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Volume 272, Number 38,
Issue of September 19, 1997
pp. 23653-23658
©1997 by The American Society for Biochemistry and Molecular Biology, Inc.
Role of Serine/Threonine Protein Phosphatases in Insulin
Regulation of Na+/K+-ATPase Activity in
Cultured Rat Skeletal Muscle Cells*
(Received for publication, April 28, 1997, and in revised form, June 30, 1997)
Louis
Ragolia
§,
Basil
Cherpalis
§,
Malathi
Srinivasan
§ and
Najma
Begum
§¶
From The Diabetes Research Laboratory, Winthrop
University Hospitol, Mineola, New York 11501 and the
§ School of Medicine, State University of New York, Stony
Brook, New York 11704
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENT
REFERENCES
ABSTRACT
In this study, we examined the potential role of
serine/threonine protein phosphatase-1 (PP-1) and PP-2A in the
mechanism of Na+/K+-ATPase activation by
insulin in the rat skeletal muscle cell line L6. Incubation of L6 cells
with insulin caused a time- and dose-dependent stimulation
of ouabain-sensitive plasma membrane Na+/K+-ATPase activity. Pretreatment with
okadaic acid (OA; 0.1-1 µM) or calyculin A (1 µM) blocked insulin's effect on
Na+/K+-ATPase activation. Low concentrations of
OA that specifically inhibit PP-2A were ineffective.
Immunoprecipitation of the enzyme from 32P-labeled cells
with an antibody directed against the -1 subunit of the enzyme
revealed a 60% decrease in 110-kDa protein phosphorylation in
insulin-treated cells. The presence of calyculin A blocked insulin-mediated dephosphorylation of
Na+/K+-ATPase, whereas low concentrations of OA
were ineffective. To further confirm the role of PP-1, we used L6 cell
lines that overexpress the glycogen/SR-associated regulatory subunit of
PP-1, PP-1G. Overexpression of PP-1G resulted
in a 3-fold increase in insulin-stimulated PP-1 catalytic activity.
This was accompanied by a 30% increase in basal
Na+/K+-ATPase activity and a >2-fold increase
in insulin's effect on pump activity. Inhibition of
phosphatidylinositol-3 kinase with wortmannin blocked
insulin-stimulated PP-1 activation as well as the dephosphorylation and
activation of Na+/K+-ATPase. We conclude that
insulin regulates the activity of Na+/K+-ATPase
by promoting dephosphorylation of the subunit via an insulin-stimulated PP-1 and that phosphatidylinositol-3
kinase-generated signals may mediate insulin activation of PP-1 and
Na+/K+-ATPase.
INTRODUCTION
Na+/K+-ATPase is a ubiquitous enzyme
essential for the maintenance of electrolyte balance, preservation of
membrane potential, and control of cellular volume in all tissues (1).
This enzyme is composed of a 112-kDa catalytic subunit, of which
there are three isoforms, and a 35-kDa subunit (two isoforms)
responsible for targeting the subunit to the plasma membrane and
maintaining its structural integrity (1-2). Recent studies on purified
enzyme preparations as well as intact cells indicate that
Na+/K+-ATPase activity may be regulated acutely
by phosphorylation/dephosphorylation reactions (2-5), whereas the
long-term regulation exerted by certain hormones is mediated by changes
in gene expression (2, 6). Thus, phosphorylation of the catalytic
subunit ( subunit) of Na+/K+-ATPase by
protein kinase A and protein kinase C
(PKC)1 in vitro
inhibits enzymatic activity (7). The exact link between the
physiological effects and the direct phosphorylation of
Na+/K+-ATPase is unclear, however.
