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Diabetes Research Laboratory, Winthrop University Hospital, Mineola, New York 11501; and the School of Medicine, State University of New York, Stony Brook, New York 11794
Address all correspondence and requests for reprints to: Dr. Najma Begum, Diabetes Research Laboratory, Winthrop University Hospital, 259 First Street, Mineola, New York 11501. E-mail: diabetes96{at}aol
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Recent evidence suggests that the subcellular location, substrate specificity, and regulation of PP-1 activity in vivo are determined by its interaction with targeting subunits (3). The glycogen-associated form of PP-1 is the best characterized phosphatase to date. The holoenzyme consists of a highly conserved 37-kDa catalytic subunit (PP-1C) complexed to a 160-kDa glycogen-targeting subunit (PP-1G) (3). This subunit directs PP-1C to glycogen-protein particles as well as membranes of the sarcoplasmic reticulum, thereby facilitating the dephosphorylation of glycogen-metabolizing enzymes and sarcoplasmic reticulum proteins (4). In vitro studies with purified preparations of rabbit skeletal muscle PP-1 holoenzyme suggest that this form of PP-1 can be activated or inhibited depending upon site-specific serine phosphorylation of the PP-1G subunit by an insulin-stimulated protein kinase (ISPK) or a cAMP-dependent protein kinase (3, 5, 6). Site 1 phosphorylation of the PP-1G subunit by ISPK stimulates PP-1, causing the activation of glycogen synthase (GS) and concomitant inactivation of phosphorylase kinase (7). In contrast, phosphorylation of site 2 by a cAMP-dependent kinase in response to adrenaline may result in the dissociation of catalytic subunit from regulatory subunit, thereby decreasing PP-1 activity (3, 5, 6). GS kinase-3 can also phosphorylate the PP-1G subunit at yet another serine residue (site 3) in vitro (7). The occurrence and physiological relevance of the above site-specific phosphorylations in terms of enzymatic activation and in vivo insulin action are unclear.
Recent studies from this laboratory have shown that the PP-1G holoenzyme constitutes 40–45% of the cellular PP-1 activity in cultured rat skeletal muscle cells and freshly isolated rat adipocytes (8, 9). Acute exposure of rat skeletal muscle cells and isolated rat adipocytes to insulin results in a rapid activation of the membranous PP-1G (8, 9). In both cell types, PP-1 stimulation is accompanied by increased phosphorylation of the 160-kDa regulatory subunit (8, 9). These observations together with the recent suggestion that the PP-1G subunit may be a candidate gene for inherited insulin resistance (10) prompted us to examine the exact role and importance of the PP-1G subunit on the catalytic and regulatory functions of PP-1 in response to insulin and its impact on glycogen synthesis and glucose transport.
In the present study, we have generated stable L6 rat skeletal muscle cell lines that either overexpress or lack the PP-1G subunit. Our results show that PP-1G subunit overexpression results in increased insulin-stimulated glucose uptake, GS activation, and glycogen synthesis compared to those in neo control cells, and these increases are due to increased activation of the PP-1 catalytic subunit that is bound to the PP-1G subunit. In contrast, the depletion of endogenous PP-1G subunit, using antisense RNA, results in an inhibition of insulin-stimulated glucose uptake, presumably due to impaired PP-1 activation.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-32P]ATP (SA, >3000 Ci/mmol),
[125I]protein A,
D-[U-14C]glucose,
2-deoxy-D-[3H]glucose, and
[U-14C]uridine diphosphoglucose
([U-14C]UDP-glucose) were purchased from DuPont-New
England Nuclear (Boston, MA). Electrophoresis and Bradford protein
assay reagents were obtained from Bio-Rad (Richmond, CA). Bicinchoninic
acid protein assay reagent was purchased from Pierce Chemical Co.
(Rockford, IL). The LacSwitch Inducible Mammalian Expression System,
anti-lac repressor antibody, and
oligo(deoxythymidine)-primed complementary DNA library were purchased
from Stratagene (La Jolla, CA). Okadaic acid was obtained from Moana
Bioproducts (Honolulu, HI). Restriction endonucleases and hygromycin B
were purchased from Boehringer Mannheim (Indianapolis, IN). All other
reagents were obtained from Sigma Chemical Co. (St. Louis, MO). Porcine
insulin was a gift from Eli Lilly Co. (Indianapolis, IN). PP-1C
antibody was purchased from Upstate Biotechnologies (Lake Placid, NY).
