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INTRODUCTION |
GTP
S,1 like insulin,
has been found to activate GLUT4 translocation and/or glucose transport
in rat adipocytes (1, 2) and 3T3/L1 adipocytes (3). The mechanisms
whereby GTP
S and insulin activate GLUT4 translocation and glucose
transport, however, are unclear. In 3T3/L1 cells, unlike insulin,
GTP
S was not found to activate cytosolic phosphatidylinositol (PI)
3-kinase (3), and this suggested that (a) PI 3-kinase was
not essential for the activation of glucose transport and
(b) GTP
S may operate through different or more distal
processes. In keeping with the latter possibility, small G-proteins in
the Rab group are present in GLUT4 vesicles, appear to translocate or
mobilize in response to insulin stimulation (4), and could act as
direct mediators for GTP
S stimulation of glucose transport;
accordingly, GTP
S-stimulated glucose transport is only partly
inhibited by the PI 3-kinase inhibitor, wortmannin, in 3T3/L1
adipocytes (5), and GTP
S therefore appears to act, at least
partially, independently of PI 3-kinase in 3T3/L1 cells. On the other
hand, we have recently found that the small G-protein, RhoA, is
activated by insulin in rat adipocytes, and, based upon
Clostridium botulinum C3 transferase sensitivity, Rho
appears to be required for insulin-stimulated glucose transport in
these cells (6). Further, it seems clear that Rho is directly activated
by GTP
S in rat adipocytes, since it was found that the addition of
GTP
S to rat adipocyte homogenates stimulates the translocation of
Rho to plasma membranes and Rho-dependent activation of
phospholipase D (6). Of further note, it has been reported that GTP-Rho
directly activates PI 3-kinase in some (7), but not all (8, 9),
cell-free systems. Presently, we examined the possibility that GTP
S
activates PI 3-kinase via Rho in rat adipocytes. We also examined the
role of Rho, PI 3-kinase, and protein kinases that are known to be
downstream of Rho and PI 3-kinase (e.g. PKN and protein
kinase C-
(PKC-
)) in the activation of glucose transport during
treatment of rat adipocytes with GTP
S or insulin. Our findings
suggested that (a) both GTP
S and insulin, albeit by
different mechanisms, activate Rho, PI 3-kinase, and PKN; and
(b) each of these factors may be required for, and may contribute to, the activation of GLUT4 translocation and glucose transport in rat adipocytes.
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EXPERIMENTAL PROCEDURES |
Rat Adipocytes (Preparation, Incubation, and
Electroporation)--
Rat adipocytes were prepared by collagenase
digestion of epididymal fat pads obtained from male Sprague-Dawley rats
weighing approximately 200-250 g, as described previously (6). The
adipocytes were suspended in glucose-free Krebs Ringer phosphate (KRP)
buffer containing 1% bovine serum albumin for acute incubations, or in Dulbecco's modified Eagle's medium (DMEM; Life Technologies, Inc.) containing 1% bovine serum albumin for overnight incubations. GTP
S
(Sigma or ICN) and C. botulinum C3 transferase (List) were introduced into adipocytes (in 25 or 50% suspensions, cell
volume/total volume) by electroporation (Bio-Rad Gene Pulsar; 350 V and
960 microfarads with a time constant of 12 ms) in either an
intracellular buffer (118 mM KCl, 5 mM NaCl,
0.38 mM CaCl2, 1 mM EGTA, 1.2 mM Mg2SO4, 1.2 mM
KH2PO4, 3 mM sodium pyruyate, 25 mM HEPES, and 20 mg/ml bovine serum albumin) or in DMEM,
respectively, as described in the text.
Glucose Transport Studies in Rat Adipocytes--
Glucose
transport was measured in adipocytes suspended (6%, cell volume/total
volume) in glucose-free KRP buffer as described by Karnam et
al. (6). Where indicated, inhibitor (wortmannin, Sigma; LY294002,
Biomol; RO 31-8220, Alexis) was added to the incubation 15 min prior to
agonist addition. The cells were then treated with vehicle alone
(control), insulin (Elanco), or the indicated concentrations of
GTP
S. In the case of GTP
S treatment, immediately following
electroporation in intracellular buffer (see above), the cells were
diluted with glucose-free KRP buffer containing indicated inhibitor
concentrations. After 30 min of treatment with vehicle (controls), 10 nM insulin, or 0-500 µM GTP
S, the uptake
of 2-[3H]deoxyglucose (2-DOG; 0.1 mM; NEN
Life Science Products) was measured over a 1-min period as described
(6). In these assays, it should be noted that cytochalasin B blank
values, which reflect trapping of medium or nonspecific uptake, were
relatively small (approximately 10% of stimulated values) and were not
influenced significantly by electroporation or overnight incubation. In
cells that were assayed directly without electroporation,
insulin-induced increases in the uptake of 2-DOG generally ranged from
4- to 10-fold. In cells that were electroporated and then immediately
assayed, there usually was a small, but variable, increase in basal
2-DOG uptake, with little or no change in maximal insulin-stimulated values; consequently, the relative effect of insulin on 2-DOG uptake
generally tended to be slightly less, i.e. approximately 2-3-fold, in electroporated adipocytes (this is illustrated in Fig.
8). Overnight incubation of adipocytes also increased basal transport
activity, and the relative insulin effect was similarly diminished to
approximately 2-3-fold in these cells. However, the effect of combined
electroporation and overnight incubation on 2-DOG uptake was not
significantly different from that of either electroporation or
overnight incubation alone, since insulin effects on 2-DOG uptake were
also approximately 2-3-fold in these cells.
