DHEA improves glucose uptake via activations of protein kinase
C and phosphatidylinositol 3-kinase
Tatsuo
Ishizuka,
Kazuo
Kajita,
Atsushi
Miura,
Masayoshi
Ishizawa,
Yoshinori
Kanoh,
Satomi
Itaya,
Mika
Kimura,
Naoya
Muto,
Tomoatsu
Mune,
Hiroaki
Morita, and
Keigo
Yasuda
The Third Department of Internal Medicine, Gifu University School of
Medicine, Gifu 500, Japan
 |
ABSTRACT |
We have examined the
effect of adrenal androgen, dehydroepiandrosterone (DHEA), on glucose
uptake, phosphatidylinositol (PI) 3-kinase, and protein kinase C (PKC)
activity in rat adipocytes. DHEA (1 µM) provoked a twofold increase
in 2-[3H]deoxyglucose
(DG) uptake for 30 min. Pretreatment with DHEA increased
insulin-induced
2-[3H]DG uptake
without alterations of insulin specific binding and autophosphorylation
of insulin receptor. DHEA also stimulated PI 3-kinase activity.
[3H]DHEA bound to
purified PKC containing PKC-
, -
, and -
. DHEA provoked the
translocation of PKC-
and -
from the cytosol to the membrane in
rat adipocytes. These results suggest that DHEA stimulates both PI
3-kinase and PKCs and subsequently stimulates glucose uptake. Moreover,
to clarify the in vivo effect of DHEA on Goto-Kakizaki (GK) and Otsuka
Long-Evans fatty (OLETF) rats, animal models of non-insulin-dependent
diabetes mellitus (NIDDM) were treated with 0.4% DHEA for 2 wk.
Insulin- and 12-O-tetradecanoyl phorbol-13-acetate-induced
2-[3H]DG uptakes of
adipocytes were significantly increased, but there was no significant
increase in the soleus muscles in DHEA-treated GK/Wistar or
OLETF/Long-Evans Tokushima (LETO) rats when compared with untreated
GK/Wistar or OLETF/LETO rats. These results indicate that in vivo DHEA
treatment can result in increased insulin-induced glucose uptake in two
different NIDDM rat models.
dehydroepiandrosterone; non-insulin-dependent diabetes mellitus; 2-deoxyglucose
 |
INTRODUCTION |
GLUCOCORTICOID EXCESS causes insulin resistance.
However, the mechanisms involved are unknown (1). Studies have shown
that glucocorticoids affect specific insulin binding (27), tyrosine kinase activity (20, 35), and/or glucose transporter (7). Recently, dose-response inhibition of insulin-induced
2-[3H]deoxyglucose
(2-DG) uptake by dexamethasone and prednisolone in rat adipocytes has
been observed, and glucocorticoids have been shown to stimulate the
protein kinase C (PKC) isoform via binding to the regulatory subunit of
PKC (17, 18). On the other hand, adrenal androgen,
dehydroepiandrosterone (DHEA; 3
-hydroxy-5-androsten-17-one), and its
sulfate derivative are found in abundance in the human (28), although
their physiological roles are still unknown. Serum concentrations of
DHEA in 60-yr-old men show a gradual decrease compared with young men
aged 25-30 yr. This decrease occurs as the incidence of
atherosclerosis, obesity, and diabetes increases, suggesting that
administration of DHEA may protect against the development of these
disorders (2). In genetically diabetic (db/db) mice, DHEA
administration prevents the development of diabetes mellitus (5).
Recently, it has been reported that DHEA treatment reduces fat
accumulation and protects against insulin resistance via an increase in
phosphatidylinositol (PI) 3-kinase after immunoprecipitation with
insulin receptor substrate-1 (IRS-1) in male rats (10). We have
investigated the effects of DHEA on insulin-induced glucose uptake in
vivo and in vitro in Wistar, Goto-Kakizaki (GK; see Refs. 8 and 9), and
Otsuka Long-Evans fatty (OLETF) rat (16, 21) adipocytes.
 |
MATERIALS AND METHODS |
Materials. Pork insulin was obtained
from Novo (Copenhagen, Denmark).
2-[1,2-3H]DG was
purchased from New England Nuclear (Boston, MA). Phenylmethylsulfonyl fluoride (PMSF), leupeptin,
12-O-tetradecanoyl phorbol-13-acetate (TPA), BSA, phosphatidylserine, diolein, histone (type III-S), D-glucose, ATP, dexamethasone, prednisolone, and DHEA were
purchased from Sigma (St. Louis, MO). Silicone oil was obtained from
Aldrich Chemical (Milwaukee, WI).
[
-32P]ATP (3,000 Ci/mmol), 2-[1,2-3H]DG
(50 Ci/mmol),
L-[1-14C]glucose
(47 mCi/mmol),
[6,7-3H]dexamethasone
(50 Ci/mmol), and
[1,2-3H]DHEA (60 Ci/mmol) were purchased from New England Nuclear (Tokyo, Japan).
RU-38486 (17
hydroxy-11
,4-dimethylaminophenyl-17
-propynyl estra 4,9 diene-3-one) was generously donated by the Roussel-Uclaf Research Center. All other chemicals were of reagent grade or better.
Polypropylene plastic tubes were used in each experiment, unless
otherwise stated.
Adipocyte experiments. Male Wistar and
GK rats, a non-insulin-dependent diabetes mellitus (NIDDM) animal model
(8, 9) weighing 150-200 g (8 wk of age), and Long-Evans Tokushima
(LETO, control) and OLETF rats (16 wk of age; see Refs. 16, 21) were fed with CE2 (Japan Clea, Tokyo, Japan) ad libitum, treated with CE2
powder containing 0.4% DHEA for 2 wk, and then killed by decapitation. As shown in Table 1, in vivo treatment with
0.4% DHEA for 2 wk resulted in significant increases in plasma DHEA
concentration in GK/Wistar and OLETF/LETO rats. Isolated adipocytes
were obtained by collagenase digestion of rat epididymal fat pads (26,
29) in Krebs-Ringer phosphate (KRP) buffer (pH 7.4) containing 127 mM
NaCl, 12.3 mM
NaH2PO4,
5.1 mM KCl, 1.3 mM MgSO4, 1.4 mM
CaCl2, 3% BSA, and 2.5 mM
glucose. Adipocytes were washed two times, preincubated at 37°C in
glucose-free KRP buffer containing 1% BSA for 30 min, and then
incubated with or without (control)
10
6 M DHEA (dissolved in
<0.01% ethanol) for 60 min, followed by incubation with 10 nM
insulin for 30 min. There was no effect of 0.01% ethanol, used for the
control, on insulin-induced glucose uptake in rat adipocytes.
2-[3H]DG (0.08 µCi)
and unlabeled 2-DG (0.05 mM) were then added to 300 µl of a 10%
(vol/vol) adipocyte suspension, and uptake of 2-[3H]DG was measured
over 1 min. Corrections for trapped buffer or non-carrier-mediated
uptake were determined by measuring uptake in the presence of 70 µM
cytochalasin, which reduced control and agonist-stimulated values (14).
PKC experiments. Adipocytes were
incubated with or without (control, 0 min) 1 µM DHEA for 5, 10, and
20 min. Reactions were terminated by the addition of 20 mM
Tris · HCl buffer (pH 7.5) containing 0.25 M sucrose,
1.2 mM EGTA, 0.1 mM PMSF, 20 µg/ml leupeptin, and 20 mM
2-mercaptoethanol (buffer I), washed
two times, and homogenized in buffer
I. The homogenates were centrifuged for 60 min at
105,000 g to obtain the cytosol and
membrane fractions. The latter was homogenized in
buffer I containing 5 mM EGTA, 2 mM
EDTA, and 1% Triton X-100. Activation of PKC in rat adipocytes was
assayed by changes in the subcellular distribution of immunoreactive PKC with methods described previously (14, 15). Equal amounts of
cytosol or membrane-associated fraction were prepared, subjected to
sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), transferred to nitrocellulose, and incubated first with polyclonal antiserum raised to synthetic peptide to PKC-
