Rapid stimulation of glucose transport by mitochondrial
uncoupling depends in part on cytosolic
Ca2+ and cPKC
Zayna A.
Khayat1,2,
Theodoros
Tsakiridis1,
Atsunori
Ueyama1,
Romel
Somwar1,2,
Yousuke
Ebina3, and
Amira
Klip1,2
1 Programme in Cell Biology,
Hospital for Sick Children, Toronto M5G 1X8;
2 Department of Biochemistry,
University of Toronto, Toronto, Ontario, Canada M5S 1A8; and
3 Division of Molecular Genetics,
Institute for Enzyme Research, University of Tokushima, Tokushima
770-8503, Japan
 |
ABSTRACT |
2,4-Dinitrophenol (DNP) uncouples the mitochondrial oxidative
chain from ATP production, preventing oxidative metabolism. The
consequent increase in energy demand is, however, contested by cells
increasing glucose uptake to produce ATP via glycolysis. In L6 skeletal
muscle cells, DNP rapidly doubles glucose transport, reminiscent of the
effect of insulin. However, glucose transport stimulation by DNP does
not require insulin receptor substrate-1 phosphorylation and is
wortmannin insensitive. We report here that, unlike insulin, DNP does
not activate phosphatidylinositol 3-kinase, protein kinase
B/Akt, or p70 S6 kinase. However, chelation of intra- and
extracellular Ca2+ with
1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic
acid-AM in conjunction with EGTA inhibited DNP-stimulated glucose
uptake by 78.9 ± 3.5%. Because
Ca2+-sensitive, conventional
protein kinase C (cPKC) can activate glucose transport in L6 muscle
cells, we examined whether cPKC may be translocated and
activated in response to DNP in L6 myotubes. Acute DNP treatment led to
translocation of cPKCs to plasma membrane. cPKC immunoprecipitated from
plasma membranes exhibited a twofold increase in kinase activity in
response to DNP. Overnight treatment with 4-phorbol 12-myristate
13-acetate downregulated cPKC isoforms
,
, and
and partially
inhibited (45.0 ± 3.6%) DNP- but not insulin-stimulated
glucose uptake. Consistent with this, the PKC inhibitor
bisindolylmaleimide I blocked PKC enzyme activity at the
plasma membrane (100%) and inhibited DNP-stimulated
2-[3H]deoxyglucose
uptake (61.2 ± 2.4%) with no effect on the stimulation of glucose transport by insulin. Finally, the selective PKC-
inhibitor
LY-379196 partially inhibited DNP effects on glucose uptake (66.7 ± 1.6%). The results suggest interfering with mitochondrial ATP
production acts on a signal transduction pathway independent from that
of insulin and partly mediated by
Ca2+ and cPKCs, of which PKC-
likely plays a significant role.
2,4-dinitrophenol; insulin; glucose uptake; glucose transporter-4
translocation; conventional protein kinase C
 |
INTRODUCTION |
IN MAMMALS, SKELETAL MUSCLE is the primary target
tissue for insulin stimulation of glucose transport, a regulatory
mechanism vital for glucose homeostasis. Insulin achieves this
regulation by signaling the translocation of preformed glucose
transporters from intracellular stores to the plasma membrane. The L6
muscle cell line has been used extensively to characterize
physiological responses in muscle such as glucose transport, since it
retains many morphological and metabolic properties of skeletal muscle (32, 61). Three glucose transporter (GLUT) isoforms are expressed in
differentiated L6 myotubes, GLUT-1, GLUT-3, and GLUT-4 (5). There is
mounting evidence that muscle cells respond to a variety of stimuli by
rapidly elevating their rate of glucose uptake (reviewed in Ref. 23).
These include, on one hand, the anabolic hormone insulin and, on the
other hand, stimuli that increase energy demand such as exercise (23),
hypoxia (8), environmental stress (11), and metabolic challenges to the
oxidative chain. The mitochondrial uncoupler 2,4-dinitrophenol (DNP), a
weak base that dissipates the H+
gradient of mitochondria, uncouples the oxidative chain from ATP
production, thus compromising energy production (4). Previous work has
shown that L6 muscle cells react to this metabolic challenge by
increasing glucose transport to boost glycolytic ATP production, reminiscent of the response to hypoxia in vivo (4).
There are numerous contrasts between insulin and energy stressors in
their mechanisms of glucose transport activation in skeletal muscle.
Insulin and exercise recruit distinct intracellular pools of glucose
transporters in skeletal muscle (13, 14), and the maximal effects of
insulin and contraction or insulin and hypoxia on glucose uptake are
additive (48, 62). Activation of phosphatidylinositol 3-kinase (PI3K)
is utilized by insulin to induce glucose transporter translocation but
does not participate in the responses to exercise or hypoxia (40, 42,
57). Moreover, insulin, but not contraction, causes a redistribution of
Rab4 (a Ras-related GTP-binding protein) from internal compartments in
skeletal muscle (53). In L6 muscle cells, insulin causes translocation
to the cell membrane of GLUT-1, GLUT-3, and GLUT-4, whereas DNP
mobilizes only GLUT-1 and GLUT-4 (57). Unlike insulin, DNP does not
require PI3K activity and an intact actin cytoskeletal network (57) to
mediate these effects. Collectively, these findings suggest that energy
stressors utilize mechanisms other than insulin to increase muscle
glucose influx; however, little is known about the mechanism by which
these factors elicit this response. The purpose of this study was to
use DNP as a model of exercise or hypoxia to investigate possible
mediators of this alternative signaling pathway. Our findings provide
evidence for the existence of a signaling system activated by metabolic challenge that regulates glucose transport in muscle cells by a
mechanism distinct from that used by insulin.
 |
MATERIALS AND METHODS |
Materials.