GLUT4 Translocation Studies in Transiently Transfected Rat
Adipocytes--
Effects of transiently transfected Rho and PKN on
GLUT4 translocation were measured in rat adipocytes co-transfected with hemagglutinin (HA)-tagged GLUT4, as described by Quon et al.
(10, 11). In brief, adipocytes (as a 50% suspension in DMEM) were electroporated in the presence of eukaryotic expression vector pCIS2
containing cDNA encoding HA-tagged GLUT4 (kindly supplied by Drs.
Michael Quon and Simeon Taylor) and either (a) pCDNA3 (Invitrogen) eukaryotic expression vector alone or pCDNA3
containing cDNA encoding (i) wild-type Rho (kindly supplied by Dr.
David Lambeth), (ii) V14 mutant, constitutive Rho (kindly supplied by Dr. Alan Hall), or (iii) dominant negative Rho (kindly supplied by Dr.
Gary Bokoch); or (b) pTB701 eukaryotic expression vector alone or pTB701 containing cDNA encoding (i) wild-type or (ii) dominant negative, kinase-inactive mutant forms of PKN (prepared by Dr.
Ono). After overnight incubation to allow time for expression of
cDNA inserts (see Refs. 10 and 11 and verified by expression of
HA-tagged forms of GLUT4, Rho, and PKN), the cells were equilibrated in
glucose-free KRP buffer and treated with or without 10 nM
insulin for 30 min, prior to the addition of 2 mM KCN and
measurement of cell count and cell-surface, HA-tagged GLUT4 as
described (10, 11); for the latter purpose, the primary anti-HA mouse
monoclonal antibody was obtained from Babco, and
125I-labeled second antibody (sheep anti-mouse IGG) was
obtained from Amersham Pharmacia Biotech. Blank values (nonspecific
binding), obtained by incubating cells transfected with vectors alone
(i.e. without the HA-GLUT4 and other inserts), were
subtracted from values observed in cells in which HA-GLUT4 was
expressed. In each experiment, a single batch of adipocytes was used,
so that the level of HA-tagged GLUT4 was identical in each experimental
group, and treatment groups could be directly compared with each other. Absolute values of 125I bound per 106 cells
varied somewhat from experiment to experiment, depending upon the
batches of antibodies used and the level of 125I in the
second antibody; nevertheless, relative changes induced by GTP
S,
insulin, and other treatments were similar in all experiments.
Stable Transfection Studies in 3T3/L1 Cells--
Effects of
stably transfected, wild-type Rho or PKN on glucose transport (2-DOG
uptake) and membrane-associated PI 3-kinase activity were evaluated in
3T3/L1 fibroblasts and/or adipocytes, using transfection methods
described previously (12). Effects of dominant negative forms of Rho
and PKN were also studied in 3T3/L1 fibroblasts, but these forms
inhibited adipogenesis, thus precluding experiments with plasmids
containing these cDNA inserts in 3T3/L1 adipocytes. In these stable
transfection experiments, all cells (untransfected controls or cells
transfected with pCDNA3 vector alone or pCDNA3 containing
cDNAs encoding Rho (supplied by Dr. David Lambeth) or PKN (supplied
by Dr. Peter Parker)) were grown, differentiated, and assayed
simultaneously. As in previous transfection studies (12), all reported
clones (selected by G418 resistance) were shown to contain
immunoreactive GLUT4 and GLUT1 levels that were indistinguishable from
those observed in untransfected control cells; consequently,
alterations in glucose transport in Rho or PKN-transfected cells could
not be ascribed to changes in levels of GLUT4 or GLUT1. In some
experiments, a tetracycline-inducible system
(CLONTECH, Tet-On kit) was used to turn on the
expression of transfected, wild-type Rho in 3T3/L1 fibroblasts 72 h prior to measurement of 2-DOG uptake (described more fully in text,
see Fig. 7).
Western Analyses--
Immunoreactive Rho, GLUT4, GLUT1, PI
3-kinase, and HA-tagged moietes were blotted using methods described
previously (6, 12, 13).
Enzyme Assays for PI 3-Kinase, PKC-
, and PKN--
PI 3-kinase
enzyme activity was measured either in total membranes or in IRS-1
immunoprecipitates of rat adipocytes or 3T3/L1 adipocytes, as described
previously (13). Immunoprecipitable PKC-
enzyme activity was
measured as described (12, 14). To measure immunoprecipitable PKN
enzyme activity, cells were lysed in buffer containing 150 mM NaCl, 250 mM sucrose, 20 mM Tris/HCl (pH, 7.5), 1.2 mM EGTA; 1 mM EDTA, 5 mM MgCl2, 20 mM
-mercaptoethanol, 25 mM NaF, 3 mM
Na4P2O7, 10 mM
Na3VO4, 20 µg/ml aprotinin, 20 µg/ml
leupeptin, 1 mM PMSF, 1% Triton X-100, and 2 µM Microcystin-LR (Calbiochem). Rabbit anti-PKN
polyclonal antibody (raised in Dr. Ono's laboratories) was added in
sufficient amounts to quantitatively precipitate PKN from 300 µg of
lysate protein. After overnight incubation at 0-4 °C, the
precipitate was collected on Sepharose A/G beads (Santa Cruz
Laboratories), washed three times with lysis buffer and twice with
assay buffer (50 mM Tris/HCl (pH, 7.5), 10 mM
MgCl2, 1 mM EGTA, 1 mM NaF, 1 mM Na3VO4, 1 mM Na4P2O7, 10 mM
-glycerophosphate, 100 µM phenylmethylsulfonyl fluoride), and then incubated at 30 °C for 6 min in the presence of
40 µM serine-25 PKC-
pseudosubstrate (Life
Technologies) and 50 µM ATP containing 2 µCi of
[
-32P]ATP (NEN Life Science Products). Aliquots of the
reaction mixture were spotted on p81 filter paper, washed in 30%
acetic acid, and counted for 32P. It may be noted that
phosphatidylserine was not present in PKN assays, and, moreover, PKN
activity was not increased by the addition of phosphatidylserine,
Ca2+, or diolein: it may therefore be surmised that PKN
immunoprecipitates were not contaminated with significant amounts of
conventional, novel, or atypical PKCs.
