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J. Biol. Chem., Vol. 276, Issue 29, 27511-27518, July 20, 2001
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From the
Received for publication, December 27, 2000, and in revised form, April 30, 2001
We previously isolated dephostatin
from Streptomyces as a novel inhibitor of CD45-associated
protein-tyrosine phosphatase. We prepared Et-3,4-dephostatin as
a stable analogue and found it to inhibit PTP-1B and SHPTP-1
protein-tyrosine phosphatases selectively but not to inhibit CD45 and
leukocyte common antigen-related phosphatase ones effectively.
Et-3,4-dephostatin increased the tyrosine phosphorylation of the
insulin receptor and insulin receptor substrate-1 with or without
insulin in differentiated 3T3-L1 mouse adipocytes. The increase of
tyrosine phosphorylation by Et-3,4-dephostatin was more prominent in
6-h than in 30-min incubation. It also increased phosphorylation and
activation of Akt with or without insulin. Et-3,4-dephostatin also
enhanced translocation of glucose transporter 4 from the cytoplasm to
the membrane and 2-deoxy-glucose transport. Et-3,4-dephostatin-induced
glucose uptake was inhibited by SB203580, a p38 inhibitor, but not by
PD98059, a MEK inhibitor, or by cycloheximide as insulin-induced
uptake. Interestingly, although LY294002, a phosphatidylinositol
3-kinase inhibitor, inhibited the insulin-induced glucose uptake
completely, it only partially inhibited the Et-3,4-dephostatin-induced uptake. It also blocked insulin-induced glucose transporter 4 translocation but not the Et-3,4-dephostatin-induced one. The increase
in c-Cbl tyrosine phosphorylation caused by Et-3,4-dephostatin was
stronger than that in insulin receptor phosphorylation. These observations indicate that a phosphatidylinositol 3-kinase-independent pathway involving c-Cbl is more important in Et-3,4-dephostatin-induced glucose uptake than in insulin-induced uptake. Et-3,4-dephostatin showed an in vivo antidiabetic effect in terms of reducing
the high blood glucose level in KK-Ay mice after oral
administration. Thus, Et-3,4-dephostatin potentiated insulin-related
signal transductions in cultured mouse adipocytes and showed an
antidiabetic effect in mice.
Type 2 diabetes mellitus is mainly characterized by impaired
signal transduction downstream of the insulin receptor in peripheral tissues, such as skeletal muscles and adipocytes (1-4). Insulin secretion from the pancreatic Thiazolidine derivatives such as troglitazone are being or have been
widely used for the treatment of type 2 diabetes mellitus (9).
Troglitazone is a ligand of peroxisome proliferator-activated receptor
The insulin receptor is a heterotetramer consisting of two Several protein-tyrosine phosphatases (PTPases) have been identified in
major insulin-sensitive tissues such as skeletal muscle, liver, and
adipose tissue. These include transmembrane PTPases such as CD45 and
leukocyte common antigen-related phosphatase (LAR)/RPTP- We previously isolated dephostatin from Streptomyces as a
novel inhibitor of T-cell receptor-associated protein-tyrosine
phosphatase CD45 (26). Since dephostatin was unstable in cell culture
media, later we synthesized Me-3,4-dephostatin as a stable
analogue (27). Me-3,4-dephostatin was shown to enhance nerve growth
factor- or epidermal growth factor-induced morphological
differentiation in rat pheochromocytoma PC12 h cells (28). This
PTPase inhibitor prolonged the tyrosine-phosphorylated and activated
state of MAP kinase. More recently, we synthesized Et-3,4-dephostatin
as another stable analogue (29).
In this report, we studied the effects of Et-3,4-dephostatin on
tyrosine phosphorylation of cellular proteins, activation of Akt, GLUT4
translocation, and hexose transport. We found that Et-3,4-dephostatin
mimicked or potentiated the various activities of insulin; especially,
it activated the PI 3-kinase-independent signaling pathway. It also
showed antidiabetic activity in mice.
Materials--
Dephostatin analogues were synthesized as
described previously (29). Insulin and p-nitrophenyl
phosphate were obtained from Sigma. Human recombinant PTP-1B, SHPTP-1,
and anti-phosphotyrosine antibody (4G10) were obtained from Upstate
Biotechnology, Inc. (Lake Placid, NY). CD45 and LAR were
purchased from Biomol (Plymouth Meeting, PA). Anti-insulin receptor Cell Culture and Differentiation--
3T3-L1 cells were
maintained in Dulbecco's modified Eagle's medium supplemented with
10% calf serum at 5% CO2. For differentiation, cells (1 × 106) were cultured in a 60-mm dish for 3 days to postconfluence in medium containing 10% fetal bovine serum,
500 µM isobutylmethylxanthine, 1 µM
dexamethasone, and 1 µg/ml insulin. Then the cells were grown in
medium containing only 10% fetal bovine serum and insulin for another
2 days. After that, the medium was changed, and the cells were cultured
in 10% fetal bovine serum medium for another 5-7 days for full
differentiation (30).
