Effect of an Asp905Tyr Mutation of the Glycogen-Associated Regulatory Subunit of Protein Phosphatase-1 on the Regulation of Glycogen Synthesis by Insulin and Cyclic Adenosine 3',5'-Monophosphate Agonists

Louis Ragolia, Noreen Duddy and Najma Begum

Diabetes Research Laboratory (L.R., N.D.) Winthrop University Hospital Mineola, New York 11501
School of Medicine (N.B.) State University of New York Stony Brook, New York 11794


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The glycogen-associated regulatory subunit of protein phosphatase-1 (PP-1G) plays a major role in insulin-stimulated glycogen synthesis and thus the regulation of nonoxidative glucose disposal in skeletal muscle. In a general population of Caucasians a polymorphism at codon 905 of PP-1G from an aspartate to tyrosine has been reported to be associated with insulin resistance and hypersecretion. In this report functional studies were performed on rat skeletal muscle L6 cells stably transfected with an Asp905Tyr mutant PP-1G to evaluate the impact of this mutation on cellular responsiveness to insulin and cAMP. Although transfection resulted in a 3-fold increase in mutant PP-1G subunit expression, basal and insulin-stimulated PP-1 catalytic activities were decreased when compared with L6 cells transfected with wild-type PP-1G. The Asp905Tyr mutation resulted in an increase in cellular sensitivity to cAMP agonist, resulting in an inhibition of insulin’s stimulatory effect on glycogen synthesis. More importantly, low concentrations of (Bu)2cAMP completely reversed insulin’s stimulatory effects on glycogen synthesis when added to insulin-treated cells expressing mutant PP-1G. This was due to a rapid activation of glycogen phosphorylase a and a simultaneous inactivation of glycogen synthase via cAMP-mediated reductions in insulin-stimulated PP-1 catalytic activities. We conclude that an Asp905Tyr mutation of PP-1G is accompanied by a relative increase in sensitivity to cAMP agonists as well as a diminished capacity of the mutant PP-1G to effectively mediate the inhibitory effects of insulin on glycogen breakdown via PP-1 activation.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Skeletal muscle is the major site of insulin-stimulated glucose uptake and nonoxidative glucose disposal (1, 2, 3, 4). Insulin mediates these metabolic effects by promoting the net dephosphorylation of key regulatory enzymes and substrates of both glucose and fat metabolism via the activation of type 1 serine/threonine phosphatase (PP-1) (5). The exact role of PP-1 in insulin action and the precise molecular mechanism by which insulin regulates PP-1 catalytic function is still unclear.

It is well known that the substrate specificity and subcellular localization of PP-1 is dictated by its targeting subunits. In mammals three tissue-specific proteins have been identified that target PP-1 to glycogen (5). PP-1G (also known as RG1) encodes a 124-kDa protein product that is expressed in skeletal muscle and heart (5, 6). GL encodes a 33-kDa protein that is expressed exclusively in liver (7). PTG encodes a protein that is expressed in all tissues except the testis, being most abundant in skeletal muscle, liver, adipose tissue, and heart (8).

The glycogen-associated form of PP-1 derived from skeletal muscle is the best characterized phosphatase to date. The PP-1 holoenzyme consists of a highly conserved 37-kDa catalytic subunit (PP-1C) and a 124-kDa regulatory subunit (PP-1G, migrating as a 160-kDa subunit on SDS-PAGE) (5, 9). The PP-1G subunit binds to glycogen with high affinity and directs PP-1C to glycogen protein particles, thereby enhancing dephosphorylation of glycogen-bound PP-1 substrates such as glycogen synthase (GS), glycogen phosphorylase kinase, and glycogen phosphorylase a (5, 9, 10). Phosphorylation at site 1 (serine46) of the PP-1G subunit in response to insulin enhances the activity of the holoenzyme toward glycogen-bound substrates, while phosphorylation of PP-1G at site-2 (serine65) in response to adrenalin causes inhibition of glycogen synthesis and stimulation of glycogenolysis (5, 9, 10, 11). Thus, the PP-1G subunit plays a key role in the control of glycogen synthesis by insulin and counterregulatory hormones, thereby participating in nonoxidative glucose disposal (5, 9, 11, 12).

We have recently demonstrated that insulin rapidly activates the glycogen-associated form of PP-1 in cultured L6 rat skeletal muscle cells (13). PP-1 activation is accompanied by an increased phosphorylation of PP-1G (13). Modulation of the levels of this subunit either by overexpression of recombinant PP-1G or by depletion of endogenous PP-1G using an antisense RNA strategy results in increased activation or inhibition of insulin-stimulated glucose uptake and glycogen synthesis (14). These alterations in insulin responsiveness are due to activation or inhibition of the PP-1 catalytic subunit that is bound to PP-1G (13, 14). Also, studies on Pima Indians indicate that insulin resistance in these subjects is accompanied by marked reductions in basal and insulin-stimulated skeletal muscle PP-1 catalytic activities and impaired GS activation despite elevations in the contents of the PP-1C subunit and GS (15, 16).

Functional alterations of PP-1 may be responsible for impaired insulin-stimulated glycogen synthesis in skeletal muscle, which is characteristic of insulin-resistant individuals. Due to conflicting reports between Caucasian subjects with the Asp905Tyr polymorphism exhibiting insulin resistance and hypersecretion of insulin (17) and recent studies in a Japanese population indicating that this mutation is found in 70% of healthy individuals and, therefore, is not associated with insulin resistance (18), we attempted to evalute the impact of the PP-1G Asp905Tyr mutation on cellular responsiveness to insulin.