Tissue culture medium, serum, and other tissue culture reagents were
obtained from Life Technologies (Burlington, ON, Canada). Human
insulin was a kind gift from Eli Lilly Canada (Toronto, ON, Canada).
DNP, 4-phorbol-12-myristate-13-acetate (PMA), and cytochalasin B were
obtained from Sigma Chemical (St. Louis, MO). Bisindolylmaleimide I
(BIM) was from Calbiochem (La Jolla, CA). The protein kinase C
(PKC)-
inhibitor LY-379196 was a kind gift from Eli Lilly
(Indianapolis, IN). Protein A- and protein G-Sepharose were from
Pharmacia Biotechnology (Uppsala, Sweden).
[
-32P]ATP and
enhanced chemiluminescence reagents were purchased from Amersham
(Oakville, ON, Canada).
2-[3H]deoxyglucose and
3-O-[methyl-3H]methylglucose
were obtained from DuPont NEN (Boston, MA). Monoclonal antibody against
PKC-
, -
, and -
used for immunoprecipitation and polyclonal
antibody to PKC-
II were kind
gifts from Kinetek Pharmaceuticals (Vancouver, BC, Canada). Polyclonal
antibodies to PKC-
, PKC-
I,
and PKC-
used for immunoblotting were purchased from Signal
Transduction Laboratories (Lexington, KY). Crosstide peptide, protein
kinase A (PKA) and PKC inhibitor peptides, and polyclonal antibodies to
PKC-
, p70 S6 kinase (p70S6K),
and protein kinase B/Akt (PKB/Akt) were obtained from Santa Cruz (Santa
Cruz, CA). PKC kinase assay kit and monoclonal anti-phosphotyrosine antibody (used for PI3K activity assay) were purchased from Upstate Biotechnology (Lake Placid, NY). Monoclonal antibody McK1 to the
1-subunit of the
Na+-K+-ATPase
was a kind gift from Dr. K. Sweadner (Massachusetts General Hospital,
Boston, MA).
Cell culture.
L6 muscle cells were maintained in myoblast monolayer culture in
-MEM containing 10% vol/vol fetal bovine serum (FBS) and 1%
vol/vol antibiotic-antimycotic solution (10,000 U/ml penicillin G, 10 mg/ml streptomycin, and 25 mg/ml amphotericin B) in an atmosphere of
5% CO2 at 37°C as described
previously (15). Cells were maintained in continuous passages by
trypsinization of subconfluent cultures using 0.25% trypsin. Myoblasts
were seeded in medium containing 2% vol/vol FBS at ~4 × 104 cells/ml in 10-cm-diameter
dishes and used 6-8 days postseeding for plasma membrane
preparations and kinase activity assays. L6 cells were seeded in
12-well or 6-well plates for glucose uptake experiments. Cells were fed
fresh medium every 48 h and used at the stage of myotubes.
L6 muscle cells expressing c-myc epitope-tagged GLUT-4 (GLUT-4-myc)
were constructed as described (29). The human c-myc epitope (14 amino
acids) was introduced into the first ectodomain of GLUT-4, and the
epitope does not affect GLUT-4 activity (29, 59). GLUT-4-myc cDNA was
subcloned into the mammalian expression vector pCXN (pCXN-GLUT-4-myc).
L6 myoblasts were transfected with pCXN-GLUT-4-myc and pSV2-bsr, a
blasticidin S deaminase expression plasmid, and selected with
blasticidin S hydrochloride (Funakoshi, Tokyo, Japan). Cell surface
GLUT-4-myc was detected by a colorimetric assay as described previously
(59).
Hexose transport determinations.
Measurements of
2-[3H]deoxyglucose and
3-O-[methyl-3H]methylglucose
uptake were carried out as previously described (34, 38). Briefly,
differentiated L6 myotube monolayers grown in 12-well plates (used for
2-deoxyglucose uptake) or 6-well plates (used for
3-O-methylglucose uptake) were rinsed
twice with HEPES-buffered saline (HBS; in mM: 140 NaCl, 20 NaHEPES, 2.5 MgSO4, 1 CaCl2, and 5 KCl, pH 7.4). Glucose
uptake was quantitated by exposing the cells to 10 µM
2-[3H]deoxyglucose (1 µCi/ml) for 5 min or 10 µM
3-O-[methyl-3H]methylglucose
(2 µCi/ml) for 2 min. Nonspecific uptake was determined by
quantitating cell-associated radioactivity in the presence of 10 µM
cytochalasin B, which blocks transporter-mediated uptake. At the end of
the 5-min period, the uptake buffer was aspirated rapidly and the cells
were washed three times with ice-cold isotonic saline (0.9% wt/vol
NaCl, containing 1 mM HgCl2 in the
case of 3-O-[methyl-3H]methylglucose
uptake assays). The cells were lysed in 0.05 N NaOH, and the associated
radioactivity was determined by liquid scintillation counting. Each
condition was assayed in triplicate for
2-[3H]deoxyglucose
assays and in duplicate for
3-O-[methyl-3H]methylglucose uptake experiments.
PI3K, Akt/PKB, and p70S6K activity
assays.
PI3K activity and p70S6K activity
were assayed exactly as described previously (17, 56).