Enzyme Assays--
PTP-1B, SHPTP-1, CD45, and LAR were assayed
in a reaction mixture (final volume 200 µl) containing 25 mM HEPES (pH 7.2), 50 mM NaCl, 5 mM
dithiothreitol, 2.5 mM EDTA, 100 µg/ml bovine serum albumin, 5 mM p-nitrophenyl phosphate, 0.5 unit
of each enzyme, and the test chemical. After incubation for 10 min at
37 °C, the reaction was terminated by the addition of 100 µl of 2 M Na2CO3, and the absorbance at 405 nm was measured.
Immunoprecipitation of Signaling Proteins--
Differentiated
3T3-L1 adipocytes were grown in 60-mm dishes. After having been treated
with test chemicals, the cells were washed with 1.5 ml of ice-cold
phosphate-buffered saline (PBS) containing 1 mM
Na3VO4, scraped off the dishes, and centrifuged at 1000 × g for 5 min. After solubilization with 300 µl of lysis buffer, the soluble fractions were incubated with a
corresponding antibody at 2 µg/300 µl of lysate overnight at
4 °C followed by incubation for 6 h with protein G-agarose. The
immunocomplexes were pelleted by centrifugation, washed four times with
IP buffer (50 mM HEPES, 150 mM NaCl, 2.5 mM EGTA, 1 mM EDTA, 1 mM
dithiothreitol, 0.1% Tween 20), and then boiled in a loading buffer.
Western Blot Analysis--
3T3-L1 adipocytes (1 × 106) were grown in a 60-mm dish for 10 days. After having
been treated with test chemicals, the cells were washed with 1.5 ml of
ice-cold PBS containing 1 mM Na3VO4 and centrifuged at 1000 × g for 5 min. After
solubilization of the cells with 200 µl of lysis buffer consisting of
50 mM HEPES (pH 7.4), 150 mM NaCl, 10 mM EDTA, 1% Triton X-100, 200 mM sodium fluoride, 10% glycerol, 20 mM sodium pyrophosphate, 2 mM phenylmethanesulfonyl fluoride, 4 mM
Na3VO4, 0.1 mg/ml leupeptin, and 15 mM benzamidine, the cell lysate was centrifuged at
14,000 × g for 10 min. The supernatant was assayed for
protein content by using a Bio-Rad protein assay kit. The proteins were
resolved by SDS-polyacrylamide gel electrophoresis and
electrotransferred to polyvinylidene difluoride membranes. After having
been blocked with 5% (w/v) nonfat dry milk in Tris-buffered
saline-Tween buffer containing 20 mM Tris (pH 7.6), 0.14 M NaCl, and 0.1% (w/v) Tween 20, the membranes were
treated with antibodies, and then the proteins were visualized by use
of an ECL Western blotting detection system.
Plasma Membrane Sheet Assay--
Plasma membrane sheets were
prepared from differentiated 3T3-L1 adipocytes as described by Robinson
et al. (31). Briefly, the cells cultured on a microcover
glass were washed once with ice-cold PBS and incubated with 500 µl of
0.5 mg/ml poly-L-lysine for 30 s. The cells were then
swollen in 500 µl of hypotonic buffer (23 mM KCl, 10 mM HEPES, pH 7.5, 2 mM MgCl2, 1 mM EGTA) with three successive rinses. The swollen cells
were sonicated for 5 s in a sonication buffer (70 mM
KCl, 30 mM HEPES, pH 7.5, 5 mM
MgCl2, 3 mM EGTA, 1 mM
dithiothreitol, 0.1 mM phenylmethanesulfonyl fluoride). The
bound plasma membrane sheets were washed three times with the
sonication buffer and used for immunofluorescence studies.
2-Deoxyglucose Uptake in 3T3-L1 Adipocytes--
Differentiated
3T3-L1 adipocytes were grown in 12-well plates. After preincubation
with the desired test chemical for 1 h, 10 nM insulin
was added to the medium. After the incubation, the cells were incubated
in assay buffer consisting of 140 mM NaCl, 2.7 mM KCl, 6.5 mM Na2HPO4,
1.5 mM KH2PO4, 0.9 mM
CaCl2, 0.5 mM MgCl2, and 1 mg/ml
bovine serum albumin, pH 7.0, at 37 °C for 10 min, and then the
cells were incubated with 0.01 µCi of
2-[3H]deoxy-D-glucose for 10 min. The
uptake was terminated by adding 500 µl of ice-cold PBS containing 0.1 mM phloretin. The cells were washed twice with ice-cold PBS
and then solubilized in 0.5 N NaOH. Thereafter, the
radioactivity was measured by a scintillation counter. Nonspecific
glucose uptake was measured by treating cells with 20 µg/ml
cytochalasin B, and this value was subtracted from all of the data.