Insulin acutely stimulates the activities of ouabain-sensitive -1
and -2 isoforms of Na+/K+-ATPase in rat
skeletal muscle and adipocytes (2, 8-10). In adipocytes, the acute
effects of insulin on the Na+/K+ pump are due
to an increase in the Vmax of the -2 isoform
and a decrease in the K0.5Na+ of the -1 isoform (8). In
skeletal muscle, insulin promotes translocation of the -2 and -1
subunits to the plasma membranes (9-10). The exact molecular mechanism by which insulin regulates Na+/K+ pump activity
in these cell types is unclear. In view of the recent reports that
Na+/K+-ATPase activity can be regulated by
phosphorylation/dephosphorylation mechanisms, we have examined
potential roles of serine/threonine protein phosphatase 1 (PP-1) and
PP-2A in the mechanism of insulin activation of
Na+/K+-ATPase in cultured L6 rat skeletal
muscle cells by using two complementary approaches. In the first
approach, L6 cells were pretreated with okadaic acid or calyculin A,
two cell-permeable inhibitors of PP-1 and PP-2A, followed by insulin
treatment and assay of Na+/K+-ATPase activity
in plasma membranes. In the second approach, cellular levels of the
glycogen/SR-associated regulatory subunit of PP-1 (PP-1G)
were altered by gene transfer techniques to stimulate PP-1 catalytic
activity, and the effect of PP-1G overexpression on
insulin-stimulated Na+/K+-ATPase activity was
examined. The premise for these studies is our recent finding that
overexpression of PP-1G increases cellular responsiveness
to insulin via the activation of PP-1 catalytic function (11). We
hypothesized that if PP-1 is indeed involved in insulin activation of
Na+/K+-ATPase, then increasing the catalytic
activity of PP-1 via its regulatory subunit should increase the
activation of Na+/K+-ATPase by enhancing
dephosphorylation of the subunit.
The results presented in this study indicate that dephosphorylation of
the subunit of Na+/K+-ATPase by
insulin-stimulated PP-1 may be one of the mechanisms by which insulin
regulates the activation of the Na+/K+ pump in
L6 cells.
MATERIALS AND METHODS
Materials
Cell culture reagents, fetal bovine serum,
LipofectAMINETM, calyculin A (caly-A), G418, phosphorylase
b, phosphorylase kinase, and antibiotics were purchased from
Life Technologies, Inc. [ -32P]ATP (specific
activity 3000 Ci/mmol), [32P]orthophosphoric
acid, and 125I-protein A were purchased from DuPont NEN.
Electrophoresis and protein assay reagents were from Bio-Rad. Okadaic
acid was from Moana Bioproducts (Honolulu, HI). Ouabain, pyruvate
kinase, lactate dehydrogenase, ATP, NADH, phospho(enol)pyruvate, and
all other reagents were from Sigma. Monoclonal antibodies against -1
(McK1) and -2 (McB2) subunits of
Na+/K+-ATPase were kindly given by Dr. Kathleen
Sweadner (Massachusetts General Hospital, Charlestown, MA). Porcine
insulin was a kind gift from Eli Lilly Co. (Indianapolis, IN). The Lac
SwitchTM inducible mammalian expression system vector pOPI3
was purchased from Stratagene (La Jolla, CA).
Experimental Procedures
Cell Culture
Spontaneously fusing L6 rat skeletal muscle
cells (a kind gift from Dr. Amira Klip, The Hospital for Sick Children,
Toronto, Canada) were seeded in 100-mm dishes at a density of 1 × 106 cells/ml and maintained in -minimum Eagle's medium
containing 2% fetal bovine serum and 1% antibiotic/antimycotic
mixture in an atmosphere of 5% CO2 at 37 °C as
described previously (12). Completely differentiated myotubes were used
for all experiments after a 16-18-h starvation in serum-free
Dulbecco's modified Eagle's medium.
Generation of Stable Cell Lines Overexpressing
PP-1G
Stable L6 cell lines overexpressing
PP-1G were generated by transfection of L6 myoblasts with
PP-1G cDNA using an
isopropyl-1-thio- -D-galactopyranoside-inducible mammalian expression vector as detailed in our recent publication (11).
Experiments were performed on myotubes after a 40-h induction with
isopropyl-1-thio- -D-galactopyranoside. Neo control
represents transfection with an empty expression vector. As detailed in
our recent publication (11), transfection per se did not
affect the extent of differentiation of L6 cells as monitored by
analysis of myogenin protein and cell morphology.
