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Molecular and Cellular Biology, January 2006, p. 334-342, Vol. 26, No. 1
0270-7306/06/$08.00+0     doi:10.1128/MCB.26.1.334-342.2006
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Central Role for Protein Targeting to Glycogen in the Maintenance of Cellular Glycogen Stores in 3T3-L1 Adipocytes

Cynthia C. Greenberg, Arpad M. Danos, and Matthew J. Brady*

Department of Medicine, Committee on Molecular Metabolism and Nutrition, University of Chicago, Chicago, Illinois

Received 31 May 2005/ Returned for modification 5 July 2005/ Accepted 7 October 2005


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ABSTRACT
 
Overexpression of the protein phosphatase 1 (PP1) subunit protein targeting to glycogen (PTG) markedly enhances cellular glycogen levels. In order to disrupt the endogenous PTG-PP1 complex, small interfering RNA (siRNA) constructs against PTG were identified. Infection of 3T3-L1 adipocytes with PTG siRNA adenovirus decreased PTG mRNA and protein levels by >90%. In parallel, PTG reduction resulted in a >85% decrease in glycogen levels 4 days after infection, supporting a critical role for PTG in glycogen metabolism. Total PP1, glycogen synthase, and GLUT4 levels, as well as insulin-stimulated signaling cascades, were unaffected. However, PTG knockdown reduced glycogen-targeted PP1 protein levels, corresponding to decreased cellular glycogen synthase- and phosphorylase-directed PP1 activity. Interestingly, GLUT1 levels and acute insulin-stimulated glycogen synthesis rates were increased two- to threefold, and glycogen synthase activation in the presence of extracellular glucose was maintained. In contrast, glycogenolysis rates were markedly increased, suggesting that PTG primarily acts to suppress glycogen breakdown. Cumulatively, these data indicate that disruption of PTG expression resulted in the uncoupling of PP1 activity from glycogen metabolizing enzymes, the enhancement of glycogenolysis, and a dramatic decrease in cellular glycogen levels. Further, they suggest that reduction of glycogen stores induced cellular compensation by several mechanisms, but ultimately these changes could not overcome the loss of PTG expression.


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INTRODUCTION
 
The proper organization of intracellular effectors and downstream targets plays a critical role in the efficient transmission of a variety of extracellular signals. A growing number of targeting proteins, which bind to enzymes and specific cellular structures, and scaffolding proteins, which assemble sequential enzymatic mediators, have been identified in recent years. In mammals, scaffolding proteins have been identified in the regulation of multiple cellular processes, including apoptosis, cardiac contraction, and epithelial trafficking pathways (1, 5, 39). Additionally, mitogen-activated protein kinase isoforms in Saccharomyces cerevisiae and mammals are organized with immediate upstream activating kinases through a variety of scaffolding proteins such as Ste5 and JNK-interacting protein (29, 35). Also, the family of A-kinase anchoring proteins acts as multivalent scaffolding proteins, assembling protein kinase A and a variety of kinases, phosphatases, and other signaling elements in specific subcellular locations in a wide array of cell types (40).

Targeting proteins also play a critical role in the regulation of glycogen metabolism by insulin (32). The two opposing enzymes controlling glycogen synthetic rates, glycogen synthase and phosphorylase, are both regulated by protein phosphorylation. Insulin markedly enhances glycogen accumulation through the coordinated dephosphorylation of these enzymes, resulting in the activation of glycogen synthase and the inactivation of glycogen phosphorylase. Although inactivation of upstream kinases such as glycogen synthase kinase 3 (GSK3) and phosphorylase kinase partially mediates the regulation of glycogen metabolism, the activation of protein phosphatase 1 (PP1) by insulin plays a critical role in the enhancement of glucose storage as glycogen (8). However, insulin treatment of cells results in the enhanced phosphorylation of a large number of proteins while simultaneously promoting the dephosphorylation of a limited number of enzymes primarily involved in lipid and glucose storage. Additionally, PP1 is involved in the regulation of numerous cellular processes that are not regulated by insulin, such as RNA processing and cell cycle progression (11). Thus, mechanisms must exist for the discrete activation of specific pools of PP1.

For regulation of glycogen metabolism, the PP1-targeting subunit protein targeting to glycogen (PTG or R5) is one of five proteins that localize PP1 to the glycogen particles (15, 34). This family of proteins has been shown to potently regulate glycogen levels through both cellular overexpression studies and transgenic and knockout animal models (4, 12, 13, 18, 20, 28, 33, 37). In addition to targeting PP1 to glycogen, PTG also directly binds to glycogen synthase and phosphorylase, thus enabling the efficient regulation of glycogen metabolism (17, 34). Overexpression of PTG in a variety of cell types and in rodent livers in vivo resulted in the dephosphorylation of glycogen synthase and phosphorylase and alteration of their enzymatic activities, which resulted in a dramatic increase in cellular glycogen accumulation (17-20, 28, 33, 34, 42). Conversely, heterozygous disruption of PTG in mice resulted in decreased tissue glycogen stores, corresponding with reduced glycogen synthase activity and glycogen synthesis (12). However, the precise role of the endogenous PTG-PP1 complex in the control of glycogen metabolism has not been elucidated. Theoretically, PTG-PP1 could act via regulation of glycogen synthase, glycogen phosphorylase, or both enzymes. In addition, the contribution of the PTG-PP1 complex versus the hormonal regulation of upstream kinases and other phosphatases to glycogen metabolism remains controversial. In particular, the inactivation of GSK3 by insulin has been proposed to be a principal regulator of glycogen synthase activity (10). Therefore, RNA interference (RNAi) against PTG was used to examine the impact of reducing PTG levels on glycogen metabolism in the highly insulin responsive and metabolically active 3T3-L1 adipocyte line. PTG knockdown resulted in decreased targeting of PP1 to glycogen, corresponding to reduced phosphatase activity against glycogen synthase and phosphorylase, increased glycogenolysis, and a marked decrease in glycogen levels. These results indicate that the PTG-PP1 complex occupies a critical role in the regulation of glycogen metabolism in 3T3-L1 adipocytes.