Immunostaining of GLUT4--
Immunostaining of GLUT4 was carried
out as previously described (32). Differentiated 3T3-L1 adipocytes were
cultured on microcover glasses in 12-well plates. After having been
treated with 10 µg/ml Et-3,4-dephostatin for 6 h, the adipocytes
were fixed in 500 µl of 2% formaldehyde in PBS for 5 min on ice and 5 min at room temperature. Then they were washed with 500 µl of 100 mM glycine in PBS for 15 min and blocked with 500 µl of
5% calf serum in PBS for 60 min. The cells were next incubated
with 2 µg/ml polyclonal anti-GLUT4 antibody in PBS overnight at
4 °C. After that, they were incubated with horseradish
peroxidase-conjugated anti-goat IgG (diluted 1:100 with PBS) at room
temperature for 60 min and then washed with TNT buffer (0.1 M Tris-HCl, pH 7.5, 0.15 M NaCl, 0.05% Tween
20) three times. The cells were then incubated with fluorophore
tyramide (diluted 1:50 with the amplification diluent; PerkinElmer Life
Sciences) for 7 min at room temperature in the dark, washed three
times, 10 min each time, in TNT buffer at room temperature, and
visualized under a fluorescence microscope.
In Vivo Blood Glucose Level--
KK-Ay mice
(10-week-old males; average weight, 40 g) were purchased from
Japan Clea Co. The test chemical was added to CE-2 diet containing 12%
sucrose, and the mice were given the drug-containing diet for 24 h. Then the blood was taken from the orbital capillary bed, and
0.01-ml aliquots of the serum were taken after centrifugation. The glucose content was measured with a glucose test kit (Wako, Tokyo).
Inhibition of PTPases by Et-3,4-dephostatin--
PTPases such as
CD45, LAR/RPTP-
First, we examined the inhibition of these PTPases by newly prepared
Et-3,4-dephostatin (Fig. 1).
Et-3,4-dephostatin strongly inhibited PTP-1B and SHPTP-1, the
IC50 values being 0.58 and 0.96 µg/ml, respectively. It
weakly inhibited CD45 but had no effect on LAR. Therefore,
Et-3,4-dephostatin showed selective inhibitory activity on PTP-1B and
SHPTP-1. 4-O-Me-Et-3,4-dephostatin (Fig. 1) was synthesized
as a negative control. As expected, it did not inhibit any of the
PTPases effectively, as shown in Table I.
Effect of Et-3,4-Dephostatin on Intracellular Tyrosine
Phosphorylation in 3T3-L1 Adipocytes--
Mouse 3T3-L1 fibroblasts
were differentiated into adipocytes for 8-12 days. Et-3,4-dephostatin
was added to the differentiated 3T3-L1 adipocytes (day 10) with or
without suboptimal 10 nM insulin; the cells were incubated
for 30 min, 2 h, or 6 h; and the tyrosine phosphorylation was
then examined by immunoprecipitation with anti-insulin receptor and
anti-IRS-1 antibodies. Insulin at 10 nM induced
tyrosine phosphorylation of the 95-kDa insulin receptor Effect of Et-3,4-dephostatin on Akt Activation--
PKB/Akt is a
downstream signal transducer of the IR-PI3K pathway that up-regulates
insulin signaling. So we examined whether Et-3,4-dephostatin could
induce Akt activation, which can be monitored by phosphorylation. We
employed a 6-h incubation, since the effect of Et-3,4-dephostatin on
tyrosine phosphorylation was more prominent in 6-h incubation. As a
result, Et-3,4-dephostatin induced Akt phosphorylation and enhanced
insulin-induced activation synergistically, as shown in Fig.
3, whereas it did not alter the amount of
Akt. As expected, either insulin-induced or Et-3,4-dephostatin-induced Akt activation was inhibited by LY294002, a PI 3-kinase inhibitor.
Effect of Et-3,4-dephostatin on GLUT4 Translocation--
Insulin
induces GLUT4 translocation from intracellular vesicles to the plasma
membrane (20), so we studied the effect of Et-3,4-dephostatin on GLUT4
translocation by employing the plasma membrane sheet assay. In this
assay, the upper membrane is removed after treatment of the cells, and
translocation to the membrane can be detected by the antibody to the
intracellular portion of GLUT4 (31). As shown in Fig.
4A, Et-3,4-dephostatin or
insulin induced translocation of GLUT4 to the membrane in 3T3-L1
adipocytes in 6 h. GLUT1 was located at the membrane without
stimulation, and the location was not altered by Et-3,4-dephostatin or
insulin (Fig. 4B). As shown in Fig. 4C, the
cellular amount of GLUT4 or GLUT1 was not changed by insulin or
Et-3,4-dephostatin. Thus, Et-3,4-dephostatin specifically induced
translocation of GLUT4, like insulin.
Effect of Et-3,4-dephostatin on
2-[3H]Deoxy-D-glucose Uptake--
Glucose
uptake is a major phenotypic change induced by insulin in the target
tissues. As shown in Fig. 5A,
insulin at 10 nM induced glucose uptake most prominently at
30 min. On the other hand, Et-3,4-dephostatin at 10 µg/ml alone
induced glucose uptake time-dependently up to 6 h.