Insulin Treatment and Isolation of Crude Plasma
Membranes
Serum-starved myotubes were fed with serum-free medium
containing 5 mM glucose and incubated at 37 °C for
1 h before treatment with insulin or other agents. Identical
dishes were pulsed with insulin (0.1-1000 nM) for 0-30
min. In some experiments, to evaluate the effects of phosphatase
inhibitors on Na+/K+-ATPase enzymatic activity,
the cells were preincubated with okadaic acid (0.1-1000
nM), caly-A (1 µM), or tautomycin (3 nM) for 30 min, followed by the addition of insulin (100 nM) for 15 min. At the end of the incubation period, the
medium was removed, and the cells were rinsed three times with ice-cold
PBS and finally scraped off the dishes in 1-2 ml of ice-cold isolation
buffer containing 15 mM Tris base; 5 mM EGTA;
300 mM D-mannitol, pH 7.0, with 10 µg/ml each
of aprotinin, leupeptin, antipain, and soybean trypsin inhibitor; 1 mM benzamidine; and 1 mM phenylmethylsulfonyl fluoride. The cells were sonicated for 10 s and centrifuged at 8000 × g for 20 min at 4 °C. The supernatants were
centrifuged at 100,000 × g for 20 min to pellet the
membrane fraction (13). The crude plasma membranes were reconstituted
in 200 µl of ATPase assay buffer containing 100 mM
imidazole, 5 mM MgCl2, 100 mM
NH4Cl, 150 mM NaCl, 1 mM EGTA, 2 mM sodium azide, and a mixture of the protease inhibitors
indicated above. Aliquots of membrane preparations were used for the
assay of proteins and Na+/K+-ATPase activity.
Unless otherwise stated, all enzymatic assays were performed
immediately after the isolation of membrane fractions.
Assay of Na+/K+-ATPase
Activity
Enzyme activity was measured by monitoring the
hydrolysis of ATP to ADP using the coupled assay of Barnett (14).
Oxidation of NADH to NAD+ was followed on a Beckman DU 640 spectrophotometer at a wavelength of 340 nm. Specific
Na+/K+-ATPase activity was taken as a
difference in activity in the absence and presence of 1 mM
ouabain. At this concentration, ouabain inhibits the subunits of
the enzyme. Results of enzyme activity are expressed as µmol of NADH
converted to NAD+/mg of protein/h.
Phosphorylation Studies
Differentiated L6 cells in 100-mm
dishes were serum-starved overnight for 16-18 h. The next day the
medium was removed and replaced with 4 ml of phosphate-free Dulbecco's
modified Eagle's medium and incubated for an additional 1 h.
[32P]Orthophosphate (1 mCi/dish) was added, and the cells
were incubated for 4 h and then exposed to OA (final
concentration, 10 nM, to inhibit PP-2A) or caly-A (1 µM, to inhibit PP-1 and PP-2A) for 30 min. Insulin (100 nM) was added for 15 min. The cells were rinsed four times
with ice-cold PBS containing protease and phosphatase inhibitors and
harvested. Crude plasma membranes were prepared as described above and
dissolved in 0.5 ml of lysis buffer containing 20 mM
triethanolamine, pH 7.2; 1 mM dithiothreitol; 0.5 mM EGTA; 2 mM sodium vanadate; 100 mM sodium pyrophosphate; 100 mM sodium fluoride; 40 mM -glycerophosphate; 1 mM
benzamidine; 0.1 mM phenylmethylsulfonyl fluoride; 10 µg/ml each of aprotinin, leupeptin, pepstatin A, antipain, and
soybean trypsin inhibitor; 100 mM NaCl; and 1% Triton X-100. The cell membrane lysates (100 µg of protein) from above were
precleared by incubation with mouse IgG (5 µg/ml coupled to
anti-mouse IgG agarose at 4 °C for 1 h. The supernatants were immunoprecipitated with anti- -1
Na+/K+-ATPase antibodies precoupled to
anti-mouse IgG agarose at room temperature for 1 h. The pellets
were washed four times with lysis buffer and resuspended in 30 µl of
2 × LSB. The samples were incubated at 37 °C for 10 min followed by
centrifugation (10,000 × g for 30 s) to pellet
down the agarose beads. Electrophoresis of the immunoprecipitates was
performed in 10% SDS-polyacrylamide gels followed by autoradiography
(15). The protein contents of the subunit of
Na+/K+-ATPase were determined by
immunoprecipitating unlabeled plasma membranes with the antibodies as
described above followed by separation of the immunoprecipitates on
SDS-polyacrylamide gel electrophoresis. After transferring the proteins
to polyvinylidene difluoride membranes, the blots were probed with
-1 subunit antibody (16).