The results of the functional studies with L6 rat skeletal cells stably expressing Asp905Tyr mutation of PP-1G indicate that an Asp905Tyr mutation of PP-1G is indeed accompanied by increased sensitivity to cAMP agonists in terms of GS activation and glycogen synthesis.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Expression of Flag-Tagged Wild-Type PP-1G and Asp905Tyr Mutant PP-1G
The results of immunoblot analysis of L6 transfected cell lines overexpressing recombinant Flag-tagged wild-type PP-1G and Flag-tagged mutant PP-1G are shown in Fig. 1Go. Immunoblot analyses with anti PP-1G subunit antibody revealed a 3-fold increase in the expression of PP-1G subunit in mutant cells (clones 3 and 11) when compared with control L6 cells (Fig. 1AGo, compare lanes 2 and 3 vs. lane 1). The level of mutant PP-1G expression is comparable to a L6 cell line (clone 211) overexpressing wild-type recombinant PP-1G subunit (Fig. 1AGo, compare lanes 2 and 3 vs. lane 4). As detailed in our earlier studies, L6 cells express wild-type PP-1G subunit upon differentiation (13). Therefore, to discriminate between the endogenous protein and the ectopically expressed recombinant PP-1G, the PP-1G subunit was tagged with a Flag marker in frame at the amino terminus and then expressed in L6 cells. The extent of wild-type and mutant PP-1G subunit expression was detected with a monoclonal anti-Flag antibody. Both the clones of mutant PP-1G (mutIV nos. 3 and 11) and a single clone of wild-type PP-1G (no. 211) expressed the Flag-tagged PP-1G as evidenced by the presence of a 160-kDa protein band corresponding to PP-1G subunit detected with anti-Flag antibody (Fig. 1BGo, lanes 2–4). Clone 3 (Fig. 1AGo, lane 2) exhibited higher amounts of Flag-tagged mutant PP-1G protein expression when compared with clone 11 (Fig. 1BGo, lane 3). As expected, this band was absent from control L6 cell extracts transfected with the empty expression vector (Fig. 1BGo, lane 1). Thus the increase observed in the level of PP-1G subunit protein expression is due to the expression of recombinant PP-1G and not to clonal variability.



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Figure 1. Analyses of Flag-Tagged PP-1Gwild and Asp905Tyr Mutant PP-1G Subunit Expression in L6 Cells by Western Blotting and Immunoprecipitation

Equal amounts of protein extracts (20 µg) from L6 control, Flag-tagged PP-1Gwild (clone 211), and mutIV clones 3 and 11 were separated by SDS-PAGE and the proteins were transferred to PVDF membranes. In Fig. 1DGo, equal amounts of proteins (250 µg) from L6, Flag-tagged PP-1Gwild, and mutIV were immunoprecipitated overnight at 4 C with either mouse IgG or Flag antibody (10 µg) followed by separation of the immunoprecipitates by SDS-PAGE. The proteins were transferred to PVDF membrane. The membrane was probed with (A) PP-1G antibody (A), Flag antibody (B), PP-1C antibody (C), and PP-1G antibody (D). Details are given in Materials and Methods. A representative autoradiogram is shown. Similar results were obtained in multiple experiments. Lane 1, L6 (neo control); lane 2, mutIV clone 3; lane 3, mutIV clone 11; lane 4, Flag-tagged PP-1Gwild (clone 211).

 
Overexpression of the wild-type or mutant PP-1G did not alter the cellular levels of PP-1Cß subunit (Fig. 1CGo). In addition, cell growth, morphology, and extent of differentiation (as monitored by the expression of the L6 differentiation marker myogenin) were comparable between control and transfected cells expressing wild-type and mutant PP-1G (data not shown).

To test whether the Flag tag itself alters the conformation and function of the fused protein, equal amount of cell lysates from Flag-tagged wild-type PP-1G and mutant PP-1G were immunoprecipitated with a monoclonal Flag antibody followed by Western blot analyses of the immunoprecipitates with PP-1G antibody. As shown in Fig. 1DGo, the Flag antibody specifically immunoprecipitated a 160-kDa protein that reacted with PP-1G subunit antibody (Fig. 1DGo, lanes 3 and 4). This band was absent in mock immunoprecipitates in which Flag antibody was substituted by mouse IgG (Fig. 1DGo, lane 2) as well as in parent L6 cell extracts immunoprecipitated with a Flag antibody (Fig. 1DGo, lane 1). The results indicated that the Flag tag itself did not alter the conformation of the recombinant PP-1G. Mutant PP-1G clone 3 expressing the highest levels of Flag-tagged PP-1G subunit was used as a representative cell line for all of the insulin and cAMP dose-response studies on PP-1, GS activation, and glycogen synthesis, and results were compared with clone 211 overexpressing comparable levels of the recombinant Flag-tagged wild-type PP-1G and control L6 cells.

Effect of Asp905Tyr Mutant PP-1G Expression on Basal and Insulin-Stimulated PP-1 Catalytic Activities in the Extracts and PP-1G Immunoprecipitates
To determine whether expression of the mutant PP-1G results in alterations in PP-1 activity in the basal and insulin-stimulated state, we first measured the activity of bound PP-1C in PP-1G immunoprecipitates. In L6 neo controls, insulin treatment caused a 70% increase in immunoprecipitated PP-1 catalytic activity over basal values, whereas overexpression of the wild-type PP-1G resulted in a 150% increase in insulin-stimulated PP-1 enzymatic activity along with a small increase in basal PP-1 activity (Table 1Go). In contrast, despite a 3-fold increase in PP-1G subunit content, cells expressing mutant PP-1G exhibited a 20–40% decrease in basal PP-1 activity when compared with L6 neo controls and PP-1Gwild, respectively (Table 1Go). Treatment with 10 nM insulin for 5 min caused a 66% increase in PP-1 catalytic activity in the immunoprecipitates of mutant cells (Table 1Go), which is 2-fold less than the stimulation observed in cells with wild-type PP-1G. The observed decrease in bound PP-1 catalytic activity in mutant cells was not due to any difference in the efficiency of PP-1G immunoprecipitation as Western blot analysis of the immunoprecipitates with PP-1G subunit antibody detected comparable amounts of this subunit in cells overexpressing wild-type PP-1G and mutant PP-1G but rather due to reductions in the amount of PP-1C that is bound to PP-1G in mutant cells vs. wild-type PP-1G overexpressors (see also Fig. 2CGo).