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MATERIALS AND METHODS
 
Generation and screening of siRNA constructs. Four short (19-nucleotide) target sequences proposed to specifically silence gene expression of PTG were identified online using siRNA Target Finder (Ambion). Potential PTG siRNAs were termed siRNA 1 to siRNA 4, with PTG siRNA 1 corresponding to the murine PTG gene sequence (34) starting at nucleotide (nt) 391, PTG siRNA 2 starting at nt 457, PTG siRNA 3 starting at nt 544, and PTG siRNA 4 starting at nt 748. A scrambled oligonucleotide that did not correspond to any known murine gene sequence was also identified for use as a control. Oligonucleotide containing the sense sequence, a hairpin loop region, and the antisense sequence was annealed to its complement and ligated into pSIREN-Shuttle vector (BD Biosciences Clontech). Target sequences were then tested for gene silencing of transiently overexpressed PTG in 293T cells. 293T cells were cultured in Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal bovine serum (FBS), 100 U/ml penicillin, 100 U/ml streptomycin, and 0.29 mg/ml glutamine. 293T cells in 12-well dishes were transiently transfected with 0.5 µg of pcDNA3.1 vector encoding PTG and 0.5 µg of pSIREN-PTG-siRNA or pSIREN-scrambled-siRNA using Lipofectamine PLUS (Invitrogen) according to the supplier's instructions. After a 24-h recovery, cell lysates were prepared, and PTG levels were analyzed by immunoblotting to identify constructs that reduced exogenous PTG expression. PTG siRNA constructs 2 and 3 reduced exogenous PTG expression to undetectable levels. Scrambled and PTG siRNA constructs 2 and 3 were subcloned into Adeno-X viral DNA (BD Biosciences Clontech) for incorporation into adenoviral genome. In preliminary experiments, infection of 3T3-L1 adipocytes with either PTG siRNA 2 or 3 adenovirus produced similar reductions in endogenous PTG protein and cellular glycogen levels, so PTG siRNA 2 was used for all experiments reported here.

3T3-L1 adipocytes were cultured and infected as previously described (20), with the following modifications: cells plated in 12-well dishes were infected 1 or 2 days following replacement of insulin-containing growth medium with growth medium in the standard differentiation protocol. A titer of 2 x 105 viral particles per cell was diluted with 0.5 µg/ml poly-L-lysine hydrobromide (Sigma) in DMEM supplemented with 2% FBS as described. Unless otherwise indicated, cells were used 4 or 5 days following infection, with fresh growth medium provided every 48 h.

RNA and protein analyses. Total RNA was prepared from 12-well plates of scrambled or PTG siRNA-infected 3T3-L1 adipocytes using 3 ml/plate of Trizol (Gibco-BRL). Reverse transcription-PCR (RT-PCR) was performed using an iScript cDNA synthesis kit (Bio-Rad) according to the manufacturer's instructions. For real-time quantitative RT-PCR, relative expression levels were determined using iQ SYBR Green Supermix (Bio-Rad) in the iCycler system (Bio-Rad). Ten microliters of diluted cDNA was added to 12.5 µl of iQ SYBR Green Supermix (Bio-Rad), and primers were added at a concentration of 10 pM (sense, TTCCAGAAGAACCAGCGT; antisense, CTCAGTTGGAATGACACG). Water was added to bring the final volume to 25 µl. In parallel, standard curves for PTG and the 18S ribosomal subunit were generated by running reactions on a series of six serial 1:10 dilutions, initiated by the addition of 5 µl of cDNA from control cells (one-fourth volume of RT reaction mixture) to 45 µl of water. The difference between PTG and 18S threshold cycles was assessed for each sample, and these differences were compared between scrambled and PTG-siRNA-treated samples to quantify the relative difference in PTG mRNA between the samples. Thermal cycling parameters were as follows: 3 min at 95°C, followed by 40 cycles of 30 s at 60°C, 30 s at 72°C, and 60 s at 95°C. The comparative cycle threshold method was used to quantify PTG mRNA copy number in scrambled and PTG-siRNA-infected 3T3-L1 adipocytes, relative to that of the 18S ribosomal subunit.