When added together, Et-3,4-dephostatin increased the uptake additively
and time-dependently up to 6 h. Fig. 5B
shows the dose effect of Et-3,4-dephostatin on glucose uptake at 6 h. Et-3,4-dephostatin increased the uptake at 1-10 µg/ml with or
without insulin, and the effect of the combination was greater than
that of 100 nM (30.4 µg/ml) sodium vanadate. On the other
hand, 4-O-Me-Et-3,4-dephostatin only slightly enhanced the
uptake. The slight increase would be caused by the 4-O-Me compound still weakly inhibiting PTPases. Thus, Et-3,4-dephostatin stimulated glucose uptake in differentiated 3T3-L1 adipocytes with or
without a suboptimal concentration of insulin.
Inhibition of Et-3,4-dephostatin-induced
2-[3H]deoxy-D-glucose Uptake by Signal
Transduction Inhibitors--
Next we studied which signaling pathways
are involved in Et-3,4-dephostatin-induced glucose uptake in 3T3-L1
adipocytes by using enzyme inhibitors of small molecular weight. 3T3-L1
adipocytes were pretreated with various inhibitors for 1 h and
then stimulated with 10 nM insulin and/or 10 µg/ml
Et-3,4-dephostatin for 6 h. As shown in Fig.
6A, insulin- or
Et-3,4-dephostatin-stimulated glucose uptake was not affected by 50 µM PD98059, a MEK inhibitor, or 30 µg/ml cycloheximide.
SB203580, a p38 inhibitor, at 10 nM inhibited both insulin
and Et-3,4-dephostatin-stimulated glucose uptake significantly. On the
other hand, although 100 µM LY294002, a PI 3-kinase
inhibitor, completely inhibited the insulin-stimulated glucose uptake,
it only partially inhibited the Et-3,4-dephostatin-stimulated glucose
uptake. Fig. 6B shows the effect of LY294002 on insulin- and
Et-3,4-dephostatin-induced glucose uptake in 30 min. Unexpectedly, LY294002 inhibited both insulin-induced and the inhibitor-induced glucose uptake completely. Therefore, in long term incubation, Et-3,4-dephostatin may employ different signals from those of insulin
for activation of glucose transport, which are independent of PI
3-kinase.
Effect of LY294002 on Et-3,4-dephostatin-induced GLUT4
Translocation--
GLUT4 can be translocated without activation of PI
3-kinase (21). Therefore, first we examined the effect of LY294002 on Et-3,4-dephostatin-induced GLUT4 translocation in 3T3-L1 adipocytes, employing the anti-GLUT4 antibody that recognizes the extracellular domain. LY294002 inhibited the insulin-induced translocation of GLUT4
but not the Et-3,4-dephostatin-induced translocation in a 6-h
incubation, as shown in Fig. 7. On the
other hand, LY294002 inhibited Et-3,4-dephostatin-induced Akt
phosphorylation effectively (Fig. 3). Therefore, Et-3,4-dephostatin is
likely to activate a PI 3-kinase-independent pathway to mediate GLUT4
translocation and glucose uptake.
Effect of Et-3,4-dephostatin on c-Cbl
Phosphorylation--
Cellular Cbl was reported to be an important
mediator of the PI 3-kinase-independent signaling pathway induced by
insulin (21). Therefore, we studied the effect of Et-3,4-dephostatin on
c-Cbl phosphorylation in 3T3-L1 adipocytes by immunoprecipitation. As
shown in Fig. 8, insulin induced tyrosine
phosphorylation of c-Cbl at 10 min, but the phosphorylation disappeared
at 6 h. When the cells were treated with Et-3,4-dephostatin with
or without insulin, the phosphorylation was clearly observed after
6 h. This phosphorylation of c-Cbl was not inhibited by LY294002.
These results were parallel to those of 2-deoxyglucose uptake in Fig. 6. Thus, Et-3,4-dephostatin is likely to inhibit tyrosine
dephosphorylation of c-Cbl, activating PI 3-kinase-independent
signaling for GLUT4 translocation.
Effect of Et-3,4-dephostatin on Blood Glucose Level in
Vivo--
Due to the malfunction of their melanocortin receptor,
KK-Ay mice have yellow hair and show obesity and a high
blood glucose level of 400-500 mg/dl (33). When Et-3,4-dephostatin was
given to KK-Ay mice at 500 mg/kg orally with the diet, the
glucose level significantly decreased by about 50%, as shown in Fig.
9. In this model, troglitazone at 500 mg/kg also lowered the glucose level by about 50% (data not shown).
Thus, Et-3,4-dephostatin decreased the blood glucose level in
vivo.
Few PTPase inhibitors have been used as biochemical tools except
sodium vanadate. We previously developed Me-3,4-dephostatin as a stable
analogue of dephostatin (27) and showed it to enhance the effect of
nerve growth factor and epidermal growth factor on differentiation of
rat pheochromocytoma PC12 h cells (28). In this paper, we demonstrate
that an additional stable analogue, Et-3,4-dephostatin, potentiated or
mimicked the effect of insulin in differentiated 3T3-L1 adipocytes and
showed an antidiabetic effect in vivo.