Immunoprecipitation of Akt1 and kinase assay were performed as
described (37) with modifications. Cells were lysed with lysis buffer
containing 50 mM HEPES (pH 7.6), 150 mM NaCl, 10% vol/vol glycerol,
1% vol/vol Triton X-100, 30 mM sodium pyrophosphate, 10 mM NaF, 1 mM
EDTA, 1 mM phenylmethylsulfonyl fluoride (PMSF), 1 mM benzamidine,
1 mM
Na3VO4,
1 mM dithiothreitol (DTT), and 100 nM okadaic acid. Anti-Akt1 antibody
was precoupled to a mixture of protein A- and protein G-Sepharose beads
by incubating 2 µg of antibody per condition with 20 µl of the
protein A- and protein G-Sepharose beads (100 mg/ml) for a minimum of 2 h. These anti-Akt1 beads were washed twice with ice-cold PBS and once
with ice-cold lysis buffer. Akt1 was immunoprecipitated by incubating
200 µg of total cellular protein with the anti-Akt1-bead complex for 2-3 h under constant rotation (4°C). Akt1 immunocomplexes were isolated and washed four times with 1 ml of wash buffer [25 mM HEPES (pH 7.8), 10% vol/vol glycerol, 1% vol/vol Triton X-100, 0.1%
wt/vol BSA, 1 M NaCl, 1 mM DTT, 1 mM PMSF, 1 µM microcystin, and 100 nM okadaic acid] and twice with 1 ml of kinase buffer [50
mM Tris · HCl (pH 7.5), 10 mM
MgCl2, and 1 mM DTT]. This
was then incubated under constant agitation for 30 min at 30°C with 30 µl of reaction mixture (kinase buffer containing 5 µM ATP, 2 µCi [
-32P]ATP,
and 100 µM Crosstide). After the reaction, 30 µl of the supernatant
were transferred onto Whatman p81 filter paper and washed with 3 ml of
175 mM phosphoric acid four times for 10 min and once with distilled
water for 5 min. Filters were air dried and then subjected to liquid
scintillation counting.
Plasma membrane-enriched fraction and immunoblotting.
Myotube monolayers grown on 10-cm-diameter dishes were gently scraped
with a rubber policeman in 5 ml of ice-cold homogenization buffer (in
mM: 250 sucrose, 20 HEPES, 2 EGTA, and 3 NaN3, pH 7.4) containing freshly
added protease inhibitors (in µM: 200 PMSF, 1 leupeptin, and 1 pepstatin A) and homogenized in a 40-ml Dounce type A homogenizer on
ice (20 strokes). The homogenate was centrifuged at 760 g for 5 min at 4°C, and the
resultant supernatant was centrifuged at 31,000 g for 20 min to separate a plasma
membrane-enriched pellet from an intracellular microsome supernatant.
The plasma membrane fraction was resuspended in homogenization buffer.
Membrane protein content was determined by the bicinchoninic acid
method (Pierce, Rockford, IL). Fifty micrograms of protein were
separated by 7.5% SDS-PAGE, electrotransferred onto polyvinylidene
difluoride membrane, and immunoblotted for various PKC isoforms or for
the
1-subunit of the
Na+-K+-ATPase.
For monoclonal and polyclonal antibody detection, horseradish peroxidase-conjugated goat anti-mouse and goat anti-rabbit secondary antibodies were used, respectively, followed by enhanced chemiluminescence.
PKC activity assay.
Plasma membranes were resuspended in 0.5 ml of immunoprecipitation
buffer [50 mM HEPES (pH 7.8), 1% vol/vol Triton X-100, 2.5 mM
EDTA, 200 µM PMSF, 1 µM leupeptin, and 1 µM pepstatin
A] and lysed by passing through a 27-gauge syringe five times.
The homogenate was centrifuged at 12,000 g for 5 min, and the supernatant was
incubated overnight with 20 µl of anti-PKC-
,
,
monoclonal antibody at 4°C with rotary shaking. To this mixture was added 50 µl of 50% wt/vol protein A-Sepharose beads for 1 h. The Sepharose beads and attached proteins were pelleted by centrifugation and washed
three times with PBS plus 0.1% vol/vol Triton X-100. The phosphotransferase activity of PKC in immunoprecipitates from plasma
membranes was measured using a PKC assay kit. The assay is based on
phosphorylation of a specific substrate peptide
(Gln-Lys-Arg-Pro-Ser-Gln-Arg-Ser-Lys-Tyr-Leu) using the transfer of the
-phosphate of
[
-32P]ATP by PKC
kinase. The immunoprecipitated protein was diluted in 20 mM MOPS (pH
7.2) containing 25 mM
-glycerol phosphate, 1 mM sodium vanadate,
1 mM DTT, and 1 mM
CaCl2. To the enzyme preparation
were added 100 µM peptide substrate, lipid activators (0.1 mg/ml
phosphatidylserine and 0.01 mg/ml diglyceride), and kinase inhibitors
(100 nM PKA inhibitor peptide and 4 µM R-24571). The kinase reaction
was started by adding Mg2+/ATP
reaction buffer containing 15 mM
MgCl2 and 100 µM ATP (1.5 µCi
[
-32P]ATP) in assay
dilution buffer. The mixture was incubated at 30°C for 10 min. The
phosphorylated substrate was then separated from the residual
[
-32P]ATP using
Whatman p81 filter paper, washed in 175 mM phosphoric acid, air dried,
and then quantitated using a liquid scintillation counter.
Statistical analysis.
X-ray films were quantified in the linear range by densitometry using
National Institutes of Health Image software. The detection and
quantitation of [32P]phosphatidylinositol
3-phosphate (PI3P) on TLC plates were performed with a Molecular
Dynamics PhosphorImager system (Sunnyvale, CA). Statistical analysis
was performed using the ANOVA test (Fisher, multiple comparisons).
 |
RESULTS |
DNP does not activate the insulin signal transduction pathway.
The time course in Fig. 1
demonstrates that the maximal effects of DNP and insulin on glucose
transport were additive over a 60-min period after addition,
reminiscent of previous observations of additivity between insulin and
hypoxia or insulin and exercise in skeletal muscle (48, 62). This
suggests that different signals may participate in relaying the signal
from insulin and from DNP to the glucose transporters.

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Fig. 1.