Immunoprecipitation of PP-1G and Assay of Bound PP-1
Catalytic Activity
Immunoprecipitation of PP-1G and
the assay of PP-1 catalytic activity in the immunoprecipitates were
performed as described earlier (11, 12) using 32P-labeled
glycogen phosphorylase a as a substrate (12).
Protein Assay
The protein contents in the cell extracts
were determined by the bicinchoninic acid (17).
Statistics
Student's t test or analysis of
variance was used to evaluate the significance of the effects of
insulin and phosphatase inhibitors on enzyme activity.
RESULTS
Activation of Ouabain-sensitive
Na+/K+-ATPase by Insulin
In the initial
series of experiments, we studied the kinetics and insulin
dose-response of Na+/K+-ATPase activity using
the enzymatic assay described under "Materials and Methods." In L6
cell membrane preparations, ouabain-sensitive Na+/K+-ATPase activity represents 40% of
total ATPase activity. Acute exposure of L6 myotubes to 100 nM insulin for 15 min resulted in an approximate 2-fold
stimulation of ouabain-sensitive Na+/K+-ATPase
activity over the basal values (insulin versus basal values, 104.6 ± 6.6 versus 53.1 ± 4.83 µmol of
NAD+ formed/mg protein/h, respectively). Insulin treatment
did not affect ouabain-resistant enzyme activity. Kinetic studies
indicate that the half-maximal insulin effect was observed within 5 min of insulin addition, with a peak activation at 15 min with 100 nM insulin (Fig. 1). The
effect of insulin on pump activation was transient, returning to basal
values at 30 min. The stimulation of
Na+/K+-ATPase activity by insulin was
concentration dependent with an EC50 of <1 nM
insulin, and a maximum effect was seen at a concentration of 100 nM insulin (Fig. 2).
Fig. 1.
Time course of the insulin effect on
ouabain-sensitive Na+/K+-ATPase activity.
L6 cells were exposed to 100 nM insulin for 1-30 min.
Crude plasma membranes were isolated from cell extracts, and
Na+/K+-ATPase activity was assayed in the
presence and absence of ouabain. Results are the mean ± S.E. of
three experiments in duplicate. Asterisks (*) denote
p < 0.05 versus basal activity at zero
time.
[View Larger Version of this Image (13K GIF file)]
Fig. 2.
Dose-response of insulin on
Na+/K+-ATPase activity. Cells were exposed
to varying concentrations of insulin for 15 min, followed by an enzyme
assay. Results are the mean of two different experiments, each
performed on duplicate dishes.
[View Larger Version of this Image (13K GIF file)]
Protein Phosphatase Inhibitors Block Insulin-mediated Activation of
Na+/K+-ATPase Pump
To understand the
mechanism of insulin's effect on pump activation, we used specific,
cell-permeable inhibitors of serine/threonine protein phosphatases.
Preincubation of L6 cells with okadaic acid (1 µM),
caly-A (1 µM), or tautomycin (3 nM) for 30 min followed by insulin treatment for 15 min caused a greater than 85%
inhibition of insulin's effect on
Na+/K+-ATPase activation (Fig.
3). The addition of phosphatase
inhibitors 5 min after insulin treatment and a further incubation for
15 min completely reversed insulin's effect on pump activity (Fig. 3;
p < 0.05) and decreased the enzyme activity below
control levels. OA and caly-A alone caused 20% decrease in basal
Na+/K+-ATPase activity.
Fig. 3.
Phosphatase inhibitors block insulin
stimulation of Na+/K+-ATPase activity. L6
cells were pretreated with OA (1 µM), calyculin A (1 µM), or tautomycin (3 nM) for 30 min,
followed by the addition of insulin (100 nM) for 15 min.
Results are the mean ± S.E. of 10 independent experiments
performed in duplicate. Asterisks: *, p < 0.05 versus control; **, p < 0.05 versus insulin. Addition of phosphatase inhibitors at 5 min
after insulin stimulation reverses insulin's effect on
Na+/K+-ATPase activity. L6 cells were treated
with insulin for 5 min, followed by the addition of phosphatase
inhibitors for 15 min.