For immunoblotting, lysates were resolved on 10% sodium dodecyl sulfate-polyacrylamide gels and transferred to nitrocellulose (Schleicher and Schuell). Western blots were probed with antibodies against PTG (20), glycogen phosphorylase and phospho-phosphorylase (Ser 14) (18), glycogen synthase (Chemicon), pan-PP1 (sc-7482; Santa Cruz Biotechnology), phosphotyrosine (Upstate Cell Signaling Solutions), Akt, phospho-GSK3ß (Ser 9), and phospho-glycogen synthase (Ser 640) (Cell Signaling Technologies) or anti-GLUT1 and anti-GLUT4 antibodies (Alpha Diagnostic International). Blots were then incubated with horseradish peroxidase-conjugated goat anti-rabbit or goat anti-mouse immunoglobulin G (Bio-Rad) and developed using ECL reagent (Amersham Pharmacia Biotech).

Cell fractionation. Cells were washed three times with cold phosphate-buffered saline and harvested in homogenization buffer (50 mM HEPES [pH 7.4], 150 mM NaCl, 1 mM EDTA, 10 mM NaF, 10% glycerol, and protease inhibitors added just before use). Cells were lysed using a glass Dounce homogenizer and centrifuged at 1,000 x g at 4°C to pellet nuclei. The resulting postnuclear spin supernatant fraction was then subjected to sequential centrifugation at 4°C, first at 10,000 x g for 10 min and then at 100,000 x g for 30 min, to obtain the cytosolic and glycogen-enriched pellet fractions. The pellet fractions were resuspended in homogenization buffer by trituration using a 23-gauge needle. To determine levels of glycogen-targeted PP1, the pellets resulting from centrifugation at 100,000 x g were resuspended in homogenization buffer plus 40 µg/ml amylase (Sigma) and incubated at 37°C for 15 min. Samples were then centrifuged at 100,000 x g for 30 min, and PP1 levels released into the supernatant were determined by immunoblotting.

For examination of GLUT1 and GLUT4 subcellular distribution, cells were first serum starved for 2.5 h in DMEM-0.5% FBS-5 mM glucose and then lysed and processed as above. The pellets resulting from centrifugation at 10,000 x g were resuspended in homogenization buffer by trituration using a 23-gauge needle. These fractions were then combined with Laemmli sample buffer lacking ß-mercaptoethanol, heated for 10 min at 37°C, and analyzed by immunoblotting.

Metabolic and enzymatic assays. For measurement of glycogen synthase activation due to covalent modification, following serum starvation for 2.25 h, cells were washed three times with medium lacking glucose and incubated in the same medium for 15 min. Cells were then either unstimulated or stimulated with 100 nM insulin for 15 min. Lysates were prepared and reactions were carried out as previously described (27). Cellular glycogen levels, PP1 activity assays, and glycogen synthetic and lipogenic rates were determined as previously described (20). For determination of ATP levels, cells were washed three times with cold phosphate-buffered saline and scraped into 1 ml of assay buffer (100 mM Tris [pH 7.75], 4 mM EDTA). Lysates were transferred to microcentrifuge tubes, immediately quick-frozen in a dry ice-ethanol bath, and stored at –20°C. For the assay, lysates were boiled for 5 min, incubated on ice for 5 min, and then spun at 1,000 x g for 60 s at 4°C. The supernatant was transferred to a new tube and then diluted 1:100 in assay buffer. Fifty microliters of each diluted sample was assayed according to the supplier's instructions using an ATP Bioluminescence Assay Kit CLS II (Roche) and a Berthold Lumat LB 9507 luminometer. Intracellular glucose and glucose-6-phosphate levels were determined using a hexokinases/glucose-6-phosphate dehydrogenase-based kit (Sigma) according to the manufacturer's instructions.

Glycogen pulse-chase for determination of glycogenolysis. Following infection and recovery, cells were serum starved for 2.5 h in DMEM-0.5% FBS-5 mM glucose and then stimulated for 15 min with 100 nM insulin. For glycogen labeling, 2 µCi of [14C]glucose (ICN Biomedicals) was added to each well and allowed to incorporate for 30 min. After removal of glucose, cells were collected for determination of baseline labeling of glycogen. Replicate wells were incubated in growth medium for 24 h at 37°C and then collected for determination of the remaining labeled glycogen (20).

Statistical analysis. Data comparisons were analyzed by a Student's t test. Analysis was performed using Microsoft Excel XP and considered statistically significant at a P value of <0.05.