2,4-Dinitrophenol (DNP)-stimulated glucose transport is additive to
insulin. L6 myotubes were serum deprived for 5 h and then incubated for
0-60 min in -MEM with 0.5 mM DNP, 100 nM insulin (INS), or both
at 37°C. Immediately after these incubations, cells were washed
twice with HEPES-buffered saline (HBS) and
2-[3H]deoxyglucose
uptake was assayed as described in MATERIALS AND
METHODS. Transport rates were normalized with respect
to basal rate of untreated control, which was assigned a value of 1. Shown is a representative of 3 independently performed experiments.
Results are expressed as means ± SE of 3 replicates.
|
|
Recent studies have identified three kinases rapidly activated by
insulin in muscle cells: PI3K (56), PKB (Akt/PKB) (37), and
p70S6K (17). We have shown that
the selective PI3K inhibitor wortmannin does not affect the activation
of glucose transport by DNP in L6 muscle cells but abolishes the
insulin response (57). However, PI3K activity was not directly measured
in that study. Because arsenite-induced activation of glucose transport
and vanadate-mediated antilipolytic actions are wortmannin insensitive
although PI3K is activated by both agents (41, 46), it was important to test whether DNP also activates PI3K. Unlike the hormonal response of
L6 muscle cells, DNP did not activate PI3K (Fig.
2A). It
is widely held that insulin-mediated Akt/PKB and
p70S6K activation occurs
downstream of PI3K and is dependent on the lipid products of PI3K (1,
9), yet recent reports have uncovered stress-induced activation of
Akt/PKB in COS-7 cells (39) and, in cardiomyocytes, arsenite-induced
activation of p70S6K (60), which
are both PI3K independent. We therefore tested whether DNP activated
either Akt/PKB or p70S6K directly.
As shown in Fig. 2, DNP did not activate either Akt/PKB (Fig.
2B) or
p70S6K (Fig.
2C) in L6 muscle cells. Furthermore,
selective inhibition of the p70S6K
pathway by rapamycin in L6 muscle cells failed to prevent the DNP-induced activation of glucose transport (Khayat and Klip, unpublished observations).

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Fig. 2.
DNP does not activate lipid and protein kinases activated by insulin.
Serum-depleted L6 myotubes (5 h) were treated with 0.5 mM DNP or 100 nM
insulin for up to 30 min. Phosphatidylinositol 3-kinase (PI3K;
A), Akt/protein kinase B (PKB;
B), or p70 S6 kinase
(p70S6K;
C) kinase activity was assayed in
cell lysates as described in MATERIALS AND
METHODS. Observed kinase activities were normalized
relative to basal activities in untreated cells, which were assigned a
value of 1. Results are expressed as means ± SE of 3 independent
experiments.
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|
Role of intracellular
Ca2+ in
DNP-stimulated glucose uptake.
There is evidence that mitochondrial uncoupling provokes a rapid rise
in intracellular Ca2+ that
coincides with an acceleration of glucose flux in muscle and liver
cells (11, 44). To ascertain the demand for
Ca2+ in the activation of glucose
transport by DNP, L6 muscle cells were loaded with the
Ca2+ chelator
1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA)-AM incubated simultaneously with EGTA (to buffer extracellular Ca2+) before
challenge with DNP or insulin (as in Ref. 33) followed by
2-[3H]deoxyglucose
uptake measurements. The Ca2+
chelators inhibited DNP-stimulated glucose uptake by 78.9 ± 3.5% (P < 0.01), without affecting
insulin-stimulated glucose uptake (Fig. 3).
Buffering extracellular Ca2+ with
2.5 mM EGTA alone or 15 µM BAPTA-AM alone did not significantly affect the DNP response (results not shown).

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Fig. 3.
Role of Ca2+ in response to DNP.
Serum-depleted L6 myotubes (5 h) were pretreated with or without 15 µM
1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic
acid (BAPTA)-AM and 2.5 mM EGTA in
Ca2+-free HBS supplemented with 10 mM D-glucose for 10 min,
followed by stimulation for 30 min with 0.5 mM DNP or 100 nM insulin in
HBS. Cells were washed twice with HBS, and
2-[3H]deoxyglucose
uptake was measured as described in MATERIALS AND
METHODS. Transport rates were normalized with respect
to basal rate of untreated control (CON), which was assigned a value of
1. Results are expressed as means ± SE of 5 independent
experiments. * P < 0.01 vs.
control DNP.
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|
Role of
Ca2+-sensitive
PKC in DNP action.
A rise in intracellular Ca2+
triggers the activation of a variety of cellular proteins, including
Ca2+-sensitive, conventional PKC
(cPKC) (reviewed in Ref. 49). To assess the involvement of cPKC in the
glucose transport response, we utilized the potent PKC inhibitor BIM,
which inhibits Ca2+-dependent cPKC
isoforms at lower doses (<1 µM) than those affecting novel or
atypical isoforms, which do not require
Ca2+ for activation (45). At 1 µM, BIM caused a 61.2 ± 2.4% (P < 0.05) reduction in the stimulation of glucose transport by DNP (Fig.
4A), but
it did not affect the response to insulin. Furthermore, pretreatment
with 1 µM BIM did not further reduce DNP-stimulated glucose transport
beyond the 80% inhibition observed with BAPTA-EGTA pretreatment (Table
1). At a higher dose of BIM (10 µM),
which is known to inhibit novel and atypical PKC isoforms, no
additional inhibition of DNP-stimulated glucose uptake was observed;
however, insulin-stimulated glucose transport was inhibited by 50%.
The latter observation is in agreement with recent evidence supporting the involvement of atypical PKC-
in the insulin-dependent glucose transport pathway (2).

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Fig. 4.
DNP-stimulated glucose transport is reduced by the protein kinase C
(PKC) inhibitor bisindolylmaleimide I (BIM).