Antibodies against PP-1C and PP-1C
were provided by David Mott
(NIDDK, Phoenix, AZ). Antibody against PP-1G subunit were
generated by immunizing rabbits with a synthetic peptide corresponding
to the sequence surrounding site 1 of rabbit skeletal muscle
PP-1G (SPQPSRRGSESSEE). Affinity purification and
characterization of the antibody were performed according to the
protocol described by Hubbard and Cohen (11). Details are also given in
our earlier publications (8, 9).
Cloning and construction of expression vector with
PP-1G complementary DNA (cDNA)
The entire coding sequence of the PP-1G subunit was
isolated from two different rabbit skeletal muscle cDNA libraries using
synthetic oligonucleotides. Initial screening of the
oligo(deoxythymidine) [oligo(dT)]-primed library yielded four
positive clones. A second rabbit skeletal muscle 5'-stretch random
hexamer cDNA library was screened with two synthetic oligonucleotide
probes corresponding to the 5'-terminal nucleotide sequences of the
cDNA. One of the nine positive clones had an in-frame ATG initiation
site and an overlapping 3'-sequence of one of the previous clones
isolated from the oligo(dT)-primed library. Subcloning of the
PP-1G gene into the Lac Switch Inducible Mammalian
Expression System vector pOPI3 was performed by standard techniques,
yielding plasmid pOPI3/PP-1G. This expression vector was
chosen because PP-1G subunit overexpression could be
induced in differentiated cells with the addition of IPTG to the
culture medium.
Transfection and selection of stable cell lines over expressing
PP-1G
The spontaneously fusing rat skeletal muscle cell line, L6, was
a gift from Dr. Amira Klip (The Hospital for Sick Children, Toronto,
Canada). L6 myoblasts at second passage were grown and maintained in
MEM containing 10% FBS. Cells were initially transfected with the
eukaryotic lac repressor expressing vector pP3'SS according
to the manufacturers instructions. Briefly, cells at approximately 40%
confluence were transfected with a mixture of 15 µg pP3'SS DNA and 50
µl Lipofectamine reagent for 15 h. Then, 6 ml MEM were added
to the cells, and the medium was brought to 10% in FBS. After a 6-h
incubation, the medium was removed, and the cells were incubated with
MEM containing 10% FBS and 1% antibiotic/antimycotic mixture.
Approximately 72 h from the start of transfection, the cells were
passaged 1:5 into medium containing 600 µg/ml hygromycin B for
selection. Single stable clones were picked up with cloning discs and
passaged into several 24-well plates for initial amplification.
After 2 additional rounds of amplification, the clones were screened by
immunoblot analysis for expression of the lac repressor
protein using anti-lac antibody. A single clone with the
highest lac repressor protein expression was used as the
parent cell line for transfection with pOPI3/PP-1G as
described above, with the exception of 2 mg/ml G418 used for selection
of stable clones. Initial screening for PP-1G subunit
protein expression was performed by immunoblot analysis of cell
extracts prepared from myotubes (12–14 days in culture, after a 40-h
induction in the absence or presence of IPTG) using PP-1G
site 1 antibody, described in Materials and Methods
and in our previous publications (8, 9). As we indicated previously,
this subunit is expressed in L6 cells upon fusion and differentiation
and is absent in proliferating myoblasts (8). Neo control represents
transfection with the empty expression vector. Transfection per
se did not affect the extent of differentiation of L6 cells, as
monitored by analysis of myogenin protein and cell morphology.
Cell culture
Transfected cell lines were grown and maintained in MEM
containing 10% FBS, 400 µg/ml G418, 150 µg/ml hygromycin B, and
1% antibiotic/antimycotic mixture in an atmosphere of 5%
CO2 at 37 C as previously described (8). Unless otherwise
stated, myotubes were used for all experiments after a 40-h induction
with IPTG and a 15-h starvation in serum free MEM.