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Table 1. Effect of Asp905Tyr Mutation of PP-1G Subunit on Bound PP-1 Catalytic Activity and Insulin Responsiveness

 


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Figure 2. Kinetics of Insulin-Mediated PP-1 Activation in Extracts Isolated from L6 Controls, Flag-PP-1Gwild, and Mutant PP-1G Cells

A, Differentiated cells were incubated in the presence and the absence of 10 nM insulin for 0–20 min followed by the extraction of PP-1 as detailed in the text. Cellular PP-1 activity was measured on 1 µg of extract proteins using 32P-labeled phosphorylase a as a substrate. Results are the mean ± SEM of three to four experiments, each performed in duplicate. *, P < 0.05 vs. Flag-tagged PP-1Gwild and L6 cells. B, Effect of (Bu)2cAMP on insulin-mediated PP-1 activation in cell extracts. Cells were pretreated with and without (Bu)2cAMP (1 mM) for 30 min followed by insulin (10 nM) treatment for 5 min. Cellular PP-1 activity was measured as detailed in the text. Results are the mean ± SEM of four independent experiments performed in duplicate. *, P < 0.05 vs. respective controls; **, P < 0.05 vs. insulin; ***, P < 0.05 vs. cAMP + insulin-treated L6 and Flag-tagged PP-1Gwild; ****, P < 0.05 vs. L6 and Flag-tagged PP-1Gwild controls. C, Effect of insulin and (Bu)2cAMP on the content of PP-1C bound to the G-subunit in L6, Flag-tagged PP-1Gwild, and mutant cells expressing asp905tyr PP-1G subunit. Cells were treated with (Bu)2cAMP (1 mM) for 30 min followed by insulin (10 nM) for 5 min. The cell extracts were centrifuged at 10,000 x g for 10 min to sediment cell debris, and the supernatants with equal amounts of protein (100 µg) were centrifuged at 100,000 rpm for 30 min in a mini ultracentrifuge to pellet down glycogen. The glycogen pellets were resuspended in 100 µl sample loading buffer followed by SDS-PAGE and immunoblot analysis of PP-1G and the associated PP-1C subunit as detailed in Fig. 1Go, A and C. A representative autoradiogram is shown. Similar results were obtained in four different experiments.

 
Next, we measured PP-1 activities in cell extracts. As seen in the immunoprecipitates, basal cellular PP-1 activity was 20–30% lower in extracts isolated from the mutant cells when compared with neo controls and cells expressing wild-type PP-1G (Fig. 2AGo). In all the three cell types studied, the stimulation by insulin was rapid occurring within 2 min with a maximum response seen in 5 min. The activity returned to basal levels in 20 min in L6 controls and cells with mutant PP-1G, whereas in cells with wild-type PP-1G the activation was sustained for 20 min (Fig. 2AGo). Overall, the stimulation by insulin was of a lower magnitude in PP-1G mutant cells when compared with cells expressing wild-type PP-1G and neo controls. As reported in our earlier studies (13, 14), the percent insulin effect on PP-1 stimulation in cell extracts vs. PP-1G immunoprecipitates is lower due to the presence of the other forms of PP-1 that are not activated by insulin. Insulin dose-response studies indicated a maximal stimulation at {approx}1 nM insulin. The effect was sustained up to 10 nM insulin (data not shown). As reported earlier by our group as well as others (13, 14, 19), higher concentrations of insulin (100 nM) inhibit PP-1 activation.

Given the knowledge that the PP-1G subunit plays a major role in the regulation of PP-1 catalytic activity in response to insulin as well as counterregulatory hormones, we next examined whether the Asp905Tyr mutation of PP-1G affects cellular responsiveness of PP-1 to cAMP agonists. Serum-starved cells were pretreated with 1 mM (Bu)2cAMP (a cell-permeable analog of cAMP) for 30 min followed by insulin treatment (10 nM) for 5 min and assayed for PP-1 enzymatic activity. In cells expressing recombinant Flag-tagged wild-type PP-1G as well as L6 controls, cAMP agonist had little effect on the basal PP-1 activity but decreased insulin-mediated PP-1 activation by 40–50% (Fig. 2BGo). In contrast, the Asp905Tyr mutation of PP-1G caused a 60% decrease in the basal PP-1 activity upon exposure to (Bu)2cAMP. Furthermore, cAMP agonist completely blocked insulin’s effect on PP-1 activation and decreased insulin-stimulated PP-1 activity below the basal values in these mutant cells (Fig. 2BGo). Western blot analysis of glycogen pellets isolated from control and mutant cells indicated that despite a 3-fold increase in PP-1G content, the amount of PP-1C that was bound to PP-1G in mutant cell lines was markedly less when compared with cells expressing Flag-tagged wild-type PP-1G (Fig. 2CGo). Insulin and cAMP did not significantly alter the amount of PP-1C that was associated with PP-1G in all the three cell types studied (Fig. 2CGo). This is in contrast to earlier reports from Cohen’s laboratory documenting that cAMP treatment results in dissociation of catalytic subunit from the regulatory subunit (10). The reason for the discrepancy between our in vivo studies and those reported earlier is not clear at present.

