng/ml insulin and [14C]glucose for
2 h in 5% CO2 incubator. [14C]Glucose
incorporation into glycogen is expressed as % of the basal value of
nontransfected cells. The graph shows the means ± S.E.
of three experiments. * indicates the difference from basal values in
the cells with control virus at p < 0.01. ** indicates
the difference from insulin-stimulated values in the cells with control
virus at p < 0.001.
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Fig. 6.
Effect of PTP1B overexpression on MAP kinase
phosphorylation in L6 myocytes and Fao cells. The differentiated
L6 myocytes (A and B) and Fao cells (C
and D) were infected with Ad5-ctrl (ctrl) or
Ad5-PTP1B (PTP1B) at 50 or 10 m.o.i. for 1 or 2 h,
respectively. Following infection, cells were serum-starved (16 h) and
incubated in the absence or presence of insulin (100 ng/ml) for 10 min.
Total cell lysates (20 µg) were subjected to SDS-PAGE and
immunoblotted with phospho-specific MAP kinase antibody (A
and C). The membrane was stripped and re-blotted with
anti-Erk2 antibody (B and D). Each Western blot
is a representative of four independent experiments.
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Fig. 7.
Effect of PTP1B overexpression on
PDGF-stimulated receptor and Akt phosphorylation in L6 myocytes.
The differentiated L6 myocytes were infected with Ad5-ctrl
(ctrl) or Ad5-PTP1B (PTP1B) at 50 m.o.i. for
1 h. Following infection, the cells were serum-starved (16 h) and
incubated in the absence or presence of PDGF (30 ng/ml) for 5 min.
Total cell lysates (20 µg) were subjected to SDS-PAGE and
immunoblotted with phosphotyrosine antibody (RC20H) (A) or
phospho-specific Akt antibody (C). The membrane was stripped
and re-blotted with anti-PDGF receptor antibody (B) or
anti-Akt1 antibody (D). The Western blot is representative
of three independent experiments.
DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
or another phosphatase might be important in adipose tissue.
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ACKNOWLEDGEMENTS |
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We thank Dr. A. Klip (The Hospital for Sick Children, Toronto, Canada) and Dr. C. R. Kahn (Joslin Diabetes Center, Boston, MA) for donating L6 cells and Fao cells, respectively.
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FOOTNOTES |
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* This work was supported in part by a grant-in-aid from the Ministry of Education, Science, Sports and Culture of Japan (to H. M.).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: Third Dept. of Medicine, Shiga University of Medical Science, Seta, Otsu, Shiga 520-2192, Japan. Tel.: 81-77-548-2222; Fax: 81-77-543-3858; E-mail: maegawa@belle.shiga-med.ac.jp.
Supported by a research grant from the American Diabetes
Association, a Merit Review award from the Veterans Affairs Research Service, and recipient of National Institutes of Health Clinical Research Center Grant RR-00211.
Published, JBC Papers in Press, January 2, 2001, DOI 10.1074/jbc.M009489200
2 J. Wang, A. T. Cheung, J. K. Kolls, W. W. Starks, A. Martinez, D. Dietzen, and M. Bryer-Ash, manuscript in preparation.
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ABBREVIATIONS |
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The abbreviations used are: IRS, insulin receptor substrate; MAP kinase, mitogen-activated protein kinase; PTPase, protein-tyrosine phosphatase; PAGE, polyacrylamide gel electrophoresis; PI 3-kinase, phosphatidylinositol 3'-kinase; PDGF, platelet-derived growth factor; FCS, fetal calf serum; DME, Dulbecco's modified Eagle's; m.o.i., multiplicity of infection; BSA, bovine serum albumin.
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