Extraction and assay of cellular PP-1 activity
Serum-starved cells were fed serum-free medium containing 5
mM glucose and incubated at 37 C for 1 h. Identical
dishes in triplicate were incubated in the absence and presence of
insulin (0.1 nM to 1 µM) for 10 min. At the
end of the incubation period, the medium was removed, and the cells
were rinsed three times with ice-cold PBS followed by extraction with
PP-1 extraction buffer as detailed previously (8, 9).
Assay of PP-1 activity
The assay was performed as previously described (8, 9) using
32P-labeled glycogen phosphorylase a as a
substrate (12, 13).
Immunoprecipitation of PP-1G and assay of
bound PP-1 catalytic activity
Control and insulin-treated cells were harvested in
ice-cold lysis buffer containing 50 mM Tris (pH
7.4); 1 mM EDTA; 0.5 mM
EGTA; 0.1 mM PhMeSO2F; 10
µg/ml each of leupeptin, aprotinin, antipain, soybean trypsin
inhibitor, and pepstatin A; 100 mM NaCl; and 1% Triton
X-100. The cell lysates were centrifuged at 14,000 x g
for 10 min to remove cell debris. Extraction with Triton X-100 resulted
in complete recovery of the PP-1G subunit in the
supernatant. Cell lysate protein (100 µg) was diluted to 1 ml with
lysis buffer and precleared by incubation with rat IgG (5 µg/ml,
coupled to protein-A Sepharose) at 4 C for 1 h. The supernatants
were immunoprecipitated with nonimmune IgG or PP-1G site 1
antibody (10 µg/ml) for 1 h at 4 C, followed by treatment with
50 µl protein A-Sepharose CL6B (50%, vol/vol) for 1 h. In some
experiments, the antibody was preincubated with the competing peptide
before its addition to cell lysates. The supernatants were saved for
PP-1 assay. The pellets were washed four times with 1 ml wash buffer
(14), resuspended in the same buffer to its original volume, and
incubated with the site 1 peptide (15 µg/ml) for 1 h at 4 C to
release the bound enzyme (8, 9). PP-1 activity was measured on 5 µl
precleared supernatant, the immunodepleted supernatants described
above, and the immunoprecipitates. The activity lost from the
immunodepleted supernatant was comparable to the PP-1 activity
recovered in the immunoprecipitates.
Measurement of 2-deoxy-D-glucose uptake
Uptake of 2-deoxy-D-glucose was measured as
previously described (15) using
2-deoxy-D-[3H]glucose.
Assay of GS activity
GS activity was measured in the presence and absence of 50
mM glucose-6-phosphate (Glc6P) with 0.7 mM
[U-14C]UDP-glucose as a substrate (16). The radioactivity
present on the filters was counted using a ß-scintillation counter.
Insulin-stimulated GS activity (nanomoles of
[U-14C]UDP-glucose incorporated into glycogen per min/mg
protein) was expressed as the fractional activity measured in the
absence of Glc6P divided by the activity measured in the presence of
Glc6P.
Glucose incorporation into glycogen
Glucose incorporation into glycogen was measured using
D-[U-14C]glucose as described previously
(17).
Immunoblot analysis
Culture plates were washed four times with ice-cold PBS followed
by the addition of 200 µl cell lysis buffer containing 50
mM Tris-HCl (pH 7.6); 2.0 mM EDTA; 2.0
mM EGTA; 1.0% SDS; 1.0 mM benzamidine; 2.0
mM phenylmethylsulfonylfluoride; and 10 µg/ml each of
leupeptin, aprotinin, antipain, soybean trypsin inhibitor, and
pepstatin A. The plates were scraped, and the cell lysate was sonicated
and centrifuged at 2000 x g for 5 min. Extraction
resulted in complete recovery of proteins in the supernatant.