A: serum-depleted L6 myotubes (5 h)
were preincubated for 15 min in -MEM in absence or presence of
indicated doses of BIM. Cells were then stimulated for 30 min at
37°C with 0.5 mM DNP or 100 nM insulin in continuous presence of
BIM. At end of this period, cells were washed twice with HBS and
2-[3H]deoxyglucose
uptake was measured. Transport rates were normalized with respect to
basal rate of untreated control (UNT), which was assigned a value of 1. B: effect of 1 µM BIM on DNP-,
insulin-, and 4-phorbol-12-myristate-13-acetate (PMA; 1 µM, 30 min)-stimulated glucose uptake. Results are expressed as means ± SE
of 6 independent experiments.
* P < 0.05 vs. respective
control.
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|
The PKC-stimulating phorbol ester PMA is also able to stimulate glucose
transporter translocation (30), albeit to a lesser extent than insulin
or other stimuli. To confirm that the action of 1 µM BIM is on cPKC,
we tested the effect of BIM (1 µM) on PMA-stimulated glucose
transport in L6 muscle cells. Figure
4B demonstrates that PMA increased
glucose transport by 30% and 1 µM BIM inhibited this stimulation by
100%, whereas DNP-dependent glucose uptake was partially inhibited
(60%) by the same treatment. BIM had no effect on the basal value of
glucose transport (Fig. 4, A and
B).
To further clarify the dependence of cPKC in DNP-dependent glucose
transport activation, cPKCs were depleted from L6 cells by overnight
PMA treatment. PMA treatment (cPKC downregulation) eliminated all cPKC
isoforms but not the atypical PKC-
(Fig. 5A).
Furthermore, cPKC downregulation partially inhibited the stimulation of
glucose transport by DNP by 45.0 ± 3.6%, whereas it fully blocked
PMA-stimulated glucose uptake. PKC downregulation did not affect the
stimulation of glucose transport by insulin (Fig.
5B). The findings for insulin and
PMA were consistent with previous observations reported by
Bandyopadhyay et al. (2) in L6 muscle cells. As with 1 µM BIM, the
inhibition of DNP action by PKC depletion in combination with
Ca2+ chelation was no greater than
the effect of Ca2+ buffering alone
(Table 1).

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Fig. 5.
Conventional, Ca2+-sensitive PKC
(cPKC) downregulation reduces DNP-stimulated glucose transport. L6
myotubes were incubated for 16 h in absence or presence of 100 nM PMA
(downregulated). A: cells were lysed,
and various PKC isoforms were detected in 15 µg of cell lysates by
SDS-PAGE followed by immunoblotting.
B: cells were stimulated for 30 min
with 0.5 mM DNP, 100 nM insulin, or 1 µM PMA. At end of this period,
cells were washed and
2-[3H]deoxyglucose
transport was assayed as described in MATERIALS AND
METHODS. Transport rates were normalized with respect
to basal rate of untreated control, which was assigned a value of 1. Results are expressed as means ± SE of 5 independent experiments.
* P < 0.05 vs. respective
control.
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The extent of cPKC activation by DNP was ascertained by two methods.
The first approach involved purification of fractions enriched in
plasma membranes derived from DNP-treated L6 cells, followed by
immunoblotting for cPKC isoforms with an antibody that recognized
PKC-
, -
, and -
isoforms. DNP generated a 2.6-fold increase in
PKC-
, -
, and -
levels in the plasma membrane compared with
unstimulated cells (Fig. 6,
A and
B). PMA, on the other hand, provoked
a marked redistribution of cPKC to the plasma membrane (ninefold),
whereas insulin treatment resulted in only a modest increase in the
plasma membrane levels of cPKCs. As expected, cPKC could not be
detected in plasma membrane fractions isolated from cells pretreated
with PMA overnight that were not stimulated or were stimulated with
insulin, DNP, or PMA for 30 min (Fig. 6A). The second approach involved
measuring in vitro cPKC activity directly in plasma membrane fractions
derived from unstimulated or from DNP-, insulin-, or PMA-stimulated
cells. PKC-
, -
, and -
activity in cPKC immunoprecipitates from
plasma membranes was elevated by 200% by DNP (Fig.
7A).
This activation was completely blocked by pretreatment of cells with 1 µM BIM. Comparable with its higher stimulation of cPKC translocation
to the plasma membrane, PMA also induced a much greater activation of
cPKC activity (sevenfold) than DNP, whereas insulin elevated cPKC
activity by only 30%.

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Fig. 6.
DNP increases translocation of cPKC to plasma membrane. Serum-depleted
L6 myotubes (5 h) were stimulated with 0.5 mM DNP, 100 nM insulin, or 1 µM PMA in -MEM at 37°C for 30 min. Subcellular fractionation
was performed to obtain a plasma membrane-enriched fraction. Equal
amounts of this fraction (50 µg) were separated by 7.5% SDS-PAGE,
electrotransferred onto polyvinylidene difluoride membrane, and
immunoblotted for PKC- , - , and - . To ensure equality of
protein loading, blots were simultaneously probed with monoclonal
antibody against 1-subunit of
Na+-K+-ATPase
(results not shown). A: representative
immunoblot. B: quantitation of
immunoblots (n = 4) expressed as means ± SE relative to basal levels of PKC in plasma membrane from
untreated cells. * P < 0.05, ** P < 0.001 vs. untreated
control.
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Fig. 7.
DNP induces a cPKC activity that is inhibitable by BIM.
Serum-depleted L6 myotubes (5 h) were treated with or without 1 µM
BIM in -MEM for 15 min before stimulation with 0.5 mM DNP or 100 nM
insulin (A) or with 1 µM PMA (B) for 10 min
at 37°C. Cells were fractionated to obtain a plasma-membrane
enriched fraction. cPKC was immunoprecipitated from this fraction using
a monoclonal antibody against PKC- , - , and - . A nonspecific
rabbit antibody (IgG) was used as a background control. Kinase activity
of immunoprecipitated PKC on an exogenous substrate was measured.