[View Larger Version of this Image (37K GIF file)]
Dose-response analysis of OA on Na+/K+-ATPase
activation revealed that pretreatment of cells with low concentrations
of OA (0.1-10 nM), which are known to specifically inhibit
PP-2A (12, 18, 19), did not affect insulin's stimulation of pump
activity in L6 cells (Fig. 4). As the
concentration of OA was increased to concentrations that inhibit both
PP-1 and PP-2A, there was a reduction in insulin's effect on
Na+/K+-ATPase. At a concentration of 1 µM that is known to inhibit both PP-1 and PP-2A, OA
blocked insulin-stimulated Na+/K+-ATPase
activity by 80% (Fig. 4). This data further confirms the results shown
in Fig. 3 with tautomycin, another cell-permeable phosphatase inhibitor
that completely inhibits PP-1 at low concentrations (3 nM) without affecting PP-2A activity (19).
Fig. 4.
Effect of various concentrations of OA on
insulin-stimulated Na+/K+-ATPase activity.
L6 cells were pretreated with various doses of OA (0-1000
nM) for 30 min before insulin treatment, followed by the
assay of Na+/K+-ATPase activity in plasma
membranes. Results are the mean ± S.E. of three separate
experiments performed in duplicate and are expressed as the percentage
of insulin-stimulated Na+/K+-ATPase activity.
Asterisks (*) denote p < 0.05 versus insulin.
[View Larger Version of this Image (14K GIF file)]
Insulin-mediated Activation of
Na+/K+-ATPase Is Accompanied by a Decrease in
the Phosphorylation Status of the Subunit of
Na+/K+-ATPase
To examine whether insulin
activation of Na+/K+-ATPase is due to
dephosphorylation of the subunit, L6 cells were metabolically labeled with [32P]orthophosphate, treated with or without
caly-A (1 µM) or OA (10 nM) for 30 min, and
then treated with insulin for 15 min. Equal amounts of precleared cell
lysates (100 µg of protein) were immunoprecipitated with a monoclonal
antibody directed against the -1 subunit of
Na+/K+-ATPase (the predominant isoform present
in L6 cells; see Ref. 2). The immunoprecipitates were separated by
SDS-polyacrylamide gel electrophoresis, followed by autoradiography. As
seen in Fig. 5A, the
immunoprecipitated subunit contained 32P in the basal
state (Fig. 5A, lane 1). Insulin treatment
decreased the amount of 32P in the 110-kDa -1 subunit of
Na+/K+-ATPase when compared with that of
controls (Fig. 5A, compare lane 2 versus lane 1).
Pretreatment with caly-A (1 µM) prevented insulin-induced
decreases in phosphorylation (Fig. 5A, lane 3), whereas low concentrations of OA were ineffective in blocking insulin-induced dephosphorylation (compare lane 4 versus lane 2). The insulin-induced decrease in the phosphorylation status of
the -1 subunit seen in Fig. 5A was not due to variations
in the amount of -1 subunit protein immunoprecipitated from
insulin-treated samples (Fig. 5B). Quantitation of the
extent of phosphorylation of the subunit by densitometric analyses
of the autoradiograms from different experiments after normalization
for -1 subunit proteins revealed a 60% decrease in phosphorylation
status of the 110-kDa -1 subunit in insulin-treated cells when
compared with that of controls (Fig. 5C). Pretreatment with
caly-A prevented insulin-mediated reductions in -1 subunit
phosphorylation and caused an increase in the phosphorylation of the
catalytic subunit above the basal levels. In contrast, low
concentrations of OA did not effectively prevent insulin-stimulated
dephosphorylation.
Fig. 5.
Effect of insulin on the phosphorylation
status of the Na+/K+-ATPase -1 isoform.
32P-labeled cells were exposed to OA (10 nM) or
calyculin A (1 µM) for 30 min before treatment with
insulin (100 nM) for 15 min. Equal amounts (100 µg of
protein) of precleared cell lysates were immunoprecipitated with a
monoclonal antibody against the -1 isoform of the enzyme, followed
by SDS-polyacrylamide gel electrophoresis and autoradiography.