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RESULTS |
Studies of Glucose Transport in Rat Adipocytes--
As shown in
Fig. 1, electroporation of rat adipocytes
in the presence of increasing amounts of GTP
S led to dose-related
increases in 2-DOG uptake. At a concentration of 500 µM,
GTP
S-stimulated increases in 2-DOG uptake were in most cases
slightly less than those of insulin, as measured in cells
electroporated in parallel. We did not attempt to use higher
concentrations of GTP
S, but it may be noted that we probably did not
reach maximal transport rates with 500 µM GTP
S,
electroporation opens membrane pores only fleetingly, and intracellular
GTP
S concentrations were most likely less than those present in the
electroporation buffer. In addition to increasing glucose transport,
500 µM GTP
S provoked increases in the translocation of
HA-tagged GLUT4 to the plasma membrane (cell surface
125I-labeled anti-HA antibody (reflecting the level of
exofacial HA-tagged GLUT4) was 1772 ± 238 (n = 4)
versus 3748 ± 321 (n = 6)
cpm/106 cells (p < 0.005, t
test), control versus GTP
S, respectively); these
increases in HA-GLUT4 translocation were in some experimental groups
similar to those provoked by insulin (viz. approximately 2-fold) or, in some groups (e.g. see below), slightly
less.

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Fig. 1.
Effects of wortmannin (WORT)
(A), LY294002 (B), and C3 transferase
(C3) on GTP S- and insulin-stimulated 2-DOG uptake in rat
adipocytes. A and B, cells were treated with 100 nM wortmannin or 100 µM LY294002 for 15 min,
electroporated in intracellular buffer with or without the indicated
concentrations of GTP S, and then diluted with glucose-free KRP
medium and treated with or without 10 nM insulin as
indicated. After 30 min of treatment with GTP S or insulin, the
uptake of 2-DOG uptake was measured over 1 min. C, cells
were electroporated in DMEM with or without C3 transferase (0.5 µg/ml), and incubated overnight to deplete immunoreactive Rho in C3
transferase-treated cells (see Ref. 6). The cells were then
electroporated a second time in intracellular buffer with or without
500 µM GTP S and then diluted with glucose-free KRP
medium and treated with or without 10 nM insulin, as
indicated. After 30 min of treatment with GTP S or insulin, 2-DOG
uptake was measured over 1 min. Values are mean ± S.E. of four
determinations.
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Since PI 3-kinase is required for insulin effects on glucose transport,
it was of interest to see if inhibitors of PI-3-kinase altered the
effects of GTP
S on 2-DOG uptake. As shown in Fig. 1, A
and B, both 100 nM wortmannin and 100 µM LY294002, (concentrations that fully inhibit PI
3-kinase) fully inhibited GTP
S-stimulated, as well as
insulin-stimulated, 2-DOG uptake. These findings suggested that
GTP
S, like insulin, requires PI 3-kinase for the activation of
glucose transport in rat adipocytes.
We have previously reported that the effects of insulin on glucose
transport are blocked by C. botulinum C3 transferase, which specifically ADP-ribosylates, inhibits, and, after overnight incubation of rat adipocytes, leads to a complete loss of immunoreactive RhoA in
these cells (see Ref. 6). As shown in Fig. 1C, overnight C3
transferase treatment blocked subsequent effects of both GTP
S and
insulin on 2-DOG uptake in rat adipocytes. These results (as well as
results from transfection studies; see below) suggested that Rho is
required for the effects of GTP
S, as well as insulin, on glucose
transport in rat adipocytes.
Studies of PI 3-Kinase Activation--
Since wortmannin and
LY294002 fully blocked the effects of both GTP
S and insulin on 2-DOG
uptake in the rat adipocyte, we questioned whether GTP
S activates PI
3-kinase. As shown in Fig. 2, both
GTP
S and insulin provoked increases in membrane-associated PI
3-kinase activity in rat adipocytes. Interestingly, C3 transferase blocked the activating effect of GTP
S, but not insulin, on
membrane-associated PI 3-kinase (Fig. 2C). Also,
insulin-induced increases in PI 3-kinase immunoreactivity and enzyme
activity that are specifically associated with IRS-1, were not blocked
by C3 transferase treatment (Fig. 3).
These findings suggested that activating effects of insulin on PI
3-kinase, both in total membranes and as specifically activated through
IRS-1 in the rat adipocyte, were not dependent upon Rho. On the other
hand, activating effects of GTP
S on membrane-associated PI 3-kinase
in the rat adipocyte appeared to be fully dependent upon Rho. In
keeping with a role for Rho in PI 3-kinase activation, as described
below, stable overexpression of Rho in 3T3/L1 adipocytes led to
increases in membrane-associated PI 3-kinase activity.