Typically, 20 µg protein were mixed with Laemmli sample buffer
containing 0.1% bromophenol blue, 1.0 M
NaH2PO4 (pH 7.0), 50% glycerol, and 10% SDS;
boiled for 5 min; and loaded on a 7.5% SDS polyacrylamide gel (18). In
some experiments PP-1G was immunoprecipitated from equal
amounts of cell lysates prepared from control and insulin-treated
cells. The immunoprecipitates were separated on a 7.5%
SDS-polyacrylamide gel. The separated proteins were transferred to a
polyvinylidene difluoride membrane and probed with PP-1G
antibody followed by detection with [125I]protein A (0.2
µCi/ml) and autoradiography. The intensity of the signal was
quantitated by densitometric analysis of the autoradiograms.
Protein assay
The protein content in the cell extracts was determined with
bicinchoninic acid (19) or Bradford reagent (20).
Statistics
Student’s t test or ANOVA was used to evaluate the
significance of the effects of insulin on PP-1 activity, glucose
transport, GS activity, and glycogen synthesis. Results are expressed
as the mean ± SEM of three or four separate
experiments, each performed on three independent dishes.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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. In IPTG-induced cells, the levels of
PP-1G were 4- to 6-fold higher in all three clones compared
to those in neo control and wild-type L6 cells (compare lanes 3–5
vs. lanes 1 and 2). Transfection alone did not alter the
levels of PP-1G (compare lane 1 vs. lane 2).
Although the noninduced PP-1G level of clone S34 was
comparable to those of L6 and neo controls, clones S10 and S29 had
2-fold higher basal PP-1G levels, presumably due to
incomplete repression by the lac repressor, as observed by
other investigators (21).
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The increased PP-1G expression seen in the three clones was
not due to clonal variability for two reasons: 1) a single L6 clone
expressing the highest levels of lac repressor protein was
amplified and used for transfection with the PP-1G gene, as
demonstrated by the presence of an equal amount of lac
repressor protein in all transfected cell lines (Fig. 1B
); and 2) only
the cell lines transfected with the PP-1G gene demonstrated
increased PP-1G protein expression in response to
IPTG-induction and not the neo controls carrying an empty expression
vector (Fig. 1A).
L6 cells express three isoforms of PP-1 catalytic subunit [PP-1C,
PP-1C, and PP-1C (also called PP-1Cß)] (22). The cellular
levels of all three isoforms were unaltered in cells overexpressing the
PP-1G subunit (data not shown). In addition, cell growth,
morphology, and extent of differentiation (as monitored by the
expression of the L6 differentiation marker myogenin) were comparable
in control and transfected cell lines (data not shown).
Effect of PP-1G overexpression on basal and
insulin-stimulated PP-1 catalytic activities in
PP-1G immunoprecipitates
To determine whether PP-1G overexpression results
in an increase in the bound PP-1 catalytic activity in response to
insulin, serum-starved cells were treated in the absence and presence
of 10 nM insulin for 10 min, and the bound PP-1 catalytic
activity was assayed in the immunoprecipitates of cell lysates, as
detailed in Materials and Methods. Immunoprecipitations were
performed with an antipeptide antibody directed against the site 1
sequence of PP-1G (8, 9). Therefore, the PP-1 activity in
the immunoprecipitates represents the bound form of the enzyme in
vivo.
In neo control cells, insulin treatment caused a 77% increase in
immunoprecipitated PP-1 catalytic activity over basal values, whereas
clones S29 and S34 exhibited an approximately 200% increase in
insulin-stimulated PP-1 activity (Table 1).
Surprisingly, the basal PP-1 catalytic activities of clones S29 and S34
were not significantly different from that of the neo control even
though PP-1G overexpression did result in an increase in
the content of PP-1G bound to PP-1C (data not shown). This
suggests that the increase observed in PP-1 activity in insulin-treated
clones S29 and S34 is due to activation of bound PP-1C and not simply
to binding of the PP-1C subunit to the G-subunit.
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)
demonstrates an increase in PP-1 activation with all insulin
concentrations used (P < 0.05 vs. neo
control). Maximal stimulation was observed with 10 nM
insulin, and the effect was sustained even at high insulin
concentrations, in contrast to control cells, which displayed a
decrease in PP-1 activity at high concentrations of insulin. The reason
for the biphasic effect of insulin seen in wild-type cells in our
earlier studies (8) and in the neo control in the present study is
unclear. A similar effect was observed by Chan et al. in
insulin-treated 3T3 D1 fibroblasts (23).