Kinetic studies on cAMP-mediated inhibition of insulin’s effect on PP-1 activity is shown in Fig. 3Go. In mutant cells, half-maximal inhibition in insulin’s effect on PP-1 activity was observed at a concentration of 0.01 mM (Bu)2cAMP. Complete inhibition of insulin’s effect on PP-1 occurred at a cAMP concentration of 0.1 mM. In contrast, cells expressing wild-type PP-1G as well as control L6 cells exhibited a 20% inhibition at a concentration of {approx}0.1 mM. A 50% decrease in insulin-stimulated PP-1 activity was observed with 1 mM (Bu)2cAMP in these control cells.



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Figure 3. Dose-Response Analyses of (Bu)2cAMP-Mediated Inactivation of Insulin-Stimulated PP-1 Activation in Cells Expressing Asp905Tyr Mutant PP-1G

Cells were pretreated with various concentrations of (Bu)2cAMP (0–5 mM) for 30 min followed by the addition of insulin (10 nM) for 5 min. PP-1 activity was measured on cell extracts as detailed in the text. Insulin-stimulated PP-1 activity in the absence of (Bu)2cAMP was set at 100, and the remaining data on cAMP effect were calculated relative to insulin-stimulated PP-1 activity. Results are the mean ± SEM of three experiments, each performed in duplicate. *, P < 0.05 vs. L6 controls and Flag-tagged PP-1Gwild.

 
Effect of Asp905Tyr Mutant PP-1G Expression on Insulin-Stimulated Glycogen Synthesis and Its Reversal by cAMP Agonists
We next examined the impact of Asp905Tyr mutation of PP-1G subunit on insulin-stimulated glycogen synthesis and cellular sensitivity to cAMP agonists. In these experiments, cells were stimulated with insulin (10 nM) for 10 min, and then treated with various concentrations of (Bu)2cAMP for 30 min followed by addition of [14C]glucose for 90 min. The incorporation of [14C]glucose into glycogen was examined. Basal glycogen synthesis rates were comparable between L6 controls and cells expressing mutant PP-1G subunit (21,144 ± 413 vs. 22,486 ± 120 dpm incorporated/mg protein). Overexpression of Flag-wild-type PP-1G resulted in a small increase (15%) in basal glycogen synthesis when compared with mutant cells. Insulin treatment caused a respective 80%, 67%, and 140% increase over the basal values in glycogen synthesis in L6 neo controls, mutant PP-1G, and Flag-tagged wild-type PP-1G cells. However, when these insulin-stimulated cells were exposed to various concentrations of (Bu)2cAMP for 30 min, a differential response to cAMP was observed in cells expressing mutant PP-1G when compared with L6 controls and cells with wild-type PP-1G. In L6 controls and cells expressing Flag-tagged wild-type PP-1G, (Bu)2cAMP at concentrations of 0.01 mM caused a 10% and 8% decrease in insulin’s stimulatory effect on glycogen synthesis, respectively (Fig. 4AGo). In contrast, cells expressing mutant PP-1G exhibited a 42% decrease in insulin’s stimulatory effect at 0.01 mM (Bu)2cAMP. At 0.1 mM (Bu)2cAMP, a complete reversal of insulin’s stimulatory effect on glycogen synthesis was observed in mutant cells when compared with cells expressing wild-type PP-1G and L6 controls, which exhibited only a 25% and 60% decrease, respectively. A half-maximal inhibition in insulin-stimulated glycogen synthesis was observed at a concentration of 0.015 mM (Bu)2cAMP in mutants vs. 0.5 mM in cells with PP-1Gwild and 0.07 mM in control L6 cells. Higher concentrations of cAMP agonist (5 mM) appear to be less effective in overriding insulin’s stimulatory effect in mutant cells. Furthermore, when cells were pretreated with 0.5 mM (Bu)2cAMP, subsequent insulin effects on glycogen synthesis were inhibited by more than 90% in mutant PP-1G cells, while the inhibition was only partial (20–45%) in cells overexpressing wild-type PP-1G and control L6 cells (Fig. 4BGo). Similar results were obtained with other analogs of cAMP (i.e. SpcAMP and 8-bromo-cAMP, data not shown).



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Figure 4. Dose-Response Analysis of the Reversal of Insulin-Stimulated Glycogen Synthesis by (Bu)2cAMP

A, Cells were exposed to insulin (10 nM) for 10 min followed by addition of various doses of (Bu)2cAMP (0–5 mM) for 20 min. D-[U-14C]glucose (1 µCi/assay) was added for 90 min followed by precipitation of glycogen. The incorporation of [14C]glucose into glycogen was monitored by liquid scintillation spectrometry. To compare results from different experiments in the three cell types, insulin-stimulated glycogen synthesis values in the three cell types were set at 100, and the remaining data on cAMP effect were calculated relative to insulin-stimulated glycogen synthesis. Results are the mean of three independent experiments each performed in duplicate. B, Effect of pretreatment with (Bu)2cAMP on insulin-stimulated glycogen synthesis. Cells were treated with (Bu)2cAMP (0.5 mM) for 30 min followed by addition of insulin (10 nM) for 10 min. The incorporation of [14C]glucose into glycogen was monitored by liquid scintillation spectrometry. Results are the mean of four independent experiments each performed in duplicate. *, P < 0.05 vs. cAMP-treated Flag-tagged PP-1Gwild; **, P < 0.05 vs. basal; ***, P < 0.05 vs. insulin-treated L6 and mutIV; ****, P < 0.05 vs. insulin-stimulated glycogen synthesis.