Observed PKC activity was normalized relative to basal activity in
untreated cells. Results are expressed as means ± SE of 4-6
independent experiments. * P < 0.05, ** P < 0.001 vs.
untreated control.
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|
The availability of a selective PKC-
inhibitor, 379196 (27), allowed
us to test the participation of this isoform in DNP-stimulated glucose
transport. As shown in Fig. 8,
pretreatment of L6 cells with 379196 inhibited DNP-stimulated glucose
uptake in a dose-dependent manner. At a concentration of 100 nM 379196, which will effectively inhibit PKC-
(IC50 150 nM), the stimulation of
glucose uptake by DNP was reduced by 66.7%
(P < 0.01). Insulin-stimulated
glucose uptake was not affected by the inhibitor (results not shown).

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Fig. 8.
Inhibition of PKC- reduces DNP-mediated glucose transport.
Serum-depleted L6 myotubes were preincubated for 15 min in -MEM
containing indicated doses of the PKC- -specific inhibitor 379196. Cells were then stimulated for 30 min with 0.5 mM DNP in continuous
presence of inhibitor. At end of this period, cells were washed and
2-[3H]deoxyglucose
uptake was measured. Transport rates were normalized with respect to
basal rate of untreated control, which was assigned a value of 1. Results are expressed as means ± SE of 3 independent experiments.
* P < 0.01 vs. control DNP.
|
|
3-O-methylglucose uptake and GLUT-4
translocation also depend on
Ca2+
mobilization and cPKC.
The uptake of 2-deoxyglucose is the sum of transmembrane transport and
phosphorylation. We and others previously measured the transport rate
and found that changes in 2-deoxyglucose uptake reflect changes in
transport under the assay conditions used. Nonetheless, the effect of
ATP depletion by DNP on hexose uptake may have either triggered the
stimulation of hexose transport or modulation of the activity of
hexokinase, the enzyme that phosphorylates 2-deoxyglucose to form
2-deoxyglucose-6-phosphate. To ensure that incubation with DNP brings
about a response of hexose transport specifically, the uptake of
3-O-methylglucose (a
nonphosphorylatable analog of glucose) was also measured. Table
2 shows that DNP increased
3-O-methylglucose uptake by about
twofold relative to control cells. Furthermore, pretreatment with
BAPTA-EGTA, BIM, or cPKC downregulation partially inhibited
DNP-stimulated 3-O-methylglucose uptake (Table 2). The extent of reduction in DNP-stimulated glucose uptake closely paralleled the results using 2-deoxyglucose as the
transported sugar (Figs. 3, 4, and
5B).
To confirm that the observed effects of
Ca2+ chelation and cPKC inhibition
of DNP-stimulated glucose uptake resulted from impaired glucose
transporter translocation, we utilized L6 cells stably transfected with
a GLUT-4 protein containing an exofacial myc epitope tag (L6
GLUT-4-myc) (29). These cells were treated with or without DNP along
with various manipulations of Ca2+
or cPKC, and myc-tagged GLUT-4 was detected on the surface of intact
cells, as described in MATERIALS AND
METHODS. Pretreatment with
Ca2+ chelation, BIM (1 µM), or
cPKC downregulation decreased the GLUT-4-myc at the cell surface to 37, 47, and 46% of the DNP response, respectively (Fig.
9A). As
in wild-type L6 muscle cells, DNP-stimulated glucose uptake was also
partially inhibited in L6 GLUT-4-myc cells by pretreatment with
BAPTA-EGTA, 1 µM BIM, or cPKC downregulation (Fig.
9B). The inhibitory effects of
Ca2+ buffering and interfering
with cPKC activity on DNP-mediated GLUT-4 translocation in L6
GLUT-4-myc cells were qualitatively comparable to the observed
inhibition of DNP-stimulated 2-deoxyglucose uptake.

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Fig. 9.
DNP-stimulated glucose transporter-4 (GLUT-4)-myc translocation depends
on Ca2+ and cPKC. L6 GLUT-4-myc
cells were grown to stage of myotubes and were pretreated with BAPTA-AM
and EGTA (BAP/EGTA), 1 µM BIM (BIM), or were depleted of cPKC
[downregulated (DR)], as described for Figs. 3-5,
respectively, before treatment with 0.5 mM DNP for 30 min.
DNP-stimulated increase in GLUT-4-myc translocation
(A) or
2-[3H]deoxyglucose
uptake (B) was assigned a value of
100%. Inhibition of this value toward basal (0%) was calculated after
inhibition with BAPTA-EGTA, BIM, or downregulation.
|
|
 |
DISCUSSION |
Distinct pathways for glucose transport stimulation.
Several lines of evidence suggest that a pathway exists for the
stimulation of glucose transport into skeletal muscle by insulin that
differs from that for stimulation of energy demand (contraction or
hypoxia). For example, a combination of the two stimuli
produces an additive stimulation of glucose uptake. Furthermore,
insulin signaling requires activation of PI3K and Akt/PKB, but hypoxia and contraction do not (40, 43, 57). Here we show that the stimulation
of glucose uptake by DNP in L6 muscle cells is additive to that induced
by insulin. Also, we demonstrate that, unlike insulin, DNP does not
activate PI3K, Akt/PKB, or p70S6K.
The lack of activation of Akt/PKB by DNP is in agreement with the
recent study by Lund et al. (43) that showed muscle contraction had no
effect Akt/PKB activity. These findings support the notion that at
least two distinct pathways leading to the stimulation of glucose
uptake also exist in L6 muscle cells. The insulin-independent pathway
will hence be termed "the alternative pathway" for the purpose of
this discussion.
The Ca2+-PKC
hypothesis.