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). In contrast, in clone
S34, an insulin concentration as low as 1.0 nM resulted in
a 57% increase in GS fractional activity over the basal level. Higher
insulin concentrations of 10–1000 nM resulted in a further
increase in GS fractional activity (104–142% over the basal value).
At all concentrations of insulin tested, except 0.1 nM,
clone S34 exhibited more than a 2-fold higher insulin effect on percent
GS fractional activity compared to neo control cells, with a
significant difference seen from concentrations of 10 nM
insulin or more (Fig. 3). Clone S29 also exhibited similar insulin
dose-response effects on GS fractional activity.
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). This increase in glycogen
synthesis coincides with the observed 36% increase in
insulin-stimulated GS fractional activity shown in Fig. 3. Insulin
caused a 2- to 4-fold increase in glycogen synthesis in clones S29 and
S34, respectively, compared to that in the neo control (Table 2). This
parallels the increase in fractional GS activity observed in these two
clones. Basal glycogen synthesis rates were not significantly different
between the neo control and clones S29 and S34.
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). Preliminary studies suggest that this
increase in insulin-stimulated glucose transport is accompanied by an
increase in plasma membrane Glut-4 (data not shown).
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). Cell growth and extent of differentiation, as
monitored by myogenin expression, were not affected by the antisense
construct (Fig. 5B). This cell line was examined for insulin’s effect
on PP-1 activation, 2-deoxyglucose uptake, and GS activity. As shown in
Fig. 6, PP-1R cells demonstrated a 22% reduction in
basal PP-1 activity and a complete lack of PP-1 activation in response
to varying concentrations of insulin compared to the neo controls. The
content of cellular PP-1C was not different between the neo control
and PP-1R cells (data not shown). The lack of PP-1 activation was
accompanied by a marked decrease in basal and insulin-stimulated
2-deoxyglucose uptake, as evidenced by marked reductions in
insulin-stimulated glucose uptake with varying concentrations of
insulin (Fig. 7), and impaired activation of GS (data
not shown). Detailed studies on the effect of PP-1G
depletion on cellular responsiveness to insulin are in progress.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The increase in insulin-stimulated PP-1 activity observed in recombinant cell lines appears to be due to activation of the bound PP-1 catalytic subunit, possibly via site-specific phosphorylation of the PP-1G subunit in response to insulin. This would be expected because of the availability of an increased number of potential phosphorylation sites due to the increased number of PP-1G molecules. Thus, a 4-fold increase in PP-1G results in a 3-fold stimulation of bound PP-1 activity in insulin-treated clones compared to that in controls. Overexpression of the PP-1G subunit resulted in a corresponding increase in the content of bound PP-1C without an increase in basal PP-1 activity. The reason for this lack of increase in basal PP-1 activity in overexpressors is unclear at present, but could be the result of an adaptive response of the transfected cells.
PP-1G is a highly conserved protein among mammals (28). Earlier studies by Dent et al. (5) have shown that in rabbit skeletal muscle, insulin activates PP-1 by increasing the phosphorylation of site 1 by ISPK, a mammalian homolog of the 90-kDa ribosomal S6 kinase II (Rsk-2) (29). Increased phosphorylation of PP-1G and activation of PP-1 in wild-type L6 cells and isolated rat adipocytes in response to insulin have been recently reported by this laboratory (8, 9). A recent report suggests that insulin activation of glycogen synthesis is mediated via jun N-terminal kinase and the RSK3 pathway in skeletal muscle isolated from insulin-injected mice (30). Other studies indicate that ras/mitogen-activated protein kinase activation is not involved in insulin-stimulated glycogen synthesis and glucose uptake (31, 32). Therefore, the upstream activator of PP-1 still remains unclear.