 
Effect of Asp905Tyr Mutant PP-1G Expression on the Activation Status of GS and Phosphorylase a and Its Responsiveness to Insulin and cAMP Agonists
To investigate whether the observed increase in cAMP sensitivity with respect to inhibition of glycogen synthesis, in mutant cells, is due to altered regulation of GS via PP-1 inactivation, we examined the fractional activities of GS, the rate-limiting enzyme in glycogen synthesis. In these experiments, cells were either pretreated with (Bu)2cAMP for 30 min followed by addition of insulin for 10 min or treated first with insulin for 10 min followed by a 20-min incubation with cAMP agonist. The basal as well as insulin-stimulated GS percent fractional activities were similar between L6 control and mutant PP-1G cells, whereas cells overexpressing wild-type PP-1G exhibited a 2-fold increase in insulin-stimulated GS fractional activity when compared with L6 controls and mutant PP-1G cells. Pretreatment with 1 mM (Bu)2cAMP caused a 40%, 55%, and a 90% inhibition of insulin’s stimulatory effect on GS activation in Flag-tagged PP-1G wild-type, L6 control and mutant PP-1G cells, respectively. (Bu)2cAMP alone reduced the basal GS fractional activities by 40–50% in all the three cell types presumably by phosphorylation of GS via protein kinase A. More importantly, low concentrations of (Bu)2cAMP (0.01 mM) reversed the activating effects of insulin on GS in cells expressing mutant PP-1G (60% decrease in GS fractional activity) whereas L6 controls and cells with PP-1Gwild were not responsive to low concentrations of cAMP (Fig. 5Go). At 1 mM (Bu)2 cAMP, a 30% reversal of insulin’s effect on GS was observed in cells expressing wild-type PP-1G. Higher concentrations of (Bu)2cAMP did not further inhibit insulin-stimulated GS fractional activity in cells with mutant PP-1G. The reasons for this effect are unclear at the present time.



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Figure 5. (Bu)2cAMP-Mediated Reversal of Insulin-Stimulated GS Fractional Activity in Mutant Cells

Cells were treated with insulin (10 nM) for 10 min followed by the addition of (Bu)2cAMP for 20 min. Cell extracts were measured for GS activity as detailed in the text. Insulin-stimulated GS fractional activity from each cell line was set at 100, and the remaining data on cAMP effect were calculated relative to insulin-stimulated fractional activity. Results are the mean of three different experiments each performed in duplicate. *, P < 0.05 vs. L6 control and Flag-tagged PP-1Gwild.

 
It is well known that glycogen synthesis is regulated by the simultaneous activation of GS and inactivation of phosphorylase a and phosphorylase kinase, the two major glycogenolytic enzymes. To further understand the mechanism of increased cAMP sensitization in mutant PP-1G cells, we measured the basal and cAMP-stimulated phosphorylase a activities in cells exposed pretreated with insulin. Basal phosphorylase a fractional activities were not significantly different between L6 controls and mutant PP-1G, whereas cells expressing Flag-PP-1Gwild exhibited low phosphorylase a activity in the basal state when compared with cells with mutant PP-1G (50% decrease vs. mutant) (Fig. 6Go). Insulin treatment decreased phosphorylase a fractional activity in L6 controls and cells with PP-1Gwild to a smaller extent (15–20%). A further reduction in phosphorylase a fractional activity was observed when insulin-treated cells were exposed to 0.01 mM (Bu)2cAMP (Fig. 6Go). In other words, insulin effectively prevented the activation of phosphorylase a by low concentrations of cAMP (<=0.1 mM). At 1 mM concentration, (Bu)2cAMP was able to override insulin’s inhibitory effects and increased the activity of phosphorylase a by 20–25% over the basal values. In contrast to L6 cells and cells expressing PP-1Gwild, insulin did not decrease the activity of phosphorylase a in cells with mutant PP-1G nor did it prevent its activation by (Bu)2cAMP (Fig. 6Go). More importantly, low concentrations of (Bu)2cAMP (0.01 mM) increased the fractional activity of phosphorylase a by 25–50% when compared with L6 controls and wild-type PP-1G. The activity remained higher than basal levels at all concentrations of (Bu)2 cAMP tested.



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Figure 6. Increased cAMP-Mediated Glycogen Breakdown in Cells with asp905tyr Mutation of PP-1G Subunit Is Accompanied by Activation of Phosphorylase a. Insulin Fails to Inactivate Phosphorylase a in Cells Expressing Mutant PP-1G

Cells were treated with insulin followed by (Bu)2cAMP as detailed in Fig. 5Go. Cell extracts were analyzed for phosphorylase activity in the presence and the absence of 5'-AMP (5 mM). Phosphorylase a fractional activity was calculated by dividing the activity measured in the absence of AMP by the activity assayed in the presence of 5 mM AMP. Results are the mean ± SEM of four independent experiments performed in duplicate on duplicate dishes. *, P < 0.05 vs. L6; **, P < 0.05 vs. PP-1Gwild and L6.

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
In the present study we have demonstrated that stable expression of Flag-tagged PP-1G with an Asp905Tyr mutation in L6 cells results in decreased responsiveness to insulin and an increased sensitivity toward cAMP agonists when compared with cells overexpressing Flag-tagged wild-type PP-1G. The enhanced sensitivity to (Bu)2cAMP appears to be due to a diminished ability of the mutant PP-1G subunit to effectively mediate the inhibitory effects of insulin on glycogen breakdown via phosphorylase a inactivation. To our knowledge, this is the first report documenting the functional analysis of the PP-1G subunit variant and its role in the control of glycogen synthesis and breakdown.

While the Asp905Tyr polymorphism of PP-1G was first linked to insulin resistance in a subset of Danish population with type 2 diabetes (17), recent studies in a Japanese population found that 70% of normal healthy individuals carried this variant of PP-1G (18). These studies suggest that racial differences and genetic background of an individual may play a major role in the pathogenesis of insulin resistance in addition to this mutation. In addition, recent studies on Pima Indians have shown that this Asp905Tyr mutation of PP-1G is prevalent in this population and does not correlate with diabetes (20). These authors suggest that a polymorphism in an mRNA-stabilizing element in the 3'-noncoding region of the PP-1G subunit best correlates with insulin resistance (20). The exact reason for these seemingly discordant results is unclear at present.