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Fig. 2.
Effects of GTP S, insulin
(INS), and C3 transferase (C3) on
membrane-associated PI 3-kinase activity in rat adipocytes. A and B, cells were electroporated in
intracellular buffer with or without 500 µM GTP S and
then diluted with glucose-free KRP medium and treated with or without
(control, CON) 300 nM insulin as indicated.
After treatment for 15 min with GTP S or insulin, reactions were
stopped by chilling, and membranes were obtained by centrifugation for
60 min at 100,000 × g and assayed for PI 3-kinase
activity. Panel A shows a representative autoradiogram; note
that PI-3-PO4 migrates just below PI-4-PO4.
Panel B shows the mean ± S.E. of four determinations
of PI-3-PO4 labeling. C, cells were
electroporated in DMEM with or without 0.5 µg/ml C3 transferase and
incubated overnight to deplete immunoreactive Rho in C3
transferase-treated cells (see Ref. 6). The cells were then
electroporated a second time in intracellular buffer with or without
500 µM GTP S and then diluted with glucose-free KRP
medium and incubated with or without 300 nM insulin, as
indicated. After a 15-min treatment with GTP S or insulin, membranes
were obtained by centrifugation for 60 min at 100,000 × g and assayed for PI 3-kinase activity. Values are mean ± S.E. of four determinations.
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Fig. 3.
Effects of C3 transferase on insulin
(INS)-stimulated PI 3-kinase in IRS-1 immunoprecipitates
(IP). Adipocytes were electroporated in DMEM with or
without 0.5 µg/ml C3 transferase and incubated overnight to deplete
immunoreactive Rho (see Ref. 6) in C3 transferase-treated cells. The
cells were then equilibrated in glucose-free KRP medium and treated for
15 min with or without 300 nM insulin. After incubation,
IRS-1 was immunoprecipitated from total cell lysates, and precipitates
were examined for p85 immunoreactivity (A and C)
or PI 3-kinase enzyme activity (B and D). Shown
here are immunoblots and autoradiograms that are representative of four
determinations.
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Studies on Effects of RO 31-8220 on PKN Activity and Glucose
Transport--
Considerable evidence suggests that one or more protein
kinases, apparently distal to PI 3-kinase, is (are) required for
insulin stimulation of glucose transport. For example, we have
previously shown that the bisindolemaleimide-type PKC inhibitor, RO
31-8220, inhibits insulin-stimulated glucose transport, without
inhibiting the activation of PI 3-kinase by insulin (15). Recently, we have found (14) that (a) RO 31-8220 inhibits recombinant
PKC-
, -
1, -
2, -
, -
, -
, -
, and -
with
IC50 values of approximately 40, 20, 15, 15, 30, 100, 20 and 1000-4000 nM, respectively, and that (b) of
these PKCs inhibition of insulin-stimulated 2-DOG uptake in intact
adipocytes by RO 31-8220 correlates best with the inhibition of PKC-
(IC50 of approximately 4 µM, with nearly full
inhibition at 20-30 µM). Presently, we found that RO
31-8220 inhibited immunoprecipitated PKN with an IC50 of
approximately 30 nM (Fig. 4).
Although the exact identity of the RO 31-8220-sensitive kinase that is
required for insulin-stimulated glucose transport is not certain, it
was of interest to find that RO 31-8220 inhibited the effects of
GTP
S, as well as insulin, on 2-DOG uptake in intact rat adipocytes
(Fig. 5). However, the concentrations of
RO 31-8220 that were required to inhibit GTP
S effects on 2-DOG
uptake were considerably less (IC50 < 1 µM)
than those required for inhibition of insulin effects (IC50 = 5 µM) on 2-DOG uptake in the rat adipocyte (Fig. 5). It
therefore seems likely that different RO 31-8220-sensitive protein
kinases are required for glucose transport effects of GTP
S and
insulin in the rat adipocyte.

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Fig. 4.
Dose-related effects of RO 31-8220 on PKN
activity. PKN was precipitated from rat adipocyte lysates and
assayed in the absence or presence of increasing concentrations of RO
31-8220, as indicated.
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Fig. 5.
Dose-related effects of RO 31-8220 on
GTP S-stimulated and insulin-stimulated 2-DOG uptake in rat
adipocytes. Adipocytes were incubated in the presence of 0, 0.3, 1, 5, and 20 µM RO 31-8220 for 15 min prior to
electroporation and treatment with 10 nM insulin (open circles) or 500 µM GTP S (solid
circles). Values are means of three determinations.
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Studies on the Activation PKC-
and PKN--
Inasmuch as RO
31-8220 does not inhibit the activity or activation of PI 3-kinase in
the rat adipocyte (15), as alluded to above, it may be surmised that
one or more protein kinases, distinct from PI 3-kinase, is required
during the activation of glucose transport. Accordingly, we questioned
whether GTP
S or insulin activates PKC-
or PKN, i.e.
kinases that appear to be downstream of PI 3-kinase and/or Rho. Whereas
insulin (see Ref. 14 for more detailed studies) activated PKC-
,
GTP
S, if anything, diminished the activity of immunoprecipitable
PKC-
in intact adipocytes (Table I).