First, we found that Et-3,4-dephostatin selectively inhibits PTP-1B and
SHPTP-1, both of which are known to be involved in the regulation of
the insulin receptor (22, 23), as shown in Table I. It only weakly
inhibited CD45. The IC50 value for CD45 is larger
than that in our previous report (27). This difference occurred because
we changed the enzyme source to a commercially available preparation
from the crude preparation of Jurkat cell membranes. Et-3,4-dephostatin
may inhibit other untested PTPases; therefore, involvement of PTPases
other than PTP-1B and SHPTP-1 cannot be excluded in its cellular effects.
Et-3,4-dephostatin increased the phosphorylation of Akt, GLUT4
translocation, and hexose transport in 3T3-L1 adipocytes.
4-O-Me-Et-3,4-dephostatin did not induce these phenotypic
changes, indicating that these effects are due to the inhibition of
PTPases. Since protein synthesis was blocked by the addition of
cycloheximide, the increase of hexose transport shown in Fig. 5 should
not be due to the increase of glucose transporters. Vanadate was also
reported to prolong insulin action by increasing the tyrosine
phosphorylation of the insulin receptor (34). Vanadate activated PI
3-kinase-dependent glucose transport (35). But
Et-3,4-dephostatin appears more effective than vanadate, as is shown in
Fig. 5B.
Unexpectedly, Et-3,4-dephostatin alone mimicked insulin action to
induce several phenotypic changes. Compared with the quick action of
insulin in 30 min, Et-3,4-dephostatin alone increased the tyrosine
phosphorylation of the insulin receptor and IRS-1 rather slowly in
6 h, as shown in Fig. 2. This is because accumulation of
phosphorylation by inhibition of PTPases takes time.
Et-3,4-dephostatin strongly induced GLUT4 translocation and glucose
transport. Therefore, Et-3,4-dephostatin may inhibit not only insulin
receptor dephosphorylation but also dephosphorylation of other
components that regulate glucose uptake. In fact, Et-3,4-dephostatin
induced tyrosine phosphorylation in several other proteins, including
c-Cbl, PLC Insulin-induced glucose transport is mediated largely by PI 3-kinase
(37) and partly by p38 (38). On the other hand, the Ras/mitogen-activated protein kinase pathway and protein synthesis are
not necessary in the effect of insulin. In Fig. 6A,
insulin-induced glucose uptake was inhibited by LY294002 and SB203580
but not by a MEK inhibitor or by cycloheximide. Interestingly, LY294002 inhibited insulin-induced glucose transport and GLUT4 translocation completely in our assay system; but when Et-3,4-dephostatin was used,
the inhibitor only partially suppressed glucose transport and GLUT4
translocation in a 6-h incubation (Fig. 6A and 7). On the
other hand, in a 30-min incubation, LY294002 completely blocked the
increase of glucose transport by Et-3,4-dephostatin, as shown in Fig.
6B. Therefore, it is likely that Et-3,4-dephostatin
activates a PI 3-kinase-independent pathway in addition to the PI
3-kinase/Akt pathway only in long term incubation. Very recently,
Pessin and co-workers (21) showed the existence of a PI
3-kinase-independent c-Cbl pathway in insulin-induced signal
transduction. The proto-oncogene product c-Cbl is known to be a
component of protein-tyrosine kinase-mediated signaling cascades
downstream of the activated cell surface receptors. These receptors
include the T cell, B cell, and cytokine receptors (39). The c-Cbl
protein contains the RING finger domain, an extensive proline-rich
region that provides binding sites for the SH3 domain, and the
C-terminal leucine zipper domain that shows significant homology to
ubiquitin-associated proteins (40). In hematopoietic cells, c-Cbl is
known to be a negative regulator of Syk tyrosine kinase through the
RING finger domain (41). In 3T3-L1 cells, c-Cbl is prominently
tyrosine-phosphorylated in response to insulin in 3T3-L1 adipocytes and
not in 3T3-L1 fibroblasts (42). The tyrosine-phosphorylated c-Cbl
protein associates with Cbl-associated protein at the site of caveola, and this protein complex up-regulates GLUT4 translocation through the
PI 3-kinase independent pathway (21). Therefore, it is possible that
Et-3,4-dephostatin inhibits PTPases to protect phosphorylated c-Cbl
from dephosphorylation. Actually, Et-3,4-dephostatin markedly enhanced
tyrosine phosphorylation of c-Cbl, as shown in Fig. 8. Insulin-induced
c-Cbl tyrosine phosphorylation greatly decreased at 6 h, but when
Et-3,4-dephostatin was added, the phosphorylation strongly remained.
Et-3,4-dephostatin alone also induced phosphorylation of c-Cbl. Thus,
it is likely that Et-3,4-dephostatin potentiates both PI
3-kinase/Akt-dependent and PI 3-kinase/Akt- independent pathways including activation of c-Cbl.