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RESULTS
 
Gene silencing of PTG by siRNA treatment of 3T3-L1 adipocytes. The technique of RNAi was chosen to specifically reduce PTG levels in 3T3-L1 adipocytes. First, four different siRNA constructs were screened for their ability to induce RNAi against PTG transiently overexpressed in 293T cells (Materials and Methods). The scrambled control siRNA and PTG siRNA constructs were then subcloned into pAdeno-X adenoviral vector. After adenoviruses were packaged and purified, 3T3-L1 adipocytes were infected with scrambled or PTG siRNA at a titer of 2 x 105 viral particles per cell. Following a 4-day recovery, PTG transcript levels were determined by quantitative RT-PCR, using 18S mRNA as an internal control. PTG was detected after 13.4 ± 0.5 cycles in cells infected with scrambled siRNA, while PTG was detectable after 17.3 ± 0.7 cycles in cells infected with PTG siRNA. This difference corresponded to a >90% decrease in PTG mRNA levels in 3T3-L1 adipocytes infected with PTG siRNA (Fig. 1A), indicating both high infection efficiency and efficacy of the siRNA construct against PTG message levels. In parallel, the effects of the siRNA adenoviral constructs on cellular protein expression were examined. The postnuclear spin supernatant and cytosolic and glycogen-enriched pellet fractions were isolated from cells and analyzed by immunoblotting. Glycogen-enriched pellet fractions were prepared to separate endogenous PTG from a closely migrating, prominent, nonspecific cytosolic band recognized by the affinity-purified anti-PTG antibody used (20). In agreement with the quantitative RT-PCR results, infection of 3T3-L1 adipocytes with PTG siRNA markedly reduced PTG protein levels, compared to mock-infected cells or cells infected with the scrambled siRNA construct (Fig. 1B, lane 3 versus lanes 1 and 2). In parallel, glycogen synthase levels were unchanged, whereas glycogen phosphorylase levels (Fig. 1B) and total enzymatic activity (data not shown) were reduced by approximately 50% in cells infected with the PTG siRNA construct. Although total PP1 levels did not change (Fig. 1B, PP1), the amount of PP1 bound to glycogen particles was substantially reduced in cells infected with the PTG siRNA adenovirus (Fig. 1B, GB-PP1). These results are also consistent with previous findings in 3T3-L1 adipocytes that PTG-bound or glycogen-localized PP1 represents a minor portion of cellular PP1 levels, albeit a crucial enzymatic regulator of glycogen metabolism (7, 20). Together, these results indicated that infection of 3T3-L1 adipocytes with PTG siRNA dramatically reduced PTG transcript and protein levels and decreased PP1 localization at glycogen particles.


Figure 1
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  		FIG. 1. PTG siRNA reduces PTG transcript and protein levels. (A) 3T3-L1 adipocytes plated in 12-well dishes were infected with scrambled or PTG siRNA at a titer of 2 x 105 viral particles per cell. Following recovery, total RNA was prepared from cells. PTG transcript levels were analyzed by quantitative RT-PCR and normalization to ribosomal 18S. Results are representative of two independent determinations, each performed in quadruplicate. (B) Mock-, scrambled siRNA-, or PTG siRNA-infected cells were fractionated to obtain postnuclear spin supernatant and glycogen-enriched pellet fractions. Pellets were resuspended in homogenization buffer, and samples were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and analyzed by immunoblotting using the indicated antibodies. The postnuclear spin supernatant fractions were probed with anti-glycogen synthase ({alpha}-GS) or anti-PP1 antibodies, while the glycogen-enriched pellet fractions were analyzed by anti-PTG or glycogen phosphorylase ({alpha}-GP) immunoblotting. To determine levels of glycogen-bound PP1 (GB-PP1), the pellets resulting from centrifugation at 100,000 x g were resuspended in homogenization buffer containing 40 µg/ml amylase. Following incubation, samples were centrifuged at 100,000 x g for 30 min, and PP1 levels released into the supernatant were determined by anti-PP1 immunoblotting. Results are representative of three independent determinations.

The PTG-PP1 complex is required for the maintenance of glycogen stores. Previous work showed that PTG overexpression in 3T3-L1 adipocytes specifically enhanced PP1 activity against enzymes involved in glycogen metabolism (20). To determine the effect of reducing PTG expression on PP1 activity, lysates from scrambled and PTG siRNA-infected cells were prepared 4 days after infection, and phosphatase activity was assayed in vitro against [32P]-labeled glycogen phosphorylase or myelin basic protein. Infection of cells with scrambled siRNA showed a slight but not significant increase in PP1 activity against glycogen phosphorylase compared to mock-infected cells. However, PP1 activity was decreased by over 60% upon silencing of PTG expression compared to the two controls (Fig. 2). In contrast, PP1 activity against myelin basic protein was unchanged (Fig. 2). Cumulatively, these data suggested that reduction of PTG expression did not change total PP1 levels in the cells but, rather, specifically decreased phosphatase activity directed toward glycogen-metabolizing enzymes.


Figure 2
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  		FIG. 2. PTG siRNA reduces PP1 activity against glycogen phosphorylase. 3T3-L1 adipocytes were mock-infected or infected with scrambled or PTG siRNA. Following 4 days of recovery, cell lysates were prepared. PP1 activity in cell lysates was measured for 4 min, using 32P-labeled glycogen phosphorylase a (Phos a) or 32P-labeled myelin basic protein (MBP) as a substrate. A Student's t test analysis of data from PTG siRNA versus mock and versus scrambled siRNA was used. *, P < 0.05. Results are representative of three independent determinations, each performed in triplicate.