A, an autoradiogram from a representative experiment is
shown. Lane 1, control; lane 2, insulin;
lane 3, caly-A + insulin; and lane 4, OA + insulin. B, Western blot analysis of the immunoprecipitates
from unlabeled cells for quantitation of the amount of -1 isoform of
Na+/K+-ATPase. Lane order is identical to that
of Fig. 6A. C, quantitation of the specific
activity of the Na+/K+-ATPase -1 subunit.
Autoradiograms of 32P-labeled -1 subunit and Western
blots of -1 protein were scanned for optical density. Specific
activities in arbitrary units were calculated by dividing the values of
32P-labeled bands by those of protein bands. To compare
results from different experiments, specific activities from
preparations were assigned a value of 1, and the rest of the data were
expressed relative to control specific activity. Results are the
mean ± S.E. of four independent experiments. *,
p < 0.05 versus control.
[View Larger Version of this Image (40K GIF file)]
Overexpression of PP-1G Subunit Increases Basal and
Insulin-stimulated Na+/K+-ATPase
Activity
The results of the metabolic labeling studies of the subunit of Na+/K+-ATPase along with the results
on the effect of the phosphatase inhibitors implied that insulin's
effect on dephosphorylation and/or activation of
Na+/K+-ATPase subunit may be mediated via
an activated protein phosphatase. This observation together with our
recent studies that treatment of L6 cells with insulin results in a
rapid activation of a particulate form of protein phosphatase-1 (PP-1,
which is similar to glycogen/SR-associated PP-1 (PP-1G)
present in skeletal muscle; Ref. 20) led us to examine the exact role
of PP-1 in insulin-mediated dephosphorylation and activation of
Na+/K+-ATPase. For this, we used the recently
generated L6 cell lines (clones S29 and S34 that overexpress the
glycogen/SR-associated regulatory subunit of PP-1, PP-1G).
As detailed in our recent publication (11), overexpression of
PP-1G did not alter cell growth or morphology but resulted
in a small increase in basal PP-1 activity and a >2-fold increase in
insulin-stimulated PP-1 catalytic activity when compared with wild-type
L6 cells and neo controls carrying an empty expression vector. This
increase in PP-1 activity was accompanied by a 30% increase in the
basal Na+/K+-ATPase activity (when compared
with wild-type L6 cells and neo controls) and a 2-fold increase in
insulin-stimulated ouabain-sensitive Na+/K+-ATPase activity (Fig.
6). Ouabain-resistant
Na+/K+-ATPase activity was not affected by
overexpression of PP-1G (data not shown).
Fig. 6.
Overexpression of PP-1G results
in increased insulin-stimulated Na+/K+ATPase
activity. Results are the mean ± S.D. of two separate experiments, each performed on duplicate dishes.
Asterisks: *, p < 0.05 versus control; **, p < 0.05 versus control; ***, p < 0.05 versus insulin.
[View Larger Version of this Image (28K GIF file)]
Inhibition of PI3 Kinase by Wortmannin Abrogates Insulin-stimulated
PP-1 Activation and Na+/K+-ATPase
Activity
To gain insight into the intracellular upstream
signaling components that mediate insulin's effect on PP-1 and
Na+/K+-ATPase activation, we pretreated L6
cells with wortmannin, a selective and potent inhibitor of PI3 kinase.
Pretreatment with wortmannin blocked insulin's effects on PP-1
activation (Fig. 7A) and
prevented Na+/K+-ATPase stimulation by insulin
(Fig. 7B) as well as dephosphorylation of its subunit
(Fig. 7B, inset). Wortmannin alone did not affect the basal activities of PP-1 or Na+/K+-ATPase.
In contrast, rapamycin, a p70S6Kinase inhibitor at a
concentration of 20 ng/ml did not block insulin's effect on PP-1
activation and Na+/K+-ATPase activity (data not
shown).
Fig. 7.
Inhibition of PI3 kinase blocks insulin's
effects on PP-1 activation (A) as well as
Na+/K+ATPase activation (B).
L6 cells were treated with wortmannin (50 nM) for 30 min
before treatment with insulin (100 nM) for 5 min for PP-1
or 15 min for Na+/K+ATPase assay. PP-1 activity
was measured on cell extracts (A), whereas
Na+/K+ATPase was measured in plasma membranes.