and -
(GIBCO) and
second with goat anti-rabbit globulin complexed to alkaline phosphatase
(Sigma). This immunoblotting method detected a single major
immunoreactive band that comigrated on SDS-PAGE and blotted identically
with 80-kDa (PKC-
) and 73-kDa (PKC-
) synthetic peptides.
Soleus muscle experiments. Soleus
muscles were excised, and muscle tension was maintained by ligatures
attached to the tendons. The two solei from each rat provided one
control and one stimulated sample. Soleus muscles were incubated in
25-ml Erlenmeyer flasks under 95%
O2-5%
CO2 in 5 ml Krebs-Ringer
bicarbonate (KRB) buffer (119 mM NaCl, 4.8 mM KCl, 1 mM
KH2PO4,
1.2 mM MgSO4, 1 mM
CaCl2, 24 mM
NaHCO3, 12 mM HEPES, 0.1% BSA,
and 2 mM sodium pyruvate). For studies of glucose transport, after an
initial 30-min incubation in KRB buffer containing 5 mM glucose, the
solei were washed and incubated for 30 min in glucose-free KRB for 30 min with or without insulin.
2-[3H]DG (1 µCi),
L-[1-14C]glucose
(0.1 µCi), and 0.1 mM unlabeled 2-DG were then added, and incubation
was continued for 10 min. After incubation, tissue was removed, rapidly
rinsed in isotope-free medium, blotted, weighed, homogenized in 5%
trichloroacetic acid, and counted simultaneously for
3H and
14C. Corrections for
2-[3H]DG in tissue
samples unrelated to specific transport were determined by measurement
of radioactivity of
L-[14C]glucose.
As reported previously (15), uptake of
2-[3H]DG was linear
for at least 30 min.
DHEA binding study. For
[3H]DHEA specific
binding to purified PKC from rat brain, Mono Q column-purified PKC was
obtained using an HPLC system as described previously (14, 15), and
then the collected samples were concentrated fivefold in
buffer I containing 5 mM EGTA, 2 mM
EDTA, and 5% glycerol. Moreover, purified PKC (BIOML Research
Laboratories), 50 nM
[3H]DHEA, 1,000-fold
DHEA, and 100-fold RU-38486 were incubated for 24 h at 4°C with and
without 0.5 mM Ca2+ and were
separated by the addition of charcoal dextran. Radioactivity in the
supernatant was measured with a liquid scintillation counter. [3H]DHEA specific
binding to free adipocytes was carried out as described previously
(34). Briefly, free adipocytes (80 µl) and 50 nM
[3H]DHEA 1,000-fold
unlabeled DHEA in the presence or absence of 100-fold RU-38486 were
incubated for 60 min at 37°C, separated by passage through Whatman
filter paper (25 mm), and rinsed several times with an excess of 0.9%
saline. The radioactivity in this paper was counted with a liquid
scintillation counter.
PI 3-kinase assay. Isolated adipocytes
were treated with or without insulin (10 nM) for 0, 1, 5, 10, and 60 min at 37°C, lysed in buffer containing 1% (vol/vol) Nonidet P-40
(NP-40; see Ref. 11), and immunoprecipitated with an
anti-phosphotyrosine antibody (19) and protein A-agarose. The
immunoprecipitates were washed and subjected to the PI 3-kinase assay
as described elsewhere (11). Briefly, cells were treated with 10 nM
insulin, 1 µM TPA, or 1 µM DHEA for the indicated periods, lysed in
the buffer (20 mM Tris · HCl, pH 7.4, 1% NP-40, 0.5 mM EDTA, 5% glycerol, 1 mM orthovanadate, and 20 µM
p-amidinophenylmethanesulfonyl
fluoride hydrochloride), and sonicated. The homogenates were
centrifuged at 15,000 rpm for 20 min, and the indicated supernatants
were incubated with 4 µg of anti-phosphotyrosine antibody for 2 h at 4°C. The immunocomplexes were precipitated with 40 µl of protein A-agarose and washed with NP-40-free buffer (10 mM
Tris · HCl, pH 7.4, 100 mM NaCl, and 1 mM
dithiothreitol) two times. The immunoprecipitates were subjected to the
PI 3-kinase assay in a 50-µl reaction mixture containing 20 mM
Tris · HCl, pH 7.4, 100 mM NaCl, 10 mM
MgCl2, 0.5 mM EGTA, 100 µM PI,
100 µM phosphatidylserine, and 10 µM
[
-32P]ATP (0.1 µCi/µl). After 10 min at 30°C, the reaction was stopped by
adding 200 µl of 1 M HCl and 80 µl of chloroform-methanol (1:1, vol/vol). A 30-µl portion of the lower layer was spotted on a Silica
Gel 60 plate (Merck) and was developed in chloroform-methanol-25% NH4Cl-water (43:38:5:7,
vol/vol/vol/vol). The radioactive PI phosphate spot was detected by
autoradiography, and scrapes from the plate were counted by liquid
scintillation counting.
Immunoblot. Immunoprecipitate (500 µg protein) with an anti-phosphotyrosine antibody (5 µg) or
anti-insulin receptor antibody (5 µg; Upstate Biotechnology, Lake
Placid, NY) was separated by SDS-PAGE and transferred to nitrocellulose
paper. The paper was blocked with 3% gelatin Tris-buffered
saline and was incubated with an anti-p85 PI 3-kinase
antibody or anti-phosphotyrosine antibody (1:1,000 dilution;
Transduction Laboratories, Lexington, KY) for 4-5 days. Protein
bands were located with an enhanced chemiluminescence system (Amersham,
Tokyo, Japan). Immunoblots were quantified by laser scanning densitometry.
Insulin binding studies. Isolated
adipocytes were suspended in KRP buffer, incubated for 60 min with or
without (control) 1 µM DHEA, and then incubated with
[125I]insulin (2,000 Ci/mmol; Amersham) and unlabeled insulin (1-1,000 nM) in plastic
tubes at 25°C in a shaking water bath for 60 min as described
previously (17). Incubations were terminated by removing 300-µl
aliquots from the cells in plastic microfuge tubes to which 100 µl
silicone oil had been added. The cells were then separated by cutting a
plastic tube just over the silicone oil layer, and the radioactivity
was determined. All studies were performed in triplicate.
Incorporation of 3H in diacylglycerol.
Adipocytes were incubated for 30 min in 0.5 ml KRP buffer.
[3H]glycerol (10 µCi) was then added, and, after prelabeling adipocytes for 15 min, a
vehicle (control) or 10 nM insulin was added after pretreatment with 1 µM DHEA for 60 min. Incubation was continued for 2, 5, 10, and 20 min. Reactions were stopped by the addition of methanol (final
concentration, 50%). Samples were transferred to glass tubes.
Chloroform (2 vol) was added, and extraction of the lipids was
performed as described previously (6, 12).
Statistical analysis. Statistical
comparisons were performed using ANOVA followed by Fisher's protected
least significant difference (PLSD) test. Unless otherwise stated, all
data are expressed as the means ± SE.
 |
RESULTS |
Effect of DHEA on insulin-induced
2-[3H]DG uptake.
DHEA (1 µM) alone stimulated
2-[3H]DG uptake by
150-200% from the basal level within 30 min
(P < 0.05 by ANOVA with Fisher's PLSD) but did not stimulate androstenedione, ethiocholanolone, or
testosterone (Fig. 1). Maximal effect of DHEA
on glucose uptake was observed at 1 µM (data not shown). Pretreatment
with 1 µM DHEA resulted in a significant increase
(P < 0.01 by ANOVA with Fisher's
PLSD) in insulin-induced
2-[3H]DG uptake.
Maximal effect of DHEA on insulin-induced
2-[3H]DG uptake was
observed at 10
8 M
(Fig. 2).