To address the physiological significance of reducing levels of glycogen-bound PP1 activity, 3T3-L1 adipocytes were infected with scrambled or PTG siRNA, and total glycogen levels were determined. At baseline, both scrambled and PTG siRNA-infected cells contained 1.8 mg of glycogen/mg of protein (Fig. 3A, day 0). At days 2, 4, 6, and 9, cells infected with the scrambled siRNA construct maintained the starting level of glycogen, indicating that adenoviral-mediated siRNA delivery alone did not interfere with glucose uptake and storage as glycogen (Fig. 3A, dashed line). In contrast, infection of cells with PTG siRNA adenovirus markedly inhibited glycogen accumulation. By day 2 following infection, glycogen levels were reduced by 45%, and by day 4 they were decreased 88% (Fig. 3A, solid line). Glycogen levels remained low compared to baseline and scrambled siRNA levels for up to 9 days following infection (Fig. 3A). Interestingly, despite the marked decrease in glycogen levels 4 days after infection, there was no apparent redistribution of glycogen synthase or phosphorylase (Fig. 3B), indicating that the enzymes remained bound to the residual glycogen. Further, adenoviral-mediated PTG overexpression in the PTG siRNA-treated cells enhanced glycogen levels to above those found in the mock- and ß-galactosidase-infected cells (Fig. 3C), indicating that restoration of PTG expression was sufficient to rescue glycogen stores. Together, these data demonstrate that PTG plays a central role in the maintenance of glycogen stores in 3T3-L1 adipocytes by specifically targeting and directing PP1 activity against enzymes involved in glycogen metabolism.


Figure 3
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  		FIG. 3. PTG is required for maintenance of glycogen stores. (A) 3T3-L1 adipocytes were infected with scrambled or PTG siRNA as described. At each time point, fresh growth medium was added to cells or lysates were prepared, and glycogen and protein levels were determined. Day 0 indicates the day of infection. A Student's t test analysis of data from PTG siRNA versus scrambled siRNA was used. *, P < 0.05; **, P < 0.01; +, P < 0.06. Results are representative of three independent experiments, each performed in triplicate. (B) Four days after infection, mock-, scrambled siRNA-, or PTG siRNA-treated cells were fractionated by ultracentrifugation to obtain cytosolic and glycogen-enriched pellet (GEP) fractions. The samples were analyzed by immunoblotting using anti-glycogen synthase ({alpha}-GS) or anti-glycogen phosphorylase ({alpha}-GP) antibodies. Results are representative of three independent determinations. (C) On day 0, replicate wells of 3T3-L1 adipocytes were either mock infected (–) or infected with PTG siRNA adenovirus (+). Three days later, the indicated wells were mock infected (–) or infected with adenovirus encoding ß-galactosidase (ßGal) or PTG. Two days later, cell lysates were prepared, and cellular glycogen and protein were levels determined. Results are representative of five independent determinations from two experiments. A Student's t test analysis of data from samples versus mock-infected PTG siRNA samples was used. *, P < 0.03; **, P < 0.01; n/s, not significant.

Reduction of PTG expression increases glycogen breakdown. Cellular glycogen levels reflect the relative rates of glycogen formation and breakdown. Since reduction of PTG expression dramatically decreased glycogen storage, the phosphorylation state of glycogen phosphorylase and rates of glycogen degradation were initially examined. After infection with scrambled or PTG siRNA adenovirus and a 4-day recovery in growth medium, 3T3-L1 adipocytes were collected, glycogen-enriched pellets were prepared by ultracentrifugation, and phospho-phosphorylase levels were determined by immunoblotting (18). Reduction of PTG expression with the PTG siRNA construct strongly enhanced phospho-phosphorylase levels (Fig. 4A) and the enzyme activity ratio (data not shown). The marked increase in glycogen phosphorylase phosphorylation occurred despite a decrease in total cellular enzyme levels (Fig. 1B and 3B). In parallel, to determine glycogen degradation rates, scrambled siRNA- and PTG siRNA-infected cells were pulsed with [14C]glucose for 30 min in the presence of insulin in order to label glycogen stores. All cells were then washed three times with medium, and baseline levels of glucose incorporated into glycogen were determined in half of the wells (Fig. 4B, Day 1). After 24 h, the amount of labeled glycogen remaining in the other half of the wells was determined (Fig. 4B, Day 2). There was no significant change in amounts of radiolabeled glycogen after 24 h in cells infected with scrambled siRNA. In contrast, infection of cells with PTG siRNA reduced glycogen stores by 40% after 24 h, reflecting significantly increased glycogenolytic rates (Fig. 4B), which paralleled the changes measured in total glycogen levels (Fig. 3A). Cumulatively, these data suggest that the reduction of PTG expression uncoupled PP1 activity from phosphorylase, resulting in decreased glycogen stores due to enhanced glycogen breakdown.


Figure 4
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  		FIG. 4. Glycogenolysis is increased upon reduction of PTG expression. Cells were infected with scrambled (Scr) or PTG siRNA and allowed to recover for 4 days. (A) Glycogen-enriched pellets were prepared, and levels of phosphorylated glycogen phosphorylase (GPa) were analyzed by phospho-specific immunoblotting. (B) Cells infected with scrambled or PTG siRNA were serum starved for 2.25 h and then stimulated with 100 nM insulin for 15 min. A total of 2 µCi of [14C]glucose was added to each well to label glycogen. After 30 min, cells were washed and either used to determine glucose incorporation into glycogen (Day 1) or placed in growth medium and incubated for 24 h (Day 2), after which remaining glucose incorporated into glycogen was determined by scintillation counting. A Student's t test was used for data analysis: n/s, not significant; *, P < 0.03. Results are representative of three experiments, each performed in triplicate.