It has long been considered that the rise in intracellular
Ca2+ is a critical mediator of
increased glucose transport during skeletal muscle contraction and
hypoxia (7, 25). This has been proposed mostly on the basis of
inhibition of the stimulation of glucose transport during hypoxia and
contraction by agents that are thought to block
Ca2+ channels [e.g.,
verapamil (7)] or lower Ca2+
efflux from the sarcoplasmic reticulum [e.g., dantrolene
(25)]. Additionally, several studies have shown that rates of
glucose transport can be increased in mammalian muscle when cytoplasmic Ca2+ concentrations are raised
using agents such as W-7, caffeine, and
Ca2+ ionophores (24, 25, 47). In
contrast, insulin does not significantly affect cytosolic
Ca2+ levels (33, 35).
Ca2+ is, however, released from
mitochondria as a result of DNP dissipation of the
H+ gradient (44). We therefore
reasoned that Ca2+ may be a
trigger in the insulin-independent mechanism of glucose transport
activation. Our findings with the buffering of intra- and extracellular
Ca2+ provide more direct evidence
that Ca2+ plays a significant role
in the stimulation of glucose transport induced by DNP but not in
stimulation induced by insulin.
A rise in cytoplasmic Ca2+ levels
may facilitate the activation of key intracellular signaling molecules
that lead to increased muscle glucose transport. PKC is a
Ca2+-dependent signaling
intermediary that can be activated by increases in cellular
Ca2+. Because
Ca2+ can activate cPKCs and PMA (a
known activator of cPKC) can increase glucose by a transport mechanism
distinct from insulin (2, 18, 36, 58), we explored the potential role
of cPKC in DNP-stimulated glucose transport. On the basis of four lines
of evidence, we propose that DNP, acting through
Ca2+-sensitive PKC, can modify L6
muscle cell glucose transport. 1) The downregulation of cPKC, but not of atypical PKC protein isoforms, decreased DNP-stimulated glucose transport by 45%, with no effect on
insulin-induced glucose uptake. 2)
The DNP-induced rise in glucose transport was lowered by 60% with a
low dose of BIM (1 µM) that is known to effectively inhibit
cPKC, whereas the insulin response was only affected at a far greater
BIM concentration. 3) DNP caused a
rapid translocation of PKC-
, -
, and -
to the cell surface and
brought about their activation. It is conceivable that, in
addition to Ca2+ activation, the
kinase molecules experienced covalent modifications that contributed to
this activation. 4) Using 379196 to
selectively inhibit PKC-
, we observed a partial decrease (67%) in
the stimulation of glucose transport by DNP, which closely approximates
the inhibition observed with BIM treatment (60%). Therefore, we
propose that PKC-
may account for the cPKC isoform participating in
glucose transporter mobilization during metabolic challenge. Previous reports have revealed PKC activation during muscle contraction (12,
50). However, which of the 12 different PKC isoforms was responsible
for this effect was not determined. In light of our findings with
379196, it is plausible that PKC-
may also relay the signal to
glucose transporters in the exercising muscle. If specific antagonists
for the other Ca2+-sensitive PKC
isotypes become available, it will be possible to verify the specific
cPKC mediators of the alternative mechanism of glucose transport activation.
Because PMA-stimulated glucose transport was completely inhibited by 1 µM BIM and PMA downregulation of cPKC and yet no more than 60% of
the stimulation by DNP was inhibited by these manipulations, we
postulate that there may be a PKC-independent component to the
stimulation of glucose uptake by DNP. Conversely, PMA stimulates cPKC
activity by eightfold but is only able to induce a 50% rise in glucose
transport. Therefore, robust activation of PKC alone is not sufficient
to increase glucose transport to levels comparable to those induced by
DNP or insulin. Discrepant effects of phorbol esters, insulin, and
hypoxia on glucose transport have been noted previously (18, 20, 58).
Ca2+ chelation was more effective
than cPKC inhibition in reducing the DNP stimulation of glucose uptake.
However, even this treatment left a residual increase in glucose
uptake. Also, the effect of Ca2+
buffering on DNP action was not enhanced by simultaneous cPKC inhibition or cPKC deletion (Table 1). Assuming that all treatments were fully effective on their targets (i.e., they fully inhibited cPKC
and prevented rises in cytoplasmic
Ca2+, as appropriate), then it is
possible that three types of signals cooperate to bring about the DNP
effect on glucose uptake: cPKC activation, a secondary effect of
Ca2+, and a Ca2+-independent signal. This
concept is illustrated in Fig. 10.

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|
Fig. 10.
Ca2+- and cPKC-dependent
components of DNP-stimulated glucose transport. Whole pie graph
represents 100% of glucose transport stimulated by DNP. Hatched and
stippled regions represent
Ca2+-dependent component (80%) of
this activation. Within this
Ca2+-dependent element, we
postulate that 60% of DNP response (stippled region) is mediated by
cPKCs that are sensitive to intracellular
Ca2+ levels (likely PKC- ).
Remaining Ca2+-independent
component (~20%, open region) involves unknown mediators.
|
|
Using L6 GLUT-4-myc cells, we were able to show that the inhibition of
DNP-stimulated
2-[3H]deoxyglucose
uptake caused by Ca2+ chelation or
by interference with cPKC activation is reflected by a decrease in the
mobilization of GLUT-4-myc to the cell surface. GLUT-4-myc
translocation, assessed by a colorimetric detection assay, was impaired
by these manipulations to nearly the same extent as glucose transport
in wild-type L6 or L6 GLUT-4-myc muscle cells. It was shown previously
that the GLUT-4-myc expressed in L6 myotubes is functional for glucose
uptake and behaves like endogenous GLUT-4 (31, 59). Therefore, the
inhibitory effect of cPKC inhibition or downregulation and
Ca2+ buffering on DNP-stimulated
glucose transport occurred at a signaling step proximal to GLUT-4
translocation rather than at the level of GLUT-4 vesicle docking and
fusion or by a direct effect on glucose transporter activity.