In addition, GTP
S, unlike insulin, failed to activate PKB (data not
shown). On the other hand, GTP
S activated immunoprecipitable PKN
mildly (23%), but significantly, in intact adipocytes (Table
II) and more dramatically in adipocyte
homogenates (Table III). (Note that the
in vitro incubation of adipocyte homogenate in low
Mg2+ conditions prior to immunoprecipitation markedly
lowered basal immunoprecipitable PKN activity, and this probably
facilitated the observance of greater relative effects of GTP
S in
the cell-free system.) Similarly, insulin provoked approximately 60%
increases in immunoprecipitable PKN activity throughout a 1-10-min
treatment period in intact rat adipocytes, and these increases were
blocked by C3 transferase, but not by wortmannin (Fig.
6). Thus, in keeping with our previous
report that PI 3-kinase is not required for insulin stimulation of
GTP-loading of Rho (6), PI 3-kinase did not appear to be required for
insulin-induced activation of PKN. Also, in keeping with the
possibility that PKN is downstream of Rho, Rho appeared to be required
for insulin-induced activation of PKN.
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Table I
Effects of GTP S and insulin on immunoprecipitable PKC-
activity in rat adipocytes
All adipocytes were electroporated in intracellular buffer with or
without 500 µM GTP S and then treated with or without
10 nM insulin, as indicated. After a 10-min incubation,
cells were homogenized in ice-cold 250 mM sucrose, 20 mM Tris-HCl (pH 7.5), 1.2 mM EGTA, 20 mM -mercaptoethanol, 1 mM
phenylmethylsulfonyl fluoride, 20 µg/ml leupeptin, 20 µg/ml
aprotinin, 1 mM Na3VO4, 1 mM
Na4P2O7, and 1 mM NaF. Homogenates
were centrifuged at 500 × g for 10 min to remove
nuclei, debris, and the fat cake. After adding 1% Triton X-100, 0.5%
Nonidet, and 150 mM NaCl, PKC- was immunoprecipitated
(polyclonal antibodies from Santa Cruz Biotechnology) and assayed as
described previously (12, 14). Values are the mean ± S.E. of
n determinations (in parentheses). p was
determined by t test.
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Table II
Effects of GTP S on immunoprecipitable PKN activity in intact rat
adipocytes
Adipocytes were electroporated in intracellular buffer in the presence
or absence of 500 µM GTP S. After a 5-min incubation at
37 °C, cells were lysed, and PKN was immunoprecipitated and assayed
(see "Experimental Procedures"). Values are mean ± S.E. of
n determinations (in parentheses). p was
determined by t test.
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Table III
Effects of GTP S on immunoprecipitable PKN activity in rat
adipocyte homogenates
Adipocytes were homogenized in buffer containing 250 mM
sucrose, 20 mM Tris-HCl (pH 7.5), 1.2 mM EGTA,
1 mM EDTA, 20 µg/ml aprotinin, 20 µg/ml leupeptin, 1 mM phenylmethylsulfonyl fluoride, and 20 mM
-mercaptoethanol, 1 mM Na3VO4, 1 mM Na4P2O7, and 1 mM
NaF. Homogenates were centrifuged at 500 × g for 10 min to remove nuclei, debris, and the fat cake, incubated first for 20 min at 37 °C to facilitate the release of endogenous GDP and GTP, and then incubated for 5 min with 10 mM MgCl2, with
or without 20 µM GTP S, as indicated. After the
addition of 1% Triton X-100, 0.5% Nonidet, 150 mM NaCl,
and other substances (see "Experimental Procedures"), PKN was
immunoprecipitated and assayed (see "Experimental Procedures").
Values are the mean ± S.E. of n determinations (in parentheses).
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Fig. 6.
Effects of insulin, C3 transferase, and
wortmannin on immunoprecipitable PKN activity in rat adipocytes.
Where indicated, adipocytes were either incubated directly for 15 min
with or without 100 nM wortmannin (first six groups), or
electroporated with or without 1 µg/ml C3 transferase and then
incubated for 20 h (last two groups) as in Figs. 1C and
3. After these initial treatments, the cells were equilibrated in
glucose-free KRP medium and treated with or without 10 nM
insulin for the indicated times (1, 5, or 10 min), following which PKN
was immunoprecipitated and assayed for enzyme activity as described
under "Experimental Procedures." Shown here are mean ± S.E.
values of n determinations (shown in parentheses). Although not shown separately in the
figure, insulin effects on PKN activity were comparable in
adipocytes that were used directly or placed into primary culture for
20 h. p was determined by t test.
NS, not significant.
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Transfection Studies--
Since our findings with C3 transferase
suggested that Rho is required for (a) GTP
S- and
insulin-induced increases in glucose transport and (b)
GTP
S-induced increases in PI 3-kinase, we questioned whether Rho
itself could provoke increases in PI 3-kinase activity and glucose
transport and/or GLUT4 translocation. To pursue these questions and to
further examine the requirement for Rho in glucose transport, we used
several transfection approaches. First, in both 3T3/L1 fibroblasts and
adipocytes, the stable overexpression of wild-type Rho led to increases
in both immunoreactive Rho and basal and insulin-stimulated glucose
transport (Fig. 7); in addition, stably
transfected Rho provoked a 73 ± 10% increase (mean ± S.E.; n = 7; p < 0.001, paired t
test, Rho transfectants versus untransfected and
vector-transfected controls) in membrane-associated PI 3-kinase enzyme
activity in 3T3/L1 adipocytes. Second, in rat adipocytes, transient
transfection of wild-type and, even more so, constitutive, Rho led to
increases in the translocation of transiently co-transfected HA-tagged
GLUT4 to the plasma membrane (Fig. 8); in
contrast, dominant-negative Rho largely inhibited the effects of
insulin (Fig. 8) and fully inhibited the effects of GTP
S (Fig. 8) on HA-GLUT4 translocation. Third, we stably transfected 3T3/L1 fibroblasts with eukaryotic expression vectors (pTRE and pTet-On;
CLONTECH Tet-On kit) that (a) placed the
expression of wild-type Rho under the control of a promoter (PminCMV),
which, in turn, is controlled by a tetracycline response element, and
(b) provided the tetracycline-controlled transcription
activator, i.e. a Tet repressor (mutated to reverse its
response to tetracycline and thereby activate transcription) fused to
the VP16 activation domain of a herpes simplex virus. Upon the addition
of doxycycline to these cells (i.e. after they had become
confluent), there were increases in (a) Rho expression and
(b) control and insulin-stimulated 2-DOG uptake (Fig. 7); this approach avoided having Rho overexpressed until 72 h prior to
using cells for 2-DOG assay. Fourth, stable expression of
dominant-negative Rho partially (25-35%) inhibited insulin-stimulated
2-DOG uptake in 3T3/L1 fibroblasts (Fig.