Regardless of the mechanism of PP-1 activation, it is clear from the present study that the increased insulin activation of PP-1G, in turn, stimulates GS, resulting in a 3-fold increase in glycogen synthesis relative to that in control cells. Furthermore, insulin is also known to inhibit GS kinase-3 by phosphorylation via protein kinase B (33, 34). The combined inhibition of GS kinase-3 and activation of PP-1 (via PP-1G) may contribute to an increase in insulin-induced dephosphorylation and activation of GS, as seen in the present studies. The present findings do not rule out the possibility that insulin may inhibit the breakdown of glycogen in cells overexpressing PP-1G by inhibiting phosphorylase a.
PP-1 activity was assayed using 32P-labeled phosphorylase a as a substrate. Although GS and phosphorylase kinase have been used as substrates by Dent et al. (5) to demonstrate the activation of purified PP-1G, we used glycogen phosphorylase a for the measurement of PP-1 activation by insulin for two reasons: 1) glycogen phosphorylase is phosphorylated at a single serine site by phosphorylase kinase, thereby making it substantially less complicated to analyze the dephosphorylation kinetics of a single phosphoserine residue on phosphorylase a than the dephosphorylation of GS at multiple sites; and 2) these three substrates are dephosphorylated by the same class of phosphatase in most tissues. We recently compared the effects of insulin on PP-1 activation using all three substrates (phosphorylase a, GS, and phosphorylase kinase) (8) and showed that phosphorylase a can be used as a substrate for the measurement of PP-1G activation. Furthermore, 32P-labeled GS and phosphorylase kinase are very unstable even when stored frozen in small aliquots; hence, phosphorylase a was used as a substrate in this study as well as others (8, 9).
In addition to enhanced glycogen synthesis, PP-1G overexpression results in an increase in insulin-stimulated glucose transport. This suggests that PP-1 may play an important role in insulin-signaling pathways of glucose transport.
Although insulin-stimulated GS fractional activity and glucose transport are enhanced in cells overexpressing the G subunit, the increase is not proportional to the increase in PP-1G subunit protein content. A similar phenomenon was observed in transfected skeletal muscle cells and transgenic mice that overexpress Glut-4 in adipose tissue and skeletal muscle (35, 36). In the skeletal muscles isolated from transgenic mice, a 4-fold overexpression of Glut-4 resulted in only a 2-fold increase in insulin-stimulated glucose uptake and a 50% increase in insulin-stimulated glycogen synthesis. The basal glycogen synthesis was not different between control and Glut-4 overexpressors (36). These results suggest the possibility that each cell has a maximum capacity to activate a particular process (i.e. glucose transport or glycogen synthesis). It is known that L6 accumulate a limited amount of glycogen; besides, transfected cells adapt by compromising regulatory pathways during the long selection process used for the generation of stable cell lines.
Consistent with the above findings using overexpressors, we
observed that depletion of endogenous PP-1G by antisense
experiments results in a complete lack of insulin-activated PP-1 and
inhibition of both basal and insulin-stimulated glucose uptake. This
observation suggests that PP-1G is the major phosphatase
regulated by insulin in intact cells. Detailed studies on the impact of
PP-1G depletion on cellular actions of insulin are in
progress and will be reported separately. The results of the antisense
experiments compliment our recent observations on PP-1 inhibition in L6
cells treated with tumor necrosis factor- (15) and several reports
of insulin-resistant human subjects demonstrating reduced basal and
insulin-stimulated PP-1 activities in muscle tissue (37, 38). Thus,
impaired PP-1G activation may provide a potential mechanism
by which insulin-stimulated GS activation is reduced in these subjects
(10, 37, 38).
In summary, the results of present study indicate that PP-1G is the major GS phosphatase regulated by insulin in intact cells, and there is a positive correlation between the level of PP-1G subunit and cellular responsiveness to insulin in a normal insulin target cell. This increased insulin responsiveness may be due to increased activation of bound PP-1 mediated by PP-1G.
Studies with cell lines carrying site-specific mutations of PP-1G may help clarify the functional roles of site 1, site 2, and site 3 phosphorylation and its impact on glucose disposal. Currently, we are in the process of generating these mutants.
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Acknowledgments |
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Footnotes |
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Received January 3, 1997.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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on insulin action in cultured rat skeletal muscle cells.
Endocrinology 137:2441–2445[Abstract]
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