Kayali et al. (25) reported that phospholipase C- When Et-3,4-dephostatin was orally given to mice with high blood
glucose, it lowered their blood glucose significantly, as shown in Fig.
9. Therefore, Et-3,4-dephostatin is likely to be absorbed from the
intestine and to be stable in the body. During the in vivo
experiment, no toxicity, including the loss of body weight, was
observed. In 24 h, there was no marked difference in food uptake
between the test and control group. There was also no abnormality in
coat of fur, behavior, and feces. The methoxime-type derivative of
dephostatin (29) belongs to the PTPase inhibitors of the second
generation. Long term experiments with this compound also decreased the
blood glucose level in mice without
toxicity.2 Thiazolidine
compounds are being widely used clinically for the treatment of type 2 diabetes mellitus. Its target is known to be peroxisome
proliferator-activated receptor Thus, Et-3,4-dephostatin potentiated the insulin-related signal
transduction in cultured 3T3-L1 adipocytes and showed antidiabetic effects in mice. Especially, it was orally active in vivo.
Since the nitrosamine group in Et-3,4-dephostatin may be mutagenic and carcinogenic, a nitrosamine-free analogue of Et-3,4-dephostatin (29)
may be a prototype of new antidiabetic agents.
*
This work was supported in part by grants from the Science
Research Promotion Fund of the Promotion and Mutual Aid Corporation for
Private Schools of Japan, the Special Coordination Funds for Promotion
of Science and Technology from the Science and Technology Agency of
Japan, and the Ministry of Education, Science, Culture, and Sports of
Japan (Academic Frontier Promotion Project).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. Tel./Fax:
81-45-566-1558; E-mail: umezawa@applc.keio.ac.jp.
Published, JBC Papers in Press, May 7, 2001, DOI 10.1074/jbc.M011726200
2
I. Takei and K. Umezawa, unpublished results.
The abbreviations used are:
PI, phosphatidylinositol;
GLUT, glucose transporter;
PTPase, protein-tyrosine phosphatase;
PBS, phosphate-buffered saline;
MEK, mitogen-activated protein kinase/extracellular signal-regulated kinase
kinase;
LAR, leukocyte common antigen-related phosphatase;
IRS, insulin
receptor substrate.
Potentiation of Insulin-related Signal Transduction by
a Novel Protein-tyrosine Phosphatase Inhibitor, Et-3,4-dephostatin, on
Cultured 3T3-L1 Adipocytes*
,,
, and**
Department of Applied Chemistry, Faculty of
Science and Technology, Keio University, 3-14-1 Hiyoshi, Kohoku-ku,
Yokohama 223-0061, Japan, § Institute of Microbial
Chemistry, 3-14-23 Kamiosaki, Shinagawa-ku, Tokyo 141-0021, Japan,
¶ Kaneka Corp., 1-8 Miyamae-machi, Takasago-cho, Takasago, Hyogo
676-8688, Japan, and Clinical Laboratory and Internal Medicine,
School of Medicine, Keio University, 35 Shinanomachi, Shinjuku-ku,
Tokyo 160-8582, Japan
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-cells is also affected in many cases
of type 2 diabetes mellitus (5, 6), since the secondary effect of
impaired signal transduction may affect insulin secretion (it was
reported that long-term exposure to a high glucose concentration induced apoptosis in pancreatic -cells (7)). A functional insulin
receptor was also shown to be essential for the secretion of insulin by
cultured mouse -cells (8).
that transcriptionally regulates a number of adipose tissue-specific genes by binding to peroxisome proliferator-activated receptor response elements of these genes as a heterodimer with retinoid X receptor. These heterodimers potentiate adipose tissue differentiation in vivo (10), and their side effects include obesity. Several drugs such as sulfonylureas enhance insulin secretion. Sulfonylureas bind to receptors on pancreatic -cells to induce Ca2+ influx, thereby increasing insulin secretion (11).
Compared with the insulin therapy for type I diabetes mellitus,
chemotherapy for type 2 has been poorly developed, and less toxic
chemotherapy based on the insulin receptor-associated signal
transduction should be developed.