Results are the mean ± S.E. of four separate experiments, each
performed on duplicate dishes. Asterisks: *,
p < 0.05 versus control; **,
p < 0.05 versus insulin. Inset,
an autoradiogram showing the inhibition of insulin-mediated dephosphorylation of the -1 isoform of
Na+/K+-ATPase immunoprecipitated from L6 cells
treated with wortmannin.
[View Larger Version of this Image (52K GIF file)]
DISCUSSION
The results presented in this study indicate that the acute
stimulatory effects of insulin on Na+/K+-ATPase
activity are mediated by the dephosphorylation of its catalytic subunit. The activation mechanism observed in the present study is in
addition to the previously reported effects of insulin on the
K0.5Na+ and Vmax of the -1 and
-2 isoforms, respectively, in adipocytes and the subcellular
redistribution of the enzyme in skeletal muscle cells (7-10).
The effects of insulin on the dephosphorylation and activation of the
subunit of Na+/K+-ATPase seem to be
mediated via the activation of PP-1. This is supported by the kinetics
of insulin-activated Na+/K+-ATPase and PP-1,
insulin dose-response data, the inhibition of insulin's effect on pump
activity by high concentrations of OA and caly-A, and, most
importantly, the absence of inhibition by low concentrations of OA,
which specifically inhibit PP-2A (18, 19). Thus, these results provide
evidence that Na+/K+-ATPase catalytic activity
is regulated by insulin in vivo via a complex
dephosphorylation/phosphorylation mechanism involving PP-1 activation
and presumably inactivation of protein kinase A.
Recent work by a number of laboratories using a variety of cell types
has shown that phosphorylation of the
Na+/K+-ATPase subunit by agonists or
hormones that elevate protein kinase A, PKC, phospholipase A2, and
intracellular calcium will all inhibit its catalytic activity (21-23).
However, the inhibition observed by these agonists seems to be cell
type specific. Thus, PKC activation results in an elevation of pump
activity in rat primary skeletal muscle cells (2). Nonetheless, studies
with purified preparations of Na+/K+-ATPase subunit have shown that incubation with protein kinase A or PKC results
in phosphorylation and inactivation of the enzyme (6). In support of
our observations on the inhibitory effects of PP-1 inhibitors on
insulin-stimulated Na+/K+-ATPase, studies by Li
et al. (23) have demonstrated an inhibition of
Na+/K+-ATPase by phosphatase inhibitors and
fenoldopam, a D1 receptor agonist in rat renal tubules. These authors
suggest that inhibition of PP-1 via DARPP32 (the endogenous phosphatase
inhibitor, analogous to inhibitor 1) may be one of the mechanisms by
which dopamine inhibits the pump activity of
Na+/K+-ATPase (23-24).
Further support for a role for PP-1 in insulin-mediated activation of
the Na+/K+-ATPase pump is provided by our
studies with two L6 clonal cell lines that overexpress the regulatory
subunit of PP-1, PP-1G (11). As detailed in our earlier
studies (12), insulin stimulates the glycogen/SR-bound PP-1 in L6 cells
without affecting the other forms of phosphatases. Overexpression of
PP-1G resulted in a small but significant increase in the
basal activity of PP-1 as well as basal
Na+/K+-ATPase catalytic activity. Insulin
treatment of L6 cells overexpressing PP-1G exhibited a
>2-fold increase in ouabain-sensitive
Na+/K+-ATPase catalytic activity when compared
with neo controls and wild-type L6 cells. This increase in catalytic
activity parallels a 2-3-fold increase observed in insulin-stimulated
PP-1 activity. Furthermore, the kinetics of PP-1 activation by insulin
(see Ref. 12) precedes the kinetics of
Na+/K+-ATPase pump activation. These
observations are further strengthened by the fact that insulin
specifically activates PP-1 and simultaneously inhibits PP-2A activity
in L6 cells (12), and low concentrations of OA fail to block
insulin-stimulated pump activation. Agents that cause PP-1 activation
also cause stimulation of Na+/K+-ATPase
activity. We have recently shown that acute stimulation of L6 cells
with TPA, a PKC activator, results in PP-1 activation and that the
effects of insulin and TPA on PP-1 activation were not additive,
suggesting a common mechanism via PKC activation (25). In addition,
down-regulation of PKC by chronic treatment with TPA, as well as
inhibition of PKC with synthetic inhibitors, blocks insulin-stimulated
PP-1 activation (25). Although we did not measure
Na+/K+-ATPase activity in TPA-treated L6 cells,
recent studies with primary cultures of rat skeletal muscle have
demonstrated an activation of ouabain-sensitive
Na+/K+-ATPase by TPA and insulin (26), and
these effects could be reduced by the inhibition or down-regulation of
PKC. Thus, it seems that PP-1 not only plays a major role in glycogen
synthesis but also has a role in the regulation of ion channels. These
studies provide a new mechanism for the in vivo hormonal
regulation of intracellular Na+ and K+
homeostasis, cell volume, and resting membrane potential in skeletal muscle.