Nonetheless, several lines of evidence presented in this study suggest that an Asp905Tyr mutation of the PP-1G subunit is accompanied by altered sensitivity of PP-1 and other key regulatory enzymes of glycogen metabolism to cAMP agonists. First, treatment with (Bu)2cAMP was accompanied by marked reductions in basal PP-1 activity and a complete inhibition of insulin-mediated PP-1 activation. Second, cAMP agonist not only prevented insulin’s effects on PP-1 activation, GS, and glycogen synthesis but also reversed insulin’s stimulatory effects on these processes when added after stimulation of cells with insulin. Third, dose-response studies with cAMP agonist on GS inactivation, phosphorylase a activation, and glycogen breakdown indicate that L6 cells harboring mutant PP-1G exhibit a 2- to 5-fold increase in sensitivity toward cAMP agonists when compared with neo controls and cells expressing wild-type PP-1G. Finally, increased breakdown of glycogen in response to low concentrations of cAMP agonist in cells expressing mutant PP-1G was due to the failure of insulin to effectively block phosphorylase a activation and GS inactivation, and this may be due to cAMP-induced reductions in PP-1 activity. In the absence of cAMP, cellular responsiveness of GS and glycogen synthesis to insulin was comparable between control L6 cells and mutant cells, but markedly diminished when compared with cells overexpressing wild-type PP-1G even though the expression of the mutant PP-1G subunit resulted in a 3-fold increase in PP-1G subunit content.

Earlier studies from this laboratory have shown that insulin rapidly activates PP-1 in L6 cells, which is accompanied by increased phosphorylation of the PP-1G subunit (14). Treatment with cAMP agonist alone increased PP-1G phosphorylation but abolished insulin-mediated PP-1 activation as well as phosphorylation. Based on the in vitro studies on purified PP-1G, it was suggested that site-2 phosphorylation by PKA promotes dissociation of the PP-1C subunit and its translocation from glycogen-protein particles to the cytosol (5, 9, 10, 11). The released C subunit is 5- to 8-fold less effective than PP-1G holoenzyme in dephosphorylating GS and phosphorylase kinase. Thus, phosphorylation of the PP-1G subunit by cAMP-dependent kinase results in an immediate inhibition of glycogen synthesis and stimulation of glycogenolysis (5).

Insulin stimulates glycogen synthesis and inhibits glycogenolysis in skeletal muscle and this is mediated by the activation of PP-1G (14) as a result of the phosphorylation of site-1 on the G subunit (10, 13, 14) catalyzed by an insulin-stimulated protein kinase, Rsk2 (10, 21). However, recent studies using inhibitors of the mitogen-activated protein kinase-signaling pathway have demonstrated that this phosphorylation cascade is not involved in the regulation of glycogen synthesis (22, 23).

Asp905 of the PP-1G subunit is not located near any of the known phosphorylation sites in the primary structure of the protein. In addition, we did not observe any difference in the phosphorylation status of the PP-1G subunit immunoprecipitated from cells expressing mutant PP-1G and wild-type L6 cells, either in the basal state or after treatment with insulin and or cAMP agonist (data not shown). Thus, increased sensitivity to cAMP agonist is not due to differences in the extent of PP-1G phosphorylation in response to insulin and or cAMP agonist. Neither cAMP treatment caused dissociation of the catalytic subunit from the regulatory subunit (Fig. 2CGo) as suggested by the in vitro studies of Dent et al (10). Given that PP-1 inactivation can be mediated by inhibitors 1 and 2 and inhibitor 1 is activated by cAMP-mediated phosphorylation, we cannot exclude the possibility of increased phosphorylation and activation of inhibitor 1 in these mutant cells vs. L6 control and Flag-tagged PP-1Gwild. Inhibitor 1 activation might cause inactivation of PP-1 and activation of phosphorylase a.

It should be noted that overexpression of the mutant PP-1G subunit did not result in an increase in the amount of PP-1C that was associated with the G-subunit (see Fig. 3CGo). The simplest interpretation of these results is that this mutation of the PP-1G subunit decreases its ability to associate with the C subunit due to alterations in tertiary structure. Further structure/function studies are warranted to understand the potential binding defects.

The ability of insulin to effectively prevent cAMP-mediated phosphorylase a activation in control L6 cells and cells expressing wild-type PP-1G suggest that the process of glycogenolysis is more sensitive to insulin and the PP-1G subunit plays a dominant role in insulin-mediated inhibition of glycogenolysis. These results coincide with earlier observations in rat skeletal muscle demonstrating that glycogenolysis is more sensitive to insulin than glucose transport and glycogen synthesis (24).

In summary, the results of the present study suggest that the PP-1G subunit not only plays a dominant role in the control of PP-1 activation and glycogen synthesis but also in the control of glycogen breakdown. An Asp905Tyr mutation of the PP-1G subunit is accompanied by increased sensitivity to cAMP agonist because of PP-1 inhibition resulting in the failure of insulin to suppress glycogen breakdown in response to the cAMP agonist.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Materials
Cell culture reagents, antibiotics, FBS, Lipofectamine, Geneticin (G418), phosphorylase b, and phosphorylase kinase were purchased from Life Technologies, Inc. (Gaithersburg, MD). [{gamma}-32P]ATP (specific activity > 3000 Ci/mmol), [125I]protein A, D-[U-14C]glucose, [14C]glucose 1-phosphate, [2-3H]deoxy-D-glucose, [U-14C]uridine diphosphoglucose ([U-14C]UDP-glucose) were purchased from Dupont NEN (Boston, MA). The mammalian expression vector pcDNA3 was purchased from Invitrogen (Carlsbad, CA). Chameleon site-specific mutagenesis kit was purchased from Stratagene (La Jolla, CA). Flag expression system and anti-Flag antibodies were purchased from IBI (New Haven, CT). Okadaic acid was from Moana Bioproducts (Honolulu, Hawaii). Restriction endonucleases were from Roche Molecular Biochemicals (Indianapolis, IN). Electrophoresis and Bradford protein assay reagents were from Bio-Rad Laboratories, Inc. (Richmond, CA). Bicinchoninic acid protein assay reagent was purchased from Pierce Chemical Co. (Rockford, IL). Porcine insulin was a kind gift from the Eli Lilly & Co. (Indianapolis, IN). PP-1Cß antibody was purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). (Bu)2cAMP and all other reagents were from Sigma Chemical Co. (St. Louis, MO). Antibody against PP-1G subunit was generated and affinity purified as described in our previous publications (13, 14).