-subunits
that are entirely extracellular and two -subunits that span the
plasma membrane and contain intrinsic tyrosine kinase activity (12,
13). Binding of insulin to its receptor results in activation of the
receptor tyrosine kinase and receptor autophosphorylation, followed by
tyrosine phosphorylation of IRS-1, -2, and -3, which then bind to the
p85 regulatory subunit of phosphatidylinositol (PI)1 3-kinase. Then the p110
catalytic subunit of PI 3-kinase produces phosphatidylinositol
3,4-bisphosphate and phosphatidylinositol 3,4,3-trisphosphate, which
can bind to the PH domain of PKB()/Akt1(2), which then
up-regulate glucose transporter 4 (GLUT4) translocation. These Akts are
phosphorylated to become activated by
3-phosphoinositide-dependent protein kinase 1 or 2 (14). The
insulin-stimulated phosphorylations of Akt on threonine 308 by
3-phosphoinositide-dependent protein kinase 1 and on serine 473 by 3-phosphoinositide-dependent protein kinase 2 are required
for maximal Akt activity (15-17). Kupriyanova and Kandror (18)
reported that the phosphorylated Akt2 directly binds to
GLUT4-containing vesicles to phosphorylate GLUT4 to trigger translocation of GLUT4 from the cytoplasm to the membrane (19), resulting in an increase in cellular glucose uptake (20). On the other
hand, very recently, Pessin and co-workers (21) showed the existence of
a PI 3-kinase-independent c-Cbl pathway in insulin-induced signal
transduction. The c-Cbl protein is a proto-oncogene product that can be
tyrosine-phosphorylated. The Cbl-associated protein forms complex with
c-Cbl. This complex mediates GLUT4 translocation through the PI
3-kinase-independent pathway in 3T3-L1 adipocytes (21).
and
nontransmembrane PTPases such as SHPTP-1, SHPTP-2, PTP-1B, and PTP-1C
(22). Various PTPases are considered to be involved in the etiology of
diabetes mellitus. Especially, PTP-1B, a cytoplasmic PTPase, is known
to be a negative regulator of insulin receptor-associated signal
transduction (23). PTP1B-deficient mice showed increased insulin
sensitivity and resistance to obesity (24). Overexpression of PTP-1B in
Rat1 fibroblasts is known to reduce ligand-stimulated
autophosphorylation of the insulin receptor (23). Therefore, inhibitors
of PTP-1B may enhance the insulin sensitivity and glucose uptake .
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
subunit antibody, anti-Akt antibody, anti-GLUT1 antibody, anti-GLUT4
antibody, and horseradish peroxidase-conjugated anti-goat IgG were
obtained from Santa Cruz Biotechnology (Santa Cruz, CA).
Anti-phosphorylated Akt antibody was obtained from New England Biolabs
(Mississauga, Canada). Horseradish peroxidase-conjugated anti-mouse IgG
and anti-rabbit IgG were obtained from Amersham Pharmacia Biotech.
Western Blot Chemiluminescence Reagent and Tyramide Signal
Amplification (TSATM-Direct) were obtained from PerkinElmer
Life Sciences. 2-[3H]Deoxy-D-glucose
was obtained from American Radiolabeled Chemicals (St. Louis, MO).
LY294002 was obtained Cayman Chemical (Ann Arbor, MI).
Protein-G-agarose was obtained from Oncogene Research Products (Cambridge, MA). A Triglyceride G-Test Kit was obtained from Wako (Osaka, Japan).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
, SHPTP-1, SHPTP-2, PTP-1B, and PTP-1C are known to
be involved in the regulation of the insulin receptor (22).
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Fig. 1.
Stable dephostatin derivatives.
Inhibition of PTPases by Et-3,4-dephostatin
-subunit
most prominently in 30 min, and Et-3,4-dephostatin alone at 10 µg/ml
increased the tyrosine phosphorylation more slowly, as shown in Fig.
2. When added together, insulin and
Et-3,4-dephostatin enhanced the phosphorylation synergistically in
6 h. Insulin and Et-3,4-dephostatin did not change the cellular
amount of insulin receptor and IRS-1. In a 30-min incubation,
Et-3,4-dephostatin alone increased tyrosine phosphorylation of insulin
receptor. But unexpectedly, it even lowered the insulin-induced
tyrosine phosphorylation of insulin receptor and IRS-1 in a 30-min
incubation. In a 6-h incubation, insulin and Et-3,4-dephostatin also
increased the phosphorylation of other proteins at 135, 115, 85, and 60 kDa, which were identified to be phospholipase C, c-Cbl, PI 3-kinase regulatory subunit, and IRS-3, respectively, by the mobility of each
authentic protein in a Western blotting analysis (data not shown).
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[in a new window]
Fig. 2.
Effect of Et-3,4-dephostatin on intracellular
tyrosine phosphorylation in 3T3-L1 adipocytes. 3T3-L1 adipocytes
were treated with 10 nM insulin and/or 10 µg/ml
Et-3,4-dephostatin for the indicated periods. After immunoprecipitation
with anti-insulin receptor or anti-IRS-1 antibody, the proteins were
transferred to polyvinylidene difluoride membranes, and the proteins
were immunoblotted with anti-phosphotyrosine antibody.
View larger version (28K):
[in a new window]
Fig. 3.
Enhancement of Akt phosphorylation by
Et-3,4-dephostatin. After 3T3-L1 adipocytes had been pretreated or
not with 100 µM LY294002 for 1 h, the cells were
treated with 10 nM insulin and/or 10 µg/ml
Et-3,4-dephostatin for 6 h. Following electrophoretic transfer to
polyvinylidene difluoride membranes, the proteins were
immunoblotted with anti-phospho-Ser473 Akt antibody
(A) or with anti-Akt antibody (B).
View larger version (57K):
[in a new window]
Fig. 4.