Stimulation of glucose transport and glycogen synthesis upon depletion of cellular glycogen stores. The marked reduction of cellular glycogen stores following ablation of PTG expression could also occur via inhibition of glycogen synthesis. To address this possibility, basal and insulin-stimulated glycogen synthetic rates were compared in 3T3-L1 adipocytes following infection with scrambled or PTG siRNA adenovirus. After a 4-day recovery, cells were treated in the absence and presence of 100 nM insulin for 15 min, followed by a 30-min incubation with [14C]glucose. Cellular glycogen was then isolated, and the amounts of radiolabeled glucose incorporation were determined. The basal rate of glycogen synthesis in cells infected with the scrambled siRNA was low, and insulin stimulation resulted in a >10-fold increase (Fig. 5). Unexpectedly, in cells infected with PTG siRNA, the basal rate of glycogen synthesis was increased fourfold compared to the scrambled control, while insulin stimulation was doubled (Fig. 5). Thus, despite the marked reduction in total glycogen stores, acute glycogen synthesis rates were significantly augmented.


Figure 5
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  		FIG. 5. Glycogen synthesis rates are increased in cells lacking PTG. Scrambled or PTG siRNA-infected cells recovered for 4 days, were serum starved and then unstimulated or stimulated with 100 nM insulin for 15 min. [14C]glucose was added to all wells. After 30 min, cell lysates were prepared, glycogen was precipitated by ethanol washes, and the amount of glucose incorporated into glycogen was determined by scintillation counting. A Student's t test analysis of data from PTG siRNA versus scrambled siRNA was used. ***, P < 0.001. Results are representative of three independent determinations, each performed in triplicate.

Insulin-stimulated glycogen synthesis rates are a summation of the degree of insulin-stimulated signaling cascades resulting in increased GLUT4 translocation and glucose uptake concomitantly with glycogen synthase activation. To identify the mechanism(s) underlying the increased glycogen synthesis rates, these parameters were compared in 3T3-L1 adipocytes infected with scrambled or PTG siRNA. After infection, 4-day recovery, and serum starvation, cells were untreated or treated with insulin for 15 min, and initially cell lysates were analyzed by phospho-specific immunoblotting. Basal and insulin-stimulated phosphorylation of the insulin receptor, GSK3ß, and dephosphorylation of glycogen synthase site 3a (Ser 640) did not differ between mock-infected cells or cells infected with the scrambled or PTG siRNA adenovirus (Fig. 6). Thus, data suggest that the insulin-IRS1-GSK3 signaling pathway was unaffected by the loss of PTG expression.


Figure 6
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  		FIG. 6. Proximal insulin signaling pathways are not altered following RNAi against PTG. 3T3-L1 adipocytes were mock-infected or infected with scrambled or PTG siRNA. Following a 4-day recovery, cells were serum starved, and then left untreated or stimulated with 100 nM insulin. After 15 min, cell lysates were prepared and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting using the indicated antibodies. Tyr, tyrosine; GS, glycogen synthase. Results are representative of three independent determinations.

Next, to determine if altered glucose uptake contributed to the enhancement of glycogen synthesis in cells lacking PTG, 3T3-L1 adipocytes were infected with scrambled or PTG siRNA, allowed to recover, and then treated exactly as during the glycogen synthesis measurements, except that total glucose uptake into the cells was determined by scintillation counting. The rate of glucose transport in cells infected with the scrambled siRNA was low in the basal state and increased more than eightfold upon insulin treatment (Fig. 7A). In cells infected with PTG siRNA, the rates of both basal and insulin-stimulated glucose uptake were significantly elevated. These data indicate that increased glucose uptake may contribute to augmented rates of glycogen synthesis upon PTG knockdown.


Figure 7
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  		FIG. 7. Reduction of PTG expression increases glucose transport. Cells were infected with scrambled or PTG siRNA adenovirus as described and treated as described in the legend of Fig. 6. (A) After incubation for 30 min, cells were harvested and transferred to scintillation vials, and total glucose uptake was determined by liquid scintillation counting. A Student's t test analysis of data from PTG siRNA versus scrambled siRNA was used. *, P < 0.05; **, P < 0.01. (B) Following recovery, whole-cell lysates were prepared from triplicate wells. Alternatively, cells were treated as described above. Following a 15-min insulin stimulation, cells were collected, and plasma membrane-enriched fractions were obtained by centrifugation at 10,000 x g. Whole-cell lysates and plasma membrane-enriched fractions were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting. Results are representative of three independent determinations.

To examine the levels of facilitative glucose transporters GLUT1 and GLUT4, cells were infected with scrambled or PTG siRNA, and whole-cell lysates were prepared. Immunoblotting revealed that total GLUT1 levels were increased in cells infected with PTG siRNA compared to cells infected with scrambled siRNA (Fig. 7B, left). In contrast, there was no change in total GLUT4 levels (Fig. 7B, left). Next, to determine the ability of insulin to promote translocation of the glucose transporters to the cell surface, infected cells were serum starved and then untreated or stimulated with insulin for 15 min. Lysates were prepared and fractionated by centrifugation at 10,000 x g in order to obtain plasma membrane-enriched fractions (pellet resulting from centrifugation at 10,000 x g) that were then analyzed by immunoblotting. In cells infected with scrambled siRNA, insulin stimulation resulted in a robust increase in GLUT4 protein levels in the pellet fraction and also stimulated GLUT1 translocation (Fig. 7B, right). In cells infected with PTG siRNA, basal and insulin-stimulated GLUT1 expression in the pellet fraction was increased compared to control cells (Fig. 7B, right), while GLUT4 levels were comparable under both treatment conditions. These data suggest that upregulation of total GLUT1 expressed and increased GLUT1 levels at the cell surface may contribute to the increased glucose transport and glycogen synthetic rates seen in 3T3-L1 adipocytes infected with PTG siRNA adenovirus.