9). From these transfections studies, it
appears that (a) Rho itself can activate PI 3-kinase, GLUT4 translocation, and glucose transport; and (b) in keeping
with studies using C3 transferase, Rho is required for effects of both insulin and GTP
S on GLUT4 translocation and glucose transport in rat
adipocytes.

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Fig. 7.
Effects of stable (A)
and tetracycline-inducible (B) overexpression of Rho on
2-DOG uptake in 3T3/L1 fibroblasts and adipocytes. A,
fibroblasts were stably transfected with pCDNA3 alone (vector, V) or pCDNA3 containing cDNA encoding wild-type Rho
(R). Clones were selected by G418 resistance, and grown,
differentiated, and assayed in parallel with untransfected
(0) cells. Cells were incubated in glucose-free KRP medium
and, after treatment for 30 min, with indicated concentrations (0, 5, 100 nM) of insulin, 5-min uptake of 2-DOG was measured.
Values are the mean ± S.E. of (n) clones, each assayed
in triplicate at each insulin concentration. Insets show
increases in immunoreactivity in cells transfected (TX) with Rho (R). B, cells were stably transfected with
(a) plasmid (pTet-On) that contains cDNA encoding a
mutated tetracycline repressor fused to the VP16 activation domain of a
herpes simplex virus controlled by a constitutive Pcmv
promoter and (b) a plasmid (pTRE) containing cDNA
encoding wild-type Rho whose expression is dependent upon a
tetracycline response element that controls the activation of the
PminCMV promoter and subsequent transcription of the Rho
cDNA (prepared according to instructions in the
CLONTECH Tet-On kit). Colonies were selected both
by resistance to G418 and hygromycin, grown to confluence in 24-well
plates, induced by doxycycline for 72 h as indicated, and then
(after changing to a glucose-free KRP medium) treated with the
indicated concentrations of insulin for 30 min, prior to measurement of
2-DOG uptake over 5 min. Values are mean ± S.E. of n
(shown in parentheses) clones, each assayed in triplicate at
each insulin concentration. Results in clones treated with empty
vectors alone were indistinguishable from results in untransfected
cells, and these results were pooled (controls in panel A,
left). The insets show levels of immunoreactive
Rho in noninduced controls ( ) and tetracycline-induced (+) cells; note that increases were observed only in tetracycline-treated cells
that were transfected with plasmid containing cDNA encoding Rho.
P values (t test) were determined by comparison
of results in tetracycline-treated cells that contained and expressed
the Rho insert, relative to results in control cells that contained vectors lacking the Rho insert. Note that 2-DOG uptake was not influenced by tetracycline treatment in the control group.
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Fig. 8.
Effects of wild-type, constitutive, and
dominant negative forms of Rho (A) and dominant negative
forms of Rho and PKN (B) on translocation of HA-tagged
GLUT4 to the plasma membrane of rat adipocytes. A, cells
were co-transfected by electroporation in DMEM in the presence of
(a) pCIS2 containing cDNA encoding HA-tagged GLUT4 and
(b) pCDNA3 vector alone (V) or pCDNA3
containing cDNA encoding wild-type (WT), constitutive
(CONSTIT), or dominant negative (DN) Rho.
B, adipocytes were co-transfected with (a) pCIS2
containing cDNA encoding HA-GLUT4, along with (b)
pCDNA3 containing cDNA encoding dominant negative Rho, pTB701
containing cDNA encoding dominant-negative PKN, or vectors. After
overnight incubation to allow time for expression, cells were
equilibrated in glucose-free KRP medium for 30 min and treated with or
without 10 nM insulin (A) or 500 µM GTP S (B) as indicated, prior to the addition of 2 mM KCN (to immobilize GLUT4) and measurement
of cell number and cell surface HA-GLUT4 (reflected by 125I
labeling). Values are mean ± S.E. of n (shown in
parentheses) determinations. A, single
asterisks indicate p < 0.05 (t test), Rho transfectants versus control, non-insulin-treated,
vector-transfected cells (VEC). The double
asterisk indicates p < 0.05 (t test), DN Rho transfectants versus insulin-treated,
vector-transfected cells. B, p value determined
by t test; NS, not significant.
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Fig. 9.