View larger version (12K):
[in this window]
[in a new window]
|
Fig. 1.
Agonists stimulate
2-[3H]deoxyglucose
(2-[3H]DG) uptake in
rat adipocytes. Isolated adipocytes (10% cell suspension) were
stimulated with or without (control, Cont) 10 nM insulin (INS), 1 µM
phorbol ester 12-O-tetradecanoyl
phorbol-13-acetate (TPA), 1 µM dehydroepiandrosterone (DHEA),
androstenedione (AND), ethiocholanolone (ETH), or 1 µM testosterone
(TEST) for 30 min.
2-[3H]DG (0.05 mM,
0.08 µCi) was added to 300 µl of a 10% adipocyte suspension, and
then 2-[3H]DG uptake
was measured. Control value was set at 100%. Data are plotted as means ± SE of 6 separate experiments.
* P < 0.01, INS or TPA vs.
Cont.
|
|

View larger version (15K):
[in this window]
[in a new window]
|
Fig. 2.
Effect of DHEA on insulin-induced
2-[3H]DG uptake in rat
adipocytes. Isolated adipocytes were stimulated with 10 nM insulin
after pretreatment with 1 µM DHEA for 60 min, and then glucose uptake
was measured as shown in Fig. 1 legend. Data are plotted as the means ± SE of 5 separate experiments.
* P < 0.01 and
# P < 0.05 by Fisher's
protected least significant difference (PLSD) test.
|
|
Effect of DHEA on insulin specific binding to its
receptor and 95-kDa autophosphorylation of insulin
receptor. To clarify the effect of DHEA on insulin
specific binding activity and insulin receptor autophosphorylation
activity, which is the first insulin signaling step, we examined
[125I]insulin specific
binding to the receptor of adipocytes and 95-kDa autophosphorylation of
the insulin receptor
-subunit. As shown in Fig. 3,
there was no significant difference in
[125I]insulin specific
binding to the receptor of adipocytes (control cells bound 2.9 ± 0.2% of
[125I]insulin/tube,
cells treated with DHEA bound 2.8 ± 0.3% of
[125I]insulin/tube;
Fig. 3A), and there was also no
significant difference in 95-kDa insulin receptor autophosphorylation
(Fig. 3B) between treatment with and
without 1 µM DHEA. Densitometric analysis indicated that there was no
difference in insulin-induced 95-kDa autophosphorylation between
DHEA-treated and untreated adipocytes (925 ± 37 vs. 918 ± 46%;
Fig. 3C).