We have previously reported that insulin resistance induced by
elevations in intracellular calcium, as well as streptozoticin diabetes, is accompanied by inhibition of PP-1 activities in adipocytes and skeletal muscle (27-29). Interestingly, both of these conditions were accompanied by reductions in Na+/K+-ATPase
activity in adipocytes, skeletal muscle, and aortic tissue (13,
29-32). This lends further support to the observation that inhibition
of PP-1 results in impaired activation of
Na+/K+-ATPase, and this defect may contribute
to the early muscle fatigue associated with diabetic complications such
as neuropathy.
In intact skeletal muscle, Na+/K+-ATPase units
have been found to be present in either latent or intracellular storage
pools (2). Insulin is known to increase the number of
Na+/K+-ATPase units on the plasma membrane in
skeletal muscle but not in adipose tissue (2, 9-10). It is presently
not known whether PP-1 plays a role in insulin regulation of
subcellular distribution of Na+/K+-ATPase by
promoting reinsertion into the plasma membrane and/or increasing its
pumping efficiency. Additional studies are warranted to understand the
role of PP-1 in insulin-mediated translocation of
Na+/K+-ATPase from the intracellular
compartment to the plasma membrane.
To examine the upstream insulin signaling components that mediate
insulin's effect on PP-1 as well as
Na+/K+-ATPase activation, we pretreated L6
cells with nanomolar concentrations of wortmannin, a selective
inhibitor of PI3 kinase (33). This inhibitor not only blocked the
activation of PP-1 by insulin but also prevented insulin's effects on
Na+/K+-ATPase pump dephosphorylation and
therefore blocked activation. Thus it seems that insulin signaling via
a PI3 kinase pathway stimulates PP-1, presumably by activating PKC
and/or protein kinase B (c-Akt) (the downstream target of PI3 kinase),
which in turn may increase phosphorylation of PP-1G,
leading to activation of PP-1. Earlier studies from this laboratory
have shown that insulin- as well as TPA-mediated activation of PP-1 was
accompanied by phosphorylation of PP-1G and also that
insulin-mediated PP-1G phosphorylation/activation could be
prevented by inhibitors of PKC (25). Also, recent studies have
demonstrated a role for PKB (c-Akt) in insulin-mediated inhibition of
glycogen synthase kinase-3. It is not known presently whether or not
PKB (c-Akt) is the upstream activator of PP-1.
In summary, the results of the present studies indicate that acute
regulation of Na+/K+-ATPase by insulin in
cultured skeletal muscle cells is mediated by dephosphorylation of the
subunit via an activated PP-1G. Activation of
PP-1G and Na+/K+-ATPase by insulin
is dependent in part upon PI3 kinase-generated signals.
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. Section
1734 solely to indicate this fact.
¶
To whom correspondence should be addressed: The Diabetes
Research Laboratory, Winthrop University Hospital, 259 First St., Mineola, NY 11501. Tel.: 516-663-3915; Fax: 516-663-4780; E-mail: diabetes96{at}aol.com.
1
The abbreviations used are: PKC, protein
kinase C; PP, protein phosphatase; OA, okadaic acid; caly-A, calyculin
A; PBS, phosphate-buffered saline; PI3, phosphatidylinositol-3;
PKC, protein kinase B; TPA, 12-O-tetradecanoylphorbol-13-acetate.
ACKNOWLEDGEMENT
We thank Dr. Amira Klip for helpful
suggestions in the preparation of this manuscript.
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