Effects of stable expression of wild-type and
dominant negative forms of Rho and PKN on glucose transport in 3T3/L1
fibroblasts (A) and transient expression of wild-type and
dominant-negative PKN on translocation of HA-tagged GLUT4 in rat
adipocytes (B). A, fibroblasts were stably
transfected as in Fig. 7A, except that pCDNA3 containing
cDNAs encoding dominant negative (DN) forms of Rho and
PKN were used, along with wild-type (WT) Rho and PKN. Shown
here are data from clones containing easily discernible increases in
total immunoreactive Rho or PKN and/or expression of HA epitope-tagged
Rho or PKN. Values are mean ± S.E. of N clones, each
assayed in triplicate at each insulin concentration (0, 5, and 100 nM). B, adipocytes were transiently transfected
as in Fig. 8 with (a) pCIS2 containing cDNA encoding
HA-GLUT4 and either (b) pTB701 vector (V) or
(c) pTB701 containing cDNA encoding wild-type (WT) or dominant negative (DN) PKN. See the
legend to Fig. 8 for other details of incubation and assay. Values are
the mean ± S.E. of n (shown in parentheses)
determinations. Single asterisk, p < 0.05 (t test) versus control, non-insulin-treated,
vector-transfected cells (V). Double asterisk,
p < 0.05 versus insulin-treated,
vector-transfected cells.
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In keeping with the possibility that PKN may operate downstream of Rho
during glucose transport activation, we found that transient expression
of wild-type PKN in rat adipocytes resulted in increases in the
translocation of co-transfected HA-tagged GLUT4 to the plasma membrane
(Fig. 9); in contrast, dominant-negative PKN partially inhibited the
effects of insulin (Fig. 9) and fully inhibited the effects of GTP
S
(Fig. 8) on HA-GLUT4 translocation. Similarly, we found that stable
overexpression of PKN enhanced, and dominant negative PKN partially
(55%) inhibited, insulin effects on glucose transport in 3T3/L1
fibroblasts (Fig. 9).
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DISCUSSION |
Our findings suggested that GTP
S activates PI 3-kinase through
a Rho-dependent mechanism in intact rat adipocytes. GTP-Rho has also been found to activate PI 3-kinase in platelet homogenates (7)
but, for uncertain reasons, not in other cell-free systems (8, 9). Our
findings with wortmannin and LY294002 also suggested that PI 3-kinase
is required for the activation of glucose transport during GTP
S
stimulation of rat adipocytes. Thus, GTP
S, as presently used, did
not appear to activate glucose transport in rat adipocytes simply by
activating small G-proteins such as Rab that are thought to function
distal to PI 3-kinase in regulating Glut4 translocation (4).
Whereas GTP
S appeared to activate PI 3-kinase through Rho, insulin
effects on PI 3-kinase were largely independent of Rho. Thus, although
insulin activates Rho (6), Rho was not a major contributor to
insulin-stimulated PI 3-kinase activation, which probably occurs
largely through tyrosine phosphorylation of IRS-1 and/or other proteins
(16). Along these lines, it is pertinent to note that PI 3-kinase is
required for the translocation, but not GTP loading, of Rho during
insulin action (6); thus, PI 3-kinase can operate upstream
(e.g. during insulin action), as well as downstream
(e.g. during GTP
S activation), of Rho. During insulin
action, Rho may translocate to specific sites of PI 3-kinase-induced increases in polyphosphoinositides (accordingly, we have found that Rho
avidly binds to artificial phosphatidylcholine vesicles containing 5%
PI-3, 4-(PO4)2,
PI-3,4,5-(PO4)3, or
PI-4,5-(PO4)2),2
and this may coordinate certain actions of PI 3-kinase and Rho. During
GTP
S action, GTP
S stimulates the translocation of Rho to plasma
and microsomal membranes (6), and this may explain how
membrane-activated PI 3-kinase is activated by GTP-Rho.
In addition to requirements for Rho and PI 3-kinase, our findings with
RO 31-8220 suggested a requirement for one or more protein kinases in
the activation of glucose transport by GTP
S as well as by insulin.
In the case of insulin, the required protein kinase(s) appears to
operate distally to, or in parallel with, PI 3-kinase, since RO 31-8220 does not inhibit insulin-induced activation of either PI 3-kinase (15)
or PI 3-kinase-dependent PKB activation2;
presumably, the same situation pertains during GTP
S action, i.e. the RO 31-8220-sensitive protein kinase(s) required for
glucose transport is distal or parallel to PI 3-kinase. Although the
identity of the protein kinase is uncertain, note that both PKC-
and
PKN are activated by PI 3-kinase lipid products (i.e.
polyphosphoinositides) (17-19), and PKN is directly activated by
GTP-Rho (20, 21). Also, as reported for other bisindolemaleimides (see
Refs. 22 and 23), we have found (14) that RO 31-8220 inhibits
recombinant conventional (
,
, and
) and novel (
,
, and
) PKCs at relatively low concentrations (IC50 values of
15-100 nM) and the atypical PKC, PKC-
, at relatively
high concentrations (IC50 of 1-4 µM). Presently, we found that RO 31-8220, at relatively low concentrations (IC50 = 30 nM), inhibited immunoprecipitated
PKN. Presumably, inhibitory effects of RO 31-8220 on PKC and PKN
reflect homology in the catalytic domains of PKN and most PKCs
(24).