View larger version (13K):
[in this window]
[in a new window]

View larger version (43K):
[in this window]
[in a new window]

View larger version (49K):
[in this window]
[in a new window]
|
Fig. 3.
Effect of DHEA on insulin specific binding. Isolated adipocytes were
suspended in Krebs-Ringer phosphate buffer and incubated for 60 min
with or without (control) 1 µM DHEA and then with
[125I]insulin (2,000 Ci/mmol) and unlabeled insulin (1-1,000 nM) in plastic tubes at
25°C in a shaking water bath for 60 min. Cells were then removed
using the addition of silicone oil, and radioactivity was determined.
Data are plotted by Scatchard analysis as the mean of triplicate
determinations (A).
Immunoprecipitates with antibody against insulin receptor were
separated by SDS-PAGE. Immunoblot analysis of 100 nM insulin-induced
95-kDa tyrosine autophosphorylation of insulin receptor was performed
in DHEA-treated cells for 60 min and control cells. Representative
experiment is shown (B), and data by
densitometric analysis are plotted as the means ± SE of 3 separate
experiments (C). B/F, bound-to-free
ratio; C, control.
|
|
DHEA-induced PI 3-kinase activation.
Moreover, to clarify the effect of DHEA on PI 3-kinase, which binds to
tyrosine-phosphorylated IRS-1 via the SH2 domain of PI 3-kinase,
downstream of the insulin signaling pathway, we examined whether DHEA
stimulates PI 3-kinase in rat adipocytes. When adipocytes were
incubated with 1 µM DHEA, unexpectedly, enzyme activity (Fig.
4A) and the p85
subunit of PI 3-kinase (Fig. 4B),
after immunoprecipitation with anti-phosphotyrosine antibody, increased
for 5 and 10 min, similar to 100 nM insulin-induced PI 3-kinase
activation (Fig. 4A,
left). Densitometric data are also
shown in Fig. 4C.

View larger version (8K):
[in this window]
[in a new window]

View larger version (14K):
[in this window]
[in a new window]

View larger version (16K):
[in this window]
[in a new window]
|
Fig. 4.
DHEA-induced phosphatidylinositol (PI) 3-kinase activation. Isolated
adipocytes were incubated with or without (control: 0 min) 1 µM DHEA
for 1, 5, and 10 min, homogenized, and immunoprecipitated with
phosphotyrosine antibody. Each PI 3-kinase activity
(A) and p85 subunit of PI 3-kinase
immunoreactivity (B) were measured
as shown in MATERIALS AND METHODS. Adipocytes were
incubated with 10 8 to
10 5 M TPA or dexamethasone
for 5 min, homogenized, and immunoprecipitated with phosphotyrosine
antibody. Immunoprecipitate was immunoblotted with antibody against p85
-subunit of PI 3-kinase. Densitometric data
(C) are plotted as the means ± SE of 4 separate experiments.
* P < 0.01 by ANOVA with
Fisher's PLSD test.
|
|
Effect of DHEA on diacylglycerol
production. To resolve the DHEA-stimulated glucose
uptake mechanism, we focused on diacylglycerol-PKC signaling. When
adipocytes were labeled with
[3H]glycerol, 1 µM
DHEA alone stimulated diacylglycerol production to 150% from basal
levels (Fig. 5A).
Insulin-stimulated incorporation of
[3H]glycerol in
[3H]diacylglycerol for
2 and 20 min was markedly enhanced almost twofold by pretreatment with
1 µM DHEA for 60 min (Fig. 5B).

View larger version (12K):
[in this window]
[in a new window]

View larger version (16K):
[in this window]
[in a new window]
|
Fig. 5.
Effect of DHEA on insulin-induced diacylglycerol production. Isolated
adipocytes were prelabeled for 15 min with
[3H]glycerol. Labeled
adipocytes were stimulated with 1 µM DHEA alone for 2, 5, 10, and 20 min (A). After pretreatment with 1 µM DHEA for 60 min, labeled adipocytes were stimulated with 10 nM
insulin (B). Reaction was terminated
with methanol as shown in MATERIALS AND METHODS. Data are
plotted as the means ± SE of 4 separate experiments.
* P < 0.01 by ANOVA with
Fisher's PLSD test.
|
|
DHEA-induced PKC translocation. DHEA
stimulated both PI 3-kinase activation and diacylglycerol production as
shown in Figs. 4 and 5. We examined whether DHEA activates PKC-
,
which is thought to be downstream of PI 3-kinase (23), and PKC-
.
Isolated adipocytes were treated with or without (control: 0 min) 1 µM DHEA for 5, 10, and 20 min. Cytosolic PKC-
and -
immunoreactivities gradually decreased, and membrane-associated PKC-
and -
immunoreactivity was inversely increased (Fig.
6). Dexamethasone induced PKC-
and -
translocations from the cytosol to the membrane, as shown previously
(18).