Glycogen synthase catalyzes the rate-limiting step of glycogen synthesis. To determine the regulation of glycogen synthase by insulin in the PTG-deficient cells, enzymatic activity was assayed in vitro. Cells were infected with scrambled or PTG siRNA adenovirus, allowed to recover, and then stimulated in the absence and presence of 100 nM insulin in medium lacking glucose. Under these conditions, changes in glycogen synthase activity reflect enzymatic regulation due to covalent modification independent of the effects of increased intracellular levels of glucose metabolites, particularly glucose-6-phosphate (G6P). In cells infected with scrambled siRNA, the basal glycogen synthase activity ratio was 0.10, which doubled upon insulin stimulation (Fig. 8A). In contrast, insulin stimulation did not significantly increase the glycogen synthase activity ratio in cells lacking PTG (Fig. 8A). Despite the marked reduction in cellular glycogen levels upon infection with PTG siRNA adenovirus (Fig. 3), there was no change in total glycogen synthase levels, as measured by immunoblotting (Fig. 1B and 3B) or activity assay (data not shown). Thus, these results suggest that the PTG-PP1 complex forms the principal glycogen synthase- and phosphorylase-directed phosphatase activity in 3T3-L1 adipocytes.


Figure 8
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  		FIG. 8. Regulation of glycogen synthase activity is altered following PTG knockdown. 3T3-L1 adipocytes were infected as described. Following recovery, scrambled or PTG siRNA-infected cells were serum starved for 2.25 h. (A) Cells were then incubated in the absence of extracellular glucose for 15 min and left untreated or were stimulated with 100 nM insulin for 15 min. Cell lysates were prepared and glycogen synthase (GS) activity was measured in the absence (active) or presence (total) of 10 mM G6P and expressed as an activity ratio (active/total). Significance was determined by a Student's t test. **, P < 0.01; n/s, not significant. Results are representative of three independent determinations. (B) Cells were treated as above, but in the presence of 5 mM extracellular glucose. Significance was determined by a Student's t test. *, P < 0.03; ***, P < 0.001.

However, use of glucose-free medium does not recapitulate the physiological conditions used to measure glycogen synthesis rates, so glycogen synthase activity was also determined in cells incubated in medium containing 5 mM glucose. In cells infected with scrambled siRNA, the basal glycogen synthase activity ratio was low, and insulin stimulation increased the activity ratio to 0.17. In contrast to results obtained in the absence of extracellular glucose, the basal and insulin-stimulated glycogen synthase activity ratios in cells infected with PTG siRNA were similar to ratios in control cells (Fig. 8B). The insulin-stimulated dephosphorylation of site 3a on glycogen synthase was identical in glucose-free and glucose-containing media (data not shown), suggesting that the compensatory restoration of glycogen synthase activation in glucose-containing medium occurred through another mechanism. Additionally, the increase in glucose uptake in PTG-depleted cells (Fig. 7) may also result in further allosteric activation of glycogen synthase that is not detected under these assay conditions, potentially explaining the increases observed in acute glycogen synthesis rates (Fig. 5). Cumulatively, these data suggest that effects mediated by insulin-stimulated glucose uptake and generation of intracellular glucose metabolites compensated for the reduction of phosphatase activity targeted against glycogen synthase.

Depletion of cellular glycogen stores does not impact glucose utilization. Glucose transported into the cell is rapidly phosphorylated to form G6P, which then may be utilized to synthesize glycogen, metabolized to generate ATP, or used to generate the glycerol backbone for triglyceride synthesis. Reduction of PTG expression dramatically decreased glycogen levels (Fig. 3) and markedly increased rates of glycogen synthesis (Fig. 5). To determine the effects of reduced cellular glycogen stores on glucose utilization, G6P and ATP levels and glucose incorporation into lipid were determined in cells infected with the scrambled or PTG siRNA adenovirus. Interestingly, G6P and ATP levels and basal and insulin-stimulated lipogenic rates were not altered upon depletion of cellular glycogen levels (data not shown). These results are consistent with previous work in 3T3-L1 adipocytes that indicated that a dramatic enhancement of cellular glycogen synthetic rates and total glycogen stores upon PTG overexpression also did not alter lipogenic rates (20) or ATP levels (data not shown).