With respect to PKC-
and PKN as RO 31-8220-inhibitable protein
kinases that may be required for glucose transport during the actions
of GTP
S and insulin, the following are germane. First, PKC-
was
activated by insulin, but not by GTP
S; thus, PKC-
seems unlikely
to be involved in the action of GTP
S but may play a role during
insulin action. Second, as in other systems in which GTP-Rho directly
activates PKN (20, 21), we found that GTP
S activated both Rho and
PKN, and insulin activated PKN by a Rho-dependent mechanism; accordingly, PKN is probably activated via Rho during the
actions of both GTP
S and insulin in rat adipocytes. On the other
hand, our studies suggested that different RO 31-8220-sensitive protein
kinases were required for glucose transport effects of GTP
S and
insulin in rat adipocytes; thus, insulin required a protein kinase
sensitive to higher (IC50 of 4-5 µM)
concentrations of RO 31-8220, e.g. PKC-
, whereas GTP
S
required a protein kinase sensitive to lower (IC50 < 1 µM) concentrations of RO 31-8220, e.g. PKN.
Although these findings with RO 31-8220 might suggest that PKN is
required for glucose transport effects of GTP
S, but not insulin,
note that expression of dominant-negative PKN partially inhibited
(a) the effects of insulin as well as GTP
S on the
translocation of HA-GLUT4 to the plasma membrane in rat adipocytes and
(b) insulin effects on glucose transport in 3T3/L1
fibroblasts. It is presently uncertain if these seemingly divergent
findings reflect shortcomings in our experimental approaches
(e.g. effective local concentrations of inhibitors such as
RO 31-8220 at specific enzyme sites in situ are uncertain,
and transfections of dominant-negative proteins may cause untoward
effects).
As discussed, our findings suggested that Rho and PKN contributed to
the activation of glucose transport during GTP
S action. However,
activating effects of GTP-Rho on PKN would not explain the sensitivity
of GTP
S-stimulated 2-DOG uptake to wortmannin and LY294002, unless
PI 3-kinase, as well as Rho, needed to be co-activated, perhaps to
further activate or correctly localize PKN. Accordingly, (a)
PI-4,5-(PO4)2 and
PI-3,4,5-(PO4)3 directly activate PKN (also
known as PKC-related kinase-1 or 2 (PRK-1 or 2)) (18); and (b) Rho is translocated by
PI-4,5-(PO4)2 (Ref. 6) and
PI-3,4-(PO4)2 and
PI-3,4,5-(PO4)3 (see above), and PI 3-kinase
lipid products may correctly localize Rho and, therefore, PKN to
specific membrane compartments during the actions of both GTP
S and
insulin.
Our observation of activation of membrane-associated PI 3-kinase by
GTP
S in rat adipocytes appears to differ from that of a previous
report in which GTP
S failed to activate cytosolic PI 3-kinase in
3T3/L1 adipocytes (3); this may reflect differences in cell types or
the fact that we measured membrane, rather than cytosolic, PI 3-kinase
activity. Along these lines, note that (a) we found that
insulin and GTP
S activated membrane, but not cytosolic, PI 3-kinase
in rat adipocytes; and (b) our failure to observe increases
in cytosolic PI 3-kinase may reflect the large pool of
insulin-independent PI 3-kinase that is activated indiscriminately
during our assays of crude rat adipocyte cytosol. Although we did not
examine the effects of GTP
S on membrane PI 3-kinase activity in
3T3/L1 adipocytes, we did find that overexpression of Rho activated
membrane PI 3-kinase in these cells. Accordingly, it may be surmised
that, as in rat adipocytes, GTP
S, by activating Rho, may activate PI
3-kinase in 3T3/L1 adipocytes; this could explain why GTP
S, at least
partly (approximately 50%, as per wortmannin studies in Ref. 5)
requires PI 3-kinase for the activation of glucose transport in 3T3/L1
adipocytes; on the other hand, glucose transport effects of GTP
S
that are independent of PI 3-kinase (also approximately 50%; see Ref.
5) may be explained by direct activating effects of GTP
S on Rab (4) or other G-proteins that act distally to PI 3-kinase.
Finally, it was of interest to find that, in addition to inhibitory
effects of C3 transferase and dominant-negative forms of Rho and PKN on
GTP
S- and insulin-stimulated glucose transport and/or GLUT4
translocation, transfected Rho (particularly if constitutively activated) and its downstream kinase, PKN, provoked increases in GLUT4
translocation and/or glucose transport in rat adipocytes and 3T3/L1
cells. It therefore may be conjectured that Rho is not only required
for, but may actively participate in, the activation of GLUT4
translocation and glucose transport in the actions of insulin, GTP
S,
and other agonists.
In summary, like insulin, GTP
S provoked increases in 2-DOG uptake
and HA-GLUT4 translocation in rat adipocytes. Also, like insulin,
(a) GTP
S provoked increases in membrane-associated PI 3-kinase, and PI 3-kinase appeared to be required for GTP
S-induced activation of glucose transport; and (b) both Rho and an RO
31-8220-sensitive protein kinase appeared to be required for
GTP
S-induced activation of glucose transport. In studies of RO
31-8220-sensitive protein kinases, both GTP
S and insulin activated
PKN, and PKN appeared to be required for activation of GLUT4
translocation by GTP
S and insulin. Unlike insulin, however, GTP
S
appeared to activate PI 3-kinase primarily through Rho, rather than
through IRS-1; PKC-
was activated by insulin but not by GTP
S; and
effects of GTP
S on glucose transport were inhibited by lower
concentrations of RO 31-8220 than were effects of insulin. It may
therefore be surmised that, although there are similarities in the
signaling factors (i.e. Rho, PKN, and PI 3-kinase) that are
used by GTP
S and insulin to activate glucose transport, these agents
activate Rho and PI 3-kinase by different mechanisms and appear to use different distal protein kinases to activate glucose transport.