View larger version (49K):
[in this window]
[in a new window]
|
Fig. 6.
DHEA-induced protein kinase C (PKC)- and PKC- translocations.
Isolated adipocytes were stimulated with or without (control) 1 µM
DHEA for 5, 10, and 20 min. Cells were homogenized and centrifuged to
obtain cytosol and membrane fractions. Each cytosolic and
membrane-associated protein (40 µg) was subjected to SDS-PAGE,
transferred to the nitrocellulose membrane, and immunoblotted with
PKC- and PKC- antibodies using enhanced chemiluminescence or
alkaline phosphatase procedure.
|
|
[3H]DHEA binding to PKC
purified from rat brain.
When 10 ng purified conventional PKC (PKC-
, -
, and -
) activity
was measured using histone III-s as the substrate in the presence of
0.001-1 mM Ca2+,
phosphatidylserine, and diolein, the most effective
Ca2+ concentration was 0.5 mM. On
the other hand, in the presence of 40 µg/ml phosphatidylserine,
protein kinase activity was most activated by the addition of 1 µM
DHEA in 0.5 mM Ca2+ (Fig.
7).
Immunoprecipitate with anti-PKC-
antibody was also activated in the
presence of phosphatidylserine/diolein (data not shown). In addition to
the above results, we examined
[3H]DHEA specific
binding to conventional PKC and atypical PKC. [3H]DHEA specific
binding to conventional PKC was found to be as shown in Fig.
8. Glucocorticoid receptor antagonist RU-38486 did not
affect [3H]DHEA
binding to PKC in vitro.
[3H]DHEA specific
binding to PKC-
was also observed to a similar extent (data not
shown).

View larger version (14K):
[in this window]
[in a new window]
|
Fig. 7.
Ca2+ dependence of the ability of
DHEA and diolein (DL) with phosphatidylserine (PS) to stimulate
purified PKC (BIOML Research Laboratories). Protein kinase activity was
assayed by histone phosphorylation activity in the presence of 1 µM
DHEA or 0.4 µg/ml DL with 40 µg/ml PS at various concentrations of
Ca2+. Data are plotted as the
means of 3 separate experiments. cpm, Counts/min.
|
|

View larger version (10K):
[in this window]
[in a new window]
|
Fig. 8.
[3H]DHEA binding to
PKC purified from rat brain. Purified PKC from rat brain (specific
activity 1,530 nmol/mg; BIOML Research Laboratories) containing
PKC- , - , and - ,
[3H]DHEA, and
1,000-fold cold DHEA were incubated for 24 h at 4°C in the presence
or absence of 0.5 mM Ca2+. Total
binding, nonspecific binding, total binding in the presence of
RU-38486, and nonspecific binding in the presence of RU-38486 are
abbreviated as TB, NB, RU/TB, and RU/NB, respectively.
|
|
Effect of DHEA treatment in vivo on insulin- or
TPA-induced glucose uptake in adipocytes of GK/Wistar and OLETF/LETO
rats. At 10 wk of age, 10 nM insulin- or 1 µM
TPA-induced 2-[3H]DG
uptake in adipocytes of GK rats decreased by only 20% compared with
Wistar rats. There were no significant differences in body weight
(DHEA-treated vs. untreated Wistar rats, 270 ± 7 vs. 285 ± 3 g;
DHEA-treated vs. untreated GK rats, 252 ± 25 vs. 211 ± 11 g),
plasma glucose level (DHEA-treated vs. untreated Wistar rats, 128 ± 14 vs. 130 ± 15 mg/dl; DHEA-treated vs. untreated GK rats, 211 ± 33 vs. 234 ± 14 mg/dl), or plasma insulin level (DHEA-treated
vs. untreated Wistar rats, 83 ± 16 vs. 84 ± 18 pM; DHEA-treated
vs. untreated GK rats, 79 ± 11 vs. 78 ± 12 pM) in Wistar and GK
rats after in vivo treatment with 0.4% DHEA. In the 2-wk treatment
with 0.4% DHEA in vivo, 10 nM insulin- or 1 µM TPA-induced
2[3H]DG uptake was
significantly increased by 50-100%, but basal 2-[3H]DG uptake was
not increased compared with untreated GK and Wistar rats at 10 wk of
age. However, DHEA-induced
2-[3H]DG uptakes in
adipocytes of DHEA-treated GK and Wistar rats were not significantly
increased compared with those of untreated GK and Wistar rats (Fig.
9A).
It should be noted that glucose uptake in adipocytes of GK rats was not
markedly different from that in Wistar control rats. Therefore, we
selected another NIDDM animal model, the OLETF rat, and examined the
effect of in vivo DHEA treatment on insulin-, TPA-, or DHEA-induced
2-[3H]DG uptake. At 10 wk of age, 10 nM insulin- and 1 µM TPA-induced 2-[3H]DG uptake in
adipocytes of OLETF rats decreased by 45 and 40%, respectively, when
compared with LETO rats (control). In the 2-wk treatment with 0.4%
DHEA in vivo, there were no significant differences in body weight
(DHEA-treated vs. untreated LETO rats, 386 ± 41 vs. 420 ± 45 g;
DHEA-treated vs. untreated OLETF, 454 ± 55 vs. 517 ± 58 g) or
plasma insulin level (DHEA-treated vs. untreated LETO rats, 182 ± 43 vs. 185 ± 42 pM; DHEA-treated vs. untreated OLETF rats, 257 ± 38 vs. 252 ± 37 pM), but there was a significant difference
in plasma glucose of OLETF rats (DHEA-treated vs. untreated LETO rats,
112 ± 25 vs. 124 ± 20 mg/dl; DHEA-treated vs. untreated OLETF
rats, 144 ± 19 vs. 201 ± 21 mg/dl;
P < 0.05 by ANOVA with Fisher's
PLSD). After in vivo treatment with DHEA, insulin and TPA-induced
glucose uptakes were significantly increased by 50-100% when
compared with untreated OLETF and LETO rats, but there was no increase
in DHEA-induced glucose uptake (Fig.
9B). These results suggested that DHEA markedly increased glucose transport activity in
not only Wistar and LETO rats, but also GK and OLETF rats.