Effect of Et-3,4-dephostatin on the
localization of GLUT4 and GLUT1. 3T3-L1 adipocytes were treated
with 10 nM insulin, 10 µg/ml Et-3,4-dephostatin, insulin
and Et-3,4-dephostatin, or 10 µg/ml
4-O-Me-Et-3,4-dephostatin for 6 h. Plasma membrane
sheets were then prepared and fixed with 2% formaldehyde. The fixed
membrane sheets were stained with polyclonal anti-GLUT4 (A)
or anti-GLUT1 (B) antibody. The protein levels of GLUT4 and
GLUT1 were assayed by Western blotting (C).
View larger version (16K):
[in a new window]
Fig. 5.
Enhancement of glucose uptake in
insulin-treated or untreated 3T3-L1 adipocytes. A,
3T3-L1 adipocytes were untreated ( 
) or treated with 10 nM insulin (
), 10 µg/ml Et-3,4-dephostatin (
), or
insulin and Et-3,4-dephostatin (
) for the indicated periods.
B, dose effect of Et-3,4-dephostatin on glucose uptake.
After incubation, the cells were incubated with
2-[3H]deoxy-D-glucose for 10 min, and the
reaction was then terminated. After having been washed twice, the cells
were solubilized and neutralized, and the radioactivity was measured by
a scintillation counter. Values are means ± S.D. of triplicate
determinations.
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[in a new window]
Fig. 6.
Effect of signal transduction inhibitors on
Et-3,4-dephostatin-induced glucose uptake. A,
the cells were pretreated with various inhibitors for 1 h and then
stimulated with 10 nM insulin and/or 10 µg/ml
Et-3,4-dephostatin for 6 h. After incubation, the cells were
incubated with 2-[3H]deoxy-D-glucose for 10 min. The cell-associated radioactivity was measured by a scintillation
counter. Values are means ± S.D. of triplicate determinations.
B, the cells were pretreated with LY294002 and then
stimulated with insulin and/or 10 µg/ml Et-3,4-dephostatin for 30 min.
View larger version (56K):
[in a new window]
Fig. 7.
Effect of LY294002 on
Et-3,4-dephostatin-induced GLUT4 translocation. 3T3-L1 adipocytes
were treated with 10 nM insulin or 10 µg/ml
Et-3,4-dephostatin without or with 100 µM LY294002 for
6 h, and then the cells were fixed with 2% formaldehyde.
The fixed cells were stained with polyclonal anti-GLUT4 antibody that
binds to the extracellular portion of GLUT4.
View larger version (25K):
[in a new window]
Fig. 8.
Effect of LY294002 on insulin or
Et-3,4-dephostatin-induced intracellular c-Cbl tyrosine
phosphorylation. After 100 µM, LY294002 was
pretreated for 30 min, 3T3-L1 adipocytes were treated with 10 nM insulin and/or 10 µg/ml Et-3,4-dephostatin for 6 h, and then the cells were lysed with the lysis buffer and
immunoprecipitated with monoclonal anti-c-Cbl antibody. Following
electrophoretic transfer to the membranes, the proteins were
immunoblotted with anti-phosphotyrosine antibody.
View larger version (17K):
[in a new window]
Fig. 9.
Decrease in blood glucose level in
Et-3,4-dephostatin-treated KK-Ay mice.
KK-Ay mice were treated with a CE-2 diet containing 12%
sucrose and Et-3,4-dephostatin for 24 h. A blood sample was then
taken from the orbital capillary bed, and its glucose level was
measured. The results are representative of two independent
experiments.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
, PI 3-kinase, and IRS-3. In Fig. 2, Et-3,4-dephostatin
unexpectedly lowered the insulin-induced tyrosine phosphorylation of
insulin receptor and IRS-1 in a 30-min incubation. Insulin is known to
activate PTPases such as PTP-1B (36) that can dephosphorylate both
insulin and IRS-1. Et-3,4-dephostatin might enhance induction of the
inhibitor-insensitive PTPases by insulin in a 30-min incubation. On the
other hand, Et-3,4-dephostatin did not affect insulin-induced
activation of hexose transport even in a 30-min incubation (Fig.
5A). This is because Et-3,4-dephostatin might also
inhibit tyrosine dephosphorylation of downstream components.
is
associated with the insulin receptor and can be phosphorylated by the
receptor. Therefore, if a PI 3-kinase is not involved downstream of
phospholipase C-, activation of phospholipase C- by inhibition of
dephosphorylation may also be a possible mechanism.
, which is a unique transcription
factor for adipocyte differentiation. In our assay system shown under
"Experimental Procedures," troglitazone significantly enhanced
differentiation of 3T3-L1 cells into adipocytes. However, Et-3,4-dephostatin did not increase the differentiation significantly (data not shown). Therefore, the mechanism of the antidiabetic effect
by Et-3,4-dephostatin in vivo should be different from that
by troglitazone, and its side effects may not include obesity, unlike
the case for troglitazone.
FOOTNOTES
ABBREVIATIONS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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