Site-Directed Mutagenesis of PP-1G cDNA
The PP-1G cDNA was cloned from the rabbit skeletal muscle cDNA library as described in our recent publication (14). As detailed in this study (14), rabbit skeletal muscle PP-1G cDNA was used for site-directed mutagenesis because of its close similarity to human PP-1G. Substitution of an aspartate with a tyrosine at codon 904 of rabbit PP-1G cDNA was performed with the Chameleon site-specific mutagenesis kit, according to the manufacturers instructions using the following primer. 5'-GCC TTT AAC TCA TAC ACG AAC AGA GC-3'

The mutation was confirmed by nucleotide sequence analyses.

Construction of Expression Vectors Carrying Flag Epitope-Tagged Wild-Type and Mutant PP-1G
The mammalian expression vector pcDNA3 containing the recombinant Flag epitope-tagged wild-type and mutant PP-1G constructs were prepared by standard molecular biology techniques. Briefly, the wild-type and the mutant form of PP-1G constructs were cleaved from the Bluescript plasmid using EcoRI, tagged at the 5'-end with a Flag epitope by subcloning in-frame into a Flag expression vector (IBI). Finally, the epitope-tagged PP-1G constructs were subcloned into the mammalian expression vector, pcDNA3 using NdeI and BamHI restriction sites.

Transfection and Selection of Stable Cell Lines Expressing Recombinant Flag Epitope-Tagged Wild-Type and Mutant PP-1G Subunits
The spontaneously fusing rat skeletal muscle cell line, L6, was a kind gift from Dr. Amira Klip (The Hospital for Sick Children, Toronto, Ontario, Canada). L6 myoblasts at second passage were grown and maintained in {alpha}-MEM containing 10% FBS. Cells were transfected with the expression vectors carrying the epitope-tagged wild-type and mutant PP-1G subunit cDNA as detailed in our recent publication (14). Briefly, cells at approximately 30% confluence were transfected with a mixture of 15 µg DNA + 50 µl Lipofectamine reagent for 15 h. At approximately 72 h from the start of transfection the cells were passaged 1:5 into medium containing 2 mg/ml G418 for selection. Single stable clones were picked up after 5–6 days and passaged into several 24-well plates for initial amplification. After two further rounds of amplification, the clones were screened for the expression of Flag epitope-tagged PP-1G. Initial screening was performed by immunoblot analysis of cell extracts prepared from myotubes (12–14 days in culture) using anti-Flag antibody described in Materials and Methods. Control cells (referred to as L6 throughout the manuscript) were transfected 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. Screening with the Flag antibody led to the identification of one wild-type clone (Flag-PP-1Gwild, clone 211) and two mutant clones (mutIV nos. 3 and 11) out of the 200 G418 resistant clones that were screened for Flag-tagged wild-type and mutant PP-1G, respectively. A single mutant clone (mutIV no. 3) expressing the highest amounts of Flag-PP-1G was amplified and used for all the dose-response experiments described below along with a single wild-type PP-1G clone (no. 211, referred to as Fag-PP-1Gwild throughout the manuscript) overexpressing comparable levels of recombinant wild-type PP-1G.

Cell Culture
Transfected cell lines overexpressing Flag-tagged wild-type PP-1G (clone 211) and mutant PP-1G (mutIV) subunits were grown and maintained in {alpha}-MEM containing 2% FBS, 400 µg/ml G418, and 1% antibiotic/antimycotic mixture in an atmosphere of 5% CO2 at 37 C as previously described (14). Unless otherwise stated, myotubes were used for all experiments after a 15-h starvation in serum free {alpha}-MEM containing 5 mM glucose.

Extraction and Assay of PP-1
Serum-starved cells were fed with serum-free medium containing 5 mM glucose. Identical dishes in triplicate were incubated in the absence and presence of insulin (0.1–100 nM) for 1–20 min. In some experiments, cells were pretreated with various concentrations of (Bu)2cAMP (0.01–5 mM) or SpcAMP (10-4 M) for 30 min before insulin exposure. At the end of the incubation period, the medium was removed and the cells rinsed three times with ice-cold PBS followed by extraction with PP-1 extraction buffer as detailed in our recent publications (13, 14).

Assay of PP-1 Activity
The assay was performed as previously described (13, 14) using 32P-labeled glycogen phosphorylase a as a substrate (13). Okadaic acid (OA) at 1 nM concentration was included in the assay to inhibit PP-2A. As detailed in our earlier studies (13, 14, 25), at this concentration, OA inhibits only PP-2A activity and the remaining activity represents PP-1.