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DISCUSSION
 
Insulin is the major anabolic hormone, promoting glucose uptake and storage of glucose and fatty acid in the target organs, muscle, and adipose tissue. To exert these effects, insulin activates PP1 to mediate the dephosphorylation of key enzymes involved in glucose and lipid metabolism, namely, glycogen synthase, glycogen phosphorylase, and hormone-sensitive lipase (8). For localization of PP1 to glycogen particles, five targeting subunits have been identified. These glycogen-targeting subunits of PP1 bind to PP1 via the consensus sequence XRVXF (16, 43) and to glycogen via a central domain with significant homology to starch metabolizing enzymes (41). The first subunit identified, called RGL or GM, is expressed in skeletal and cardiac muscle and as a 124-kDa protein is significantly larger than the other four 33- to 38-kDa proteins (36, 38). RGL/GM contains two unique consensus protein kinase A sites, one of which overlaps with the conserved PP1 binding domain, and phosphorylation of this site has been proposed to mediate the glycogenolytic response to intracellular cyclic AMP elevation (22, 23). GL is primarily expressed in liver (14) and also human muscle (31) and is allosterically regulated by phospho-phosphorylase binding to a C-terminal domain (3, 14). PTG/R5 exhibits overlapping tissue distribution with RGL/GM and GL as it is expressed in all insulin-sensitive tissues (15, 34). R6 is widely expressed in tissues (2), and identification of a fifth PP1-glycogen-targeting subunit, R3E, has recently been reported (30).

Several animal models have been generated with altered expression of PP1-glycogen-targeting subunits. Two groups independently generated mouse knockout lines lacking the muscle-specific PP1-targeting subunit RGL/GM (13, 37). Both animal lines exhibited a dramatic loss of PP1 catalytic subunit and cellular glycogen levels in skeletal muscle, but the effects of ablating skeletal muscle glycogen storage on energy homeostasis were controversial. One group reported no measurable difference in plasma glucose homeostasis or body weight (37), while the second group reported the development of age-dependent obesity, insulin resistance, and glucose intolerance (13). The reasons for these discrepancies are unclear, given the similar targeting strategies and background animal strain used. Saltiel and colleagues generated a global PTG knockout line (12). Unexpectedly, the homozygous gene deletion of PTG was lethal, although the molecular mechanism for this effect has not been reported. However, heterozygous reduction of PTG in the entire animal resulted in a 40 to 50% reduction in glycogen levels in adipose tissue, liver, heart and skeletal muscle, and the development of age-dependent insulin resistance (12). In contrast to the RGL/GM transgenic animal lines, there was no change in total PP1 levels in the PTG knockout animals, suggesting that RGL/GM plays a more important role for the stabilization of PP1 protein in vivo. Conversely, several groups have also reported that overexpression of these targeting subunits in a variety of cell lines (17-20, 28), by adenoviral-mediated gene transfer in rat livers (33), and in skeletal muscle of transgenic mice (4) all markedly enhance carbon flux into glycogen. Cumulatively, these results demonstrate a critical role for PP1-glycogen-targeting subunits in the regulation of glycogen metabolism in a variety of tissue types (32).

However, the overlapping tissue distribution of five different gene products that superficially perform the same function, i.e., targeting PP1 to glycogen, suggests that each subunit confers unique regulatory properties to the bound phosphatase. To determine the molecular mechanism by which the PTG-PP1 complex regulates glycogen metabolism, 3T3-L1 adipocytes were infected with adenovirus containing siRNA constructs against PTG. This cell line was chosen for the study since PTG was originally identified from a 3T3-L1 adipocyte two-hybrid library (34), and these cells exhibit robust changes in glucose and glycogen metabolism upon insulin stimulation (6). Infection of 3T3-L1 adipocytes with PTG siRNA dramatically reduced PTG transcript and protein levels. There was a parallel reduction in the amount of PP1 targeted to glycogen, although total cellular levels of this phosphatase were unchanged, suggesting that PTG is not the principal binding partner for PP1 in these cells. Delivery of PTG siRNA had no effect on the cellular expression of several insulin-sensitive regulators of glycogen synthesis, namely GLUT4, GSK3, and glycogen synthase, and several proximal insulin receptor signaling events were likewise unaffected. However, ablation of PTG expression resulted in a >85% reduction in cellular glycogen stores, suggesting a critical role for the PTG-PP1 complex in the physiological regulation of glycogen metabolism in 3T3-L1 adipocytes.

In previous studies, the simultaneous increase in glycogen synthase and phosphorylase dephosphorylation upon PTG overexpression did not address the physiological role of the regulation of glycogenesis versus glycogenolysis in PTG action. However, the data from adenoviral-mediated delivery of PTG siRNA demonstrate that the principal function of the PTG-PP1 complex in 3T3-L1 adipocytes is to dampen glycogen phosphorylase activity and limit glycogen breakdown. Infection of 3T3-L1 adipocytes with PTG siRNA adenovirus resulted in a 60% loss of PP1 activity against glycogen phosphorylase measured in vitro from cell lysates, which corresponded with a marked increase in the cellular phosphorylation of glycogen phosphorylase, corresponding to enzymatic activation. In parallel, there was a marked increase in glycogen degradation rates in PTG-depleted cells. These data extend previous work in liver and primary hepatocytes. Cohen and colleagues assayed in vitro phosphatase activity of PTG-PP1 or GL-PP1 from rat liver against glycogen synthase or phosphorylase. They found that the glycogen synthase phosphatase/phosphorylase phosphatase activity ratio of PTG-PP1 complex was lower than that of GL-PP1, supporting the idea that PTG is primarily a glycogen phosphorylase phosphatase (9). Also, several groups have demonstrated a dose-dependent inactivation of glycogen phosphorylase with PTG overexpression in hepatocytes and showed that PTG overexpression counteracted glycogen phosphorylase activation and glycogen