View larger version (21K):
[in this window]
[in a new window]

View larger version (17K):
[in this window]
[in a new window]
|
Fig. 9.
Effect of in vivo DHEA treatment on insulin- or TPA-stimulated
2-[3H]DG uptake in
adipocytes of Goto-Kakizaki (GK)/Wistar and Otsuka Long Evans fatty
(OLETF)/Long Evans Tokushima (LETO) rats at 10 and 18 wk of age,
respectively. After treatment with 0.4% DHEA in vivo for 2 wk,
isolated adipocytes were obtained from epididymal fat pads in GK
(A) and OLETF
(B) rats. Insulin (10 nM)- and 1 µM TPA-stimulated
2-[3H]DG uptake was
measured as shown in MATERIALS AND METHODS in adipocytes of
GK and OLETF rats and Wistar and LETO rats at 10 wk of age as controls
with or without treatment with DHEA for 2 wk. Data are plotted as means ± SE of 5 separate experiments.
* P < 0.01 vs. without DHEA
treatment by ANOVA with Fisher's PLSD test.
|
|
Effect of DHEA treatment in vivo on insulin- or
TPA-induced glucose uptake in soleus muscles of OLETF/LETO
rats. As shown in Fig. 10, basal and
100 nM insulin- or 1 µM TPA-stimulated
2-[3H]DG uptake in
soleus muscles of OLETF rats decreased compared with LETO rats
(P < 0.01 by ANOVA with Fisher's
PLSD). However, in the 2-wk treatment with 0.4% DHEA, basal and 100 nM
insulin- or 1 µM TPA-stimulated
2-[3H]DG uptake values
in soleus muscles of DHEA-treated OLETF/LETO rats were not
significantly different from those in untreated OLETF/LETO rats.