Consistent with a role for PKC stimulation of GLUT-4 vesicle
translocation, numerous early studies report that agents that activate
PKC can stimulate exocytosis in a variety of cell types (26, 28).
Billiard et al. (6) observed that the exocytosis of secretory vesicles
in rat pituitary gonadotropes could be stimulated independently by
either Ca2+ elevations or PKC
activation with PMA. Because the stimulation of glucose uptake by DNP
involves incorporation of GLUT-containing vesicles into the cell
surface (57), the participation of cPKC in this step is a distinct possibility.
Other potential mediators.
The alternative pathway appears to involve
Ca2+-dependent and
Ca2+-independent signals, since
Ca2+ chelation could not fully
inhibit DNP-stimulated glucose transport. From our studies, the origin
and nature of the Ca2+-independent
pathway is not evident, but it does not include the type 1A PI3K-Akt
axis or other wortmannin-sensitive PI3Ks. This pathway probably also
does not include PKC-
, since inhibition of all known subfamilies of
PKC with 10 µM BIM did not further inhibit DNP-stimulated glucose
transport even though it reduced insulin-stimulated glucose transport,
which has been linked in part to a requirement for PKC-
(2, 3) (Fig.
4A). It is noteworthy that
activation of PKC-
by insulin probably occurs via PI3K lipid
products (2, 54).
A major unresolved issue is whether other signaling molecules, in
addition to cPKC, are responsible for mediating the effect of DNP on
glucose transport. Recently, it was proposed that
5'-AMP-activated protein kinase (AMPK) may be involved in
hypoxia- or exercise-stimulated glucose transport but not in the
insulin-dependent pathway (22, 52). Given that metabolic stressors such
as DNP are known activators of AMPK, we tested this hypothesis by
chemically activating AMPK with the AMP analog
5-aminoimidazole-4-carboxamide ribonucleoside (AICAR). Maximal
AICAR-stimulated glucose transport was not additive to insulin- or
DNP-dependent glucose uptake in L6 muscle cells (Khayat and Klip,
unpublished observations). Therefore, the participation of AMPK in the
action of DNP on glucose transport is not likely. Other signals
proposed to lead to glucose transport stimulation during muscle
contraction are nitric oxide (NO) (51) and bradykinin, acting through
the trimeric G protein Gq (31).
Whether these signals are implicated in DNP action is not presently
known. Goodyear et al. (19) have shown that exercise, a physiological
stressor, can activate the stress-activated mitogen-activated protein
kinase (MAPK) p38MAPK in rat
skeletal muscle. Similarly, we have shown rapid phosphorylation of
p38MAPK by DNP in L6 muscle cells
(55). However, DNP-stimulated glucose uptake is likely not dependent on
p38MAPK activity, since the
selective p38MAPK inhibitor
SB-203580 failed to prevent the DNP response of glucose transport
(Khayat and Klip, unpublished observations).
Three recent studies have used two chemical conditions that stimulate
glucose transport independently of insulin signals to elucidate the
molecular mechanisms underlying these distinct pathways. These are
hyperosmolarity and guanosine
5'-O-(3-thiotriphosphate) (GTP
S). In 3T3-L1 adipocytes, osmotic shock- and GTP
S-mediated elevations in GLUT-4 translocation are PI3K independent but are prevented by inhibitors of tyrosine kinases (10) or by microinjection of anti-phosphotyrosine antibodies (16, 21), suggesting that as yet
unidentified tyrosine kinases may be activated by these stimuli and
participate in glucose transport stimulation. In a previous study, we
reported that treatment of L6 cells with DNP does not alter the pattern
of tyrosine-phosphorylated proteins of myotube lysates assayed by
immunoblotting with phosphotyrosine-specific antibodies (57). Indeed,
we have tested three structurally unrelated tyrosine kinase inhibitors,
erbstatin (30 µg/ml), genistein (50 µM), and herbimycin A (50 µM), for inhibitory effects on DNP-stimulated glucose transport. None
were able to reduce the DNP stimulation of 2-deoxyglucose uptake (DNP,
100%; DNP + erbstatin pretreatment, 93.4%; DNP + herbimycin
A pretreatment, 89.2%; DNP + genistein pretreatment, 89.5%), whereas
insulin-dependent glucose uptake was blocked by all three agents
(Khayat and Klip, unpublished results). Therefore, it is not likely
that mitochondrial uncoupling engages tyrosine kinase signaling
pathway(s) similar to those of these other activators of glucose transport.
In summary, the findings presented suggest that DNP may employ
Ca2+ as a secondary messenger to
activate cPKCs, forming part of the alternative signaling system
leading to the regulation of glucose transport by energy demand in L6
muscle cells. This alternative pathway functions independently of the
PI3K signaling pathway utilized by insulin to increase muscle cell
glucose influx.
 |
ACKNOWLEDGEMENTS |
We thank Dr. Philip J. Bilan for useful discussions.
 |
FOOTNOTES |
This work was supported by grants from the Canadian Diabetes
Association and the Eli Lilly/Banting and Best Diabetes Centre (to A. Klip). Z. Khayat was supported by a Natural Sciences and Engineering
Research Council of Canada postgraduate scholarship. T. Tsakiridis was
supported by a fellowship from the Medical Research Council of Canada.
A. Ueyama received financial support from the Otsuka Pharmaceutical
Co., Ltd.
Current address of T. Tsakiridis: Department of Medicine, University of
Toronto, Toronto, ON, M5S 1A8, Canada.
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: A. Klip, Programme in Cell Biology,
Hospital for Sick Children, 555 University Ave., Toronto, ON, Canada
M5G 1X8.
Received 15 June 1998; accepted in final form September 1 1998.
 |
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