Immunoprecipitation and Assay of 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. Cell lysate protein (100 µg) was diluted to 1 ml with lysis buffer and precleared by incubation with rat or mouse IgG (5 µg/ml, coupled to protein-A Sepharose) at 4 C for 30 min. The supernatants were immunoprecipitated with 10 µg/ml anti PP-1G antibody for 1 h at 4 C, followed by treatment with 50 µl protein A/G Agarose (50% vol/vol) for 1 h. This antibody reacts very well with both phosphorylated and nonphosphorylated PP-1G as evidenced by equal amounts of PP-1G protein in the immunoprecipitates detected by Western blot analyses (13, 14). To prevent dephosphorylation during immunoprecipitation, 100 nM OA was added to cell lysates. The pellets were washed four times with 1 ml wash buffer and resuspended in the phosphatase assay buffer to the original volume. PP-1G bound to the antibody was released by incubation with excessive antigenic peptide (15 µg/ml) at 4 C for 1 h. PP-1 activity was measured on 5 µl of immunodepleted supernatants and the immunoprecipitates as detailed in our recent publication (14). To relieve the inhibition of phosphatases due to OA, the immunoprecipitates were diluted to 1:100 with phosphatase assay buffer, and 10 µl aliquots were used for the assay of PP-1 as detailed earlier (13, 14). In a duplicate experiment, the immunoprecipitates were separated by SDS/PAGE followed by immunoblot analyses of PP-1G subunit. PP-1 catalytic activity in the immunoprecipitates was normalized for variation in the contents of PP-1G in the immunoprecipitates.

Subcellular Localization of PP-1G and Its Association with the Catalytic Subunit
Control and insulin- and cAMP-treated cells were sonicated in 300 µl extraction buffer (13, 14, 25) and centrifuged at 10,000 x g for 5 min at 4 C to remove nuclei and cell debris. The supernatants were removed to fresh tubes and assayed for proteins, and equal amounts of proteins (200 µg) were centrifuged for 25 min at 100,000 x g in a mini ultracentrifuge to sediment the glycogen pellet. The supernatant was called cytosol. The glycogen pellet was resuspended to the original volume in PP-1 extraction buffer. Equal amounts of proteins were separated by SDS-PAGE followed by Western blotting with PP-1G subunit and PP-1C subunit antibodies, respectively.

Assay of GS Activity
Cells were treated with and without insulin (10 nM) for 10 min followed by (Bu)2cAMP (0.01–5 mM) for 30 min. In some experiments, cells were pretreated with cAMP (1 mM) for 30 min followed by insulin for 10 min. The cells were extracted with GS extraction buffer (14) and assayed for GS activity in the presence of low (0.1 mM) and high concentrations (50 mM) of glucose-6 phosphate (Glc6P), using 0.7 mM uridine diphospho-D-[U-14C]glucose ([U-14C]UDP-glucose) as a substrate as detailed in our recent publication (14). Insulin-stimulated GS activity (picomoles of [U-14C]UDP-glucose incorporated into glycogen/min/mg protein) was expressed as percent fractional activity measured in the presence of low Glc6P divided by the activity measured in the presence of high Glc6P.

Assay of Phosphorylase a Activity
Phosphorylase a was assayed by monitoring the conversion of [14C]glucose-1 phosphate (Glc-1-P) into glycogen according to previously published protocols (26). Briefly, cells were treated with and without insulin and cAMP as detailed in GS assay and extracted in a homogenization buffer containing 10 mM Tris-HCl, pH 7.0, 150 mM NaF, 15 mM EDTA, 15 mM 2-mercaptoethanol, 10 µg/ml each of leupeptin, antipain, aprotinin, pepstatin A, 1 mM benzamidine, and 1 mM phenymethylsulfonyl fluoride. The final assay mixture contained 75 mM Glc-1-P, 125 mM NaF, 0.6% glycogen, and labeled Glc-1-P at 0.08 µCi/assay. Glycogen phosphorylase a activity was determined in the presence and absence of 5 mM AMP. Caffeine (1 mM) was added to the assay mixture in tubes without any AMP to inhibit phosphorylase b activity.

Glucose Incorporation into Glycogen
Glucose incorporation into glycogen was measured using D-[U-14C]glucose as described previously (14, 27). To examine the effect of cAMP agonist on insulin-stimulated glycogen synthesis, cells were treated with 10 nM insulin for 10 min, and then (Bu)2cAMP (0.01–5 mM) was added for 30 min followed by the addition of [U-14C]glucose for 90 min.

Immunoblot Analysis of PP-1G and PP-1 Catalytic Subunits
Cells 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 phenylmethylsulfonyl fluoride, and 10 µg/ml each of leupeptin, aprotinin, antipain, soybean trypsin inhibitor, and pepstatin A. The cells 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 of protein were mixed with Laemmli sample buffer containing 0.1% bromophenol blue, 1.0 M NaH2PO4, pH 7.0, 50% glycerol, 10% SDS, boiled for 5 min, and loaded on a 7.5% SDS polyacrylamide gel (28). The proteins were transferred to polyvinylidene difluoride (PVDF) membrane, the membranes were probed with 1) PP-1G subunit antibody, 2) Flag antibody, and 3) PP-1Cß antibody. This was followed by incubation with [125I]protein A (0.2 µCi/ml, for PP-1G) or horseradish peroxidase-conjugated goat IgG followed by enhanced chemiluminescence and autoradiography. The intensity of the signal was quantitated by densitometric analysis of the autoradiograms.

Protein Assay
The protein content of the cell extracts was determined with either bicinchoninic acid (29) or Bradford reagent (30).

Statistics
The Student’s t test or ANOVA was used to evaluate the significance of the effect of insulin and (Bu)2cAMP on PP-1 activity, GS activity, and glycogen synthesis. Results are expressed as mean ± SEM of three to four different experiments each performed in triplicate.


    FOOTNOTES
 
Address requests for reprints to: Najma Begum, The Diabetes Research Laboratory, Winthrop University Hospital, 259 First Street, Mineola, NY 11501. e-mail: nbegum@winthrop.org.

This work was supported in part by a research grant from the American Diabetes Association and medical education funds from Winthrop University Hospital.

Received for publication September 15, 1998. Revision received June 9, 1999. Accepted for publication June 29, 1999.


    REFERENCES
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 

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