View larger version (21K):
[in this window]
[in a new window]
|
Fig. 10.
Effect of in vivo DHEA treatment on insulin- or TPA-stimulated
2-[3H]DG uptake in
adipocytes of OLETF/LETO rats at 18 wk of age. Soleus muscles in
DHEA-treated and untreated OLETF/LETO rats were incubated in 25 ml
Krebs-Ringer bicarbonate buffer for 30 min (Cont) and stimulated with
100 nM insulin (INS) or 1 µM TPA for 30 min;
2-[3H]DG,
L-[1-14C]glucose,
and 0.1 mM 2-DG were added, and the incubation was continued for 10 min. After incubation, tissue was removed, rinsed, homogenized in 5%
trichloroacetic acid, and counted simultaneously for
3H and
14C. Data are plotted as means ± SE of 3 separate experiments.
* P < 0.01, 2-[3H]DG uptake in
OLETF vs. LETO rats by ANOVA with Fisher's PLSD test.
|
|
 |
DISCUSSION |
The present findings indicate that DHEA alone stimulates glucose uptake
through diacylglycerol-PKC and PI 3-kinase signaling pathways. It has
been reported that the glucose uptake mechanism is mediated by an
IRS-1-PI 3-kinase pathway (30) and a diacylglycerol-PKC pathway (15).
Recently, the PI 3-kinase inhibitor wortmannin was shown to decrease
insulin-induced glucose uptake in rat adipocytes (25). Therefore, PI
3-kinase plays a pivotal role in the regulation of insulin. On the
other hand, insulin and phorbol esters increase membrane-associated PKC
via increases in diacylglycerol probably via phospholipase C and
phospholipase D activation (31). Moreover, atypical PKC-
, which is a
diacylglycerol-insensitive PKC, operates downstream of PI 3-kinase
(23). In addition to the PI 3-kinase-PKC-
pathway, PKC-
may be a
candidate for insulin-induced glucose transport in insulin-sensitive
tissue (33). On the basis of insulin signaling, we have found that DHEA
alone provokes diacylglycerol production by phospholipase C or
phospholipase D activation in rat adipocytes. These results suggest the
following mechanisms. First, DHEA binds to a specific membrane
receptor. Second, DHEA binds to PKC, leading to subsequent stimulation
of phospholipase D. The first suggestion has already been reported as
follows (22, 24). Meikle et al. (22) have reported that a high-affinity DHEA specific binding site can be found in the cytosol and nuclear fraction in mouse T lymphocytes. The second suggestion is based on the
time course of DHEA-induced diacylglycerol production as shown in Fig.
5. DHEA binds to conventional PKC and atypical PKC-
such as
dexamethasone, as previously reported (17), and activates conventional
PKC in the presence of Ca2+ and
atypical PKC-
in vivo and in vitro as shown in Figs. 6-8. Finally, these diacylglycerol-sensitive PKC may possibly act as a
phospholipase D activator as previously described (13, 32).
Conversely, DHEA stimulates PI 3-kinase through binding to an unknown
tyrosine phosphorylated protein unlike insulin as shown in Fig. 4. It
should be reasonable to stimulate glucose transport via activation of
PKC-
by DHEA-induced PI 3-kinase activation. In contrast,
dexamethasone does not stimulate glucose uptake, probably due to
inhibition of glucose transporter translocation. In fact, a previous
report has indicated that dexamethasone suppresses GLUT-4 translocation
in rat adipocytes (4). As shown in Fig. 9, in vivo treatment with DHEA
results in an increase in insulin-stimulated glucose uptake. The plasma
DHEA concentration reaches
10
8 to
10
7 M in 0.4% DHEA-treated
rats as shown in Table 1. This concentration of DHEA significantly
enhances insulin- or TPA-stimulated
2-[3H]DG uptake as
indicated in Fig. 2. Therefore, DHEA actually decreases insulin
resistance in GK and OLETF rats, NIDDM animal models, without affecting
body weight, glucose, or insulin levels. However, we have to consider
that there is much less DHEA in rodents than in humans (3). Therefore,
it is possible that the effect of in vivo treatment with DHEA may alter
insulin resistance dramatically in OLETF rats. On the other hand, it
should be noted that insulin- and TPA-induced glucose uptake in soleus
muscles of OLETF/LETO rats is not altered by in vivo DHEA treatment for
2 wk. This result may be due to a short period of in vivo DHEA
treatment and a weak effect of DHEA on skeletal muscle. Accordingly, it
is suggested that DHEA mainly affects adipose tissue as previously
reported (10). Further studies will be required on the effect of in
vivo treatment with DHEA on diabetic patients.
In conclusion, first, DHEA alone stimulates glucose uptake in rat
adipocytes. Second, DHEA stimulates the production of diacylglycerol. Third, pretreatment with DHEA enhances insulin-induced diacylglycerol production. Fourth, DHEA stimulates PI 3-kinase activity, conventional PKC, and atypical PKC. Fifth, specific DHEA binding activity to PKC can
be found. Sixth, histone phosphorylation activity increases with the
addition of DHEA in the presence and absence of
Ca2+. Seventh, treatment with
0.4% DHEA in vivo for 2 wk in GK and OLETF rats results in a
significant increase in insulin- and TPA-induced 2-[3H]DG uptake in
adipocytes. Finally, DHEA may be useful in overcoming insulin
resistance in NIDDM.
 |
FOOTNOTES |
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. §1734 solely to indicate this fact.
Address for reprint requests: T. Ishizuka, The Third Dept. of
Internal Medicine, Gifu Univ. School of Medicine, Tsukasamachi 40, Gifu
500, Japan.
Received 27 April 1998; accepted in final form 8 September 1998.
 |
REFERENCES |
1.
Amatruda, J. M.,
J. N. Livingston,
and
D. H. Lockwood.
Cellular mechanisms in selected states of insulin resistance: human obesity, glucocorticoid excess, and chronic renal failure.
Diabetes Metab. Rev.
1:
293-317,
1985[Medline].
2.
Barrett-Conner, E.,
K.-T. Khaw,
and
S. S. C. Yen.
A prospective study of dehydroepiandrosterone sulfate, mortality, and cardiovascular diseases.
N. Engl. J. Med.
315:
1519-1524,
1986[Abstract].
3.
Baulieu, E. E.
Dehydroepiandrosterone (DHEA): a fountain of youth?
J. Clin. Endocrinol. Metab.
81:
3147-3151,
1997[Medline].
4.
Carter-Su, C.,
and
K. Okamoto.
Effect for insulin and glucocorticoids on glucose transporters in rat adipocytes.
Am. J. Physiol.
252 (Endocrinol. Metab. 15):
E441-E453,
1987[Abstract/Free Full Text].
5.
Coleman, D. L.,
E. H. Leiter,
and
R. W. Schwizer.
Therapeutic effects of dehydroepiandrosterone (DHEA) in diabetic mice.
Diabetes
31:
830-833,
1982[Abstract].
6.
Farese, R. V.,
T. S. Konda,
J. S. Davis,
M. L. Standaert,
R. J. Pollet,
and
D. R. Cooper.
Insulin rapidly increases diacylglycerol by activating de novo phosphatidic acid synthesis.
Science
236:
596-589,
1990.
7.
Garvey, W. T.,
T. P. Huecksteadt,
R. Monzon,
and
S. Marshall.
Dexamethasone regulates the glucose transport system in primary culture adipocytes: different mechanisms of insulin resistance after acute and chronic exposure.
Endocrinology
124:
2063-2073,
1989[Abstract].
8.
Goto, Y.,
and
M. Kakizaki.
The spontaneous-diabetic rat: a model of non-insulin dependent diabetes mellitus.
Proc. Jpn. Acad.
57:
381-384,
1981.
9.
Goto, Y.,
M. Kakizaki,
and
N. Masaki.
Spontaneous diabetes produced by selective breeding of normal Wistar rats.
Proc. Jpn. Acad.
51:
80-85,
1975.
10.
Han, D.-H.,
P. A. Hansen,
M. M. Chen,
and
J. O. Holloszy.
DHEA treatment reduces fat accumulation and protects against insulin resistance in male rats.
J. Gerontol.
53:
19-24,
1998.
11.
Hayashi, H.,
S. Kamohara,
Y. Nishioka,
F. Kanai,
N. Miyake,
Y. Fukui,
F. Shibasaki,
T. Takenawa,
and
Y. Ebina.
Insulin treatment stimulates the tyrosine phosphorylation of the
type 85-kDa subunit of phosphatidylinositol 3-kinase in vivo.
J. Biol. Chem.
267:
22575-22580,
1992[Abstract/Free Full Text].
12.
Hoffman, J. M.,
T. Ishizuka,
and
R. V. Farese.
Interrelated effects of insulin and glucose on diacylglycerol-protein kinase-C signaling in rat adipocytes and solei muscle in vitro and in vivo in diabetic rats.
Endocrinology
128:
2937-2948,
1991[Abstract].
13.
Huang, C.,
and
M. C. Cabot.
Phorbol diesters stimulate the accumulation of phosphatidate, phosphatidylethanol, and diacylglycerol in three cell types
evidence for the indirect formation of phosphatidylcholine.
J. Biol. Chem.
265:
14858-14863,
1990[Abstract/Free Full Text].
14.
Ishizuka, T.,
D. R. Cooper,
and
R. V. Farese.
Insulin stimulates the translocation of protein kinase C in rat adipocytes.
FEBS Lett.
257:
337-340,
1989[Medline].
15.
Ishizuka, T.,
D. R. Cooper,
H. Hernandez,
D. Buckley,
M. Standaert,
and
R. V. Farese.
Effect of insulin on diacylglycerol-protein kinase C signaling in rat diaphragm and soleus muscles and relationship to glucose transport.
Diabetes
39:
181-190,
1990[Abstract].
16.
Ishizuka, T.,
A. Miura,
K. Kajita,
K. Yamada,
H. Wada,
S. Itaya,
K. Kanoh,
M. Ishizawa,
M. Kimura,
and
K. Yasuda.
Alterations in insulin-induced postreceptor signaling in adipocytes of the Otsuka Long-Evans Tokushima fatty rat strain.
J. Endocrinol.
156:
1-13,
1998[Abstract/Free Full Text].
17.
Ishizuka, T.,
T. Nagashima,
K. Kajita,
A. Miura,
M. Yamamoto,
S. Itaya,
Y. Kanoh,
M. Ishizawa,
H. Murase,
and
K. Yasuda.
Effect of glucocorticoid receptor antagonist RU 38486 on acute glucocorticoid-induced insulin resistance in rat adipocytes.
Metabolism
46:
996-1002,
1997.
18.
Ishizuka, T.,
M. Yamamoto,
T. Nagashima,
K. Kajita,
O. Taniguchi,
K. Yasuda,
and
K. Miura.
Effect of dexamethasone and prednisolone on insulin-induced activation of protein kinase C in rat adipocytes and soleus muscles.
Metabolism
44:
298-306,
1995[Medline].
19.
Kanai, F.,
K. Ito,
M. Todaka,
H. Hayashi,
S. Kamohara,
K. Ishii,
T. Okada,
O. Hazeki,
M. Ui,
and
Y. Ebina.
Insulin-stimulated GLUT4 translocation is relevant to the phosphorylation of IRS-1 and the activity of PI 3-kinase.
Biochem. Biophys. Res. Commun.
195:
762-768,
1993[Medline].
20.
Karasik, A.,
and
C. R. Kahn.
Dexamethasone-induced changes in phosphorylation of the insulin and epidermal growth factor receptors and their substrates in intact rat hepatocytes.
Endocrinology
123:
2214-2222,
1988[Abstract].
21.
Kawano, K.,
T. Hirashima,
S. Mori,
Y. Saitoh,
M. Kurosumi,
and
T Natori.
Spontaneous long-term hyperglycemic rat with diabetic complications. Otsuka Long-Evans Tokushima fatty strain.
Diabetes
41:
1422-1428,
1992[Abstract].
22.
Meikle, A. W.,
R. W. Dorchuck,
B. A. Araneo,
J. D. Stringham,
T. G. Evans,
S. L. Spruance,
and
R. A. Daynes.
The presence of a dehydroepiandrosterone-specific receptor binding complex in mutant T cells.
J. Steroid Biochem. Mol. Biol.
42:
293-304,
1992[Medline].
23.
Nakanishi, H.,
K. A. Brewer,
and
J. H. Exton.
Activation of the
isozyme of protein kinase C by phosphatidylinositol 3,4,5-trisphosphate.
J. Biol. Chem.
268:
13-16,
1993[Abstract/Free Full Text].
24.
Okabe, T.,
M. Haji,
R. Takayanagi,
M. Adachi,
K. Iwasaki,
F. Kurimoto,
T. Watanabe,
and
H. Nawata.
Up-regulation of high-affinity dehydroepiandrosterone binding activity by dehydroepiandrosterone in activated human T lymphocytes.
J. Clin. Endocrinol. Metab.
80:
2993-2996,
1995[Abstract].
25.
Okada, T.,
Y. Kawano,
T. Sakakibara,
O. Hazeki,
and
M. Ui.
Essential role of phosphatidylinositol 3-kinase in insulin-induced glucose transport and antilipolysis in rat adipocytes.
J. Biol. Chem.
269:
3568-3573,
1994[Abstract/Free Full Text].
26.
Olefsky, J. M.
Mechanism of the obesity of insulin to activate the glucose-transport system in rat adipocytes.
Biochem. J.
172:
127-145,
1978.
27.
Olefsky, J. M.,
J. Johnson,
F. Liu,
P. Jen,
and
G. M. Reaven.
The effects of acute and chronic dexamethasone administration on insulin binding to isolated rat hepatocytes and adipocytes.
Metabolism
24:
517-527,
1975[Medline].
28.
Orentreich, N.,
J. L. Brind,
J. H. Vogelman,
R. Ahdres,
and
H. Baldwin.
Long-term longitudinal measurements of plasma dehydroepiandrosterone sulfate in normal men.
J. Clin. Endocrinol. Metab.
75:
1002-1004,
1992[Abstract].
29.
Rodbell, M.
Metabolism of isolated fat cells.
J. Biol. Chem.
239:
375-380,
1964[Free Full Text].
30.
Ruderman, N. B.,
R. Kappeller,
M. F. White,
and
L. C. Cantley.
Activation of phosphatidylinositol 3-kinase by insulin.
Proc. Natl. Acad. Sci. USA
87:
1411-1415,
1990[Abstract].
31.
Standaert, M. L.,
A. Avignon,
K. Yamada,
G. Bandyopadhyay,
and
R. V. Farese.
The phosphatidylinositol 3-kinase inhibits insulin-induced activation of phosphatidylcholine hydrolysis and associated protein kinase C translocation in rat adipocytes.
Biochem. J.
313:
1039-1046,
1996[Medline].
32.
Standaert, M. L.,
G. Bandyopadhyay,
X. Zhou,
L. Gallowag,
and
R. V. Farese.
Insulin stimulates phospholipase D-dependent phosphatidylcholine hydrolysis, Rho translocation, de novo phospholipid synthesis, and diacylglycerol/protein kinase C signaling in L6 myotubes.
Endocrinology
137:
3014-3020,
1996[Abstract].
33.
Standaert, M. L.,
L. Galloway,
P. Karnam,
G. Bandyopadhyay,
J. Moscat,
and
R. V. Farese.
Protein kinase C-
as a downstream effector of phosphatidylinositol 3-kinase during insulin stimulation in rat adipocytes. Potential role in glucose transport.
J. Biol. Chem.
272:
30075-30082,
1997[Abstract/Free Full Text].
34.
Sjogren, J.,
M. Weck,
A. Nilsson,
M. Ottoson,
and
P. Bjorntorp.
Glucocorticoid hormone binding to rat adipocytes.
Biochim. Biophys. Acta
1224:
17-21,
1994[Medline].
35.
Trugia, J. A.,
G. R. Hayes,
and
D. H. Lockwood.
Intact adipocyte insulin receptor and in vitro tyrosine kinase activity in animal models of insulin resistance.
Diabetes
37:
147-153,
1988[Abstract].
Am J Physiol Endocrinol Metab 276(1):E196-E204
0002-9513/99 $5.00
Copyright © 1999 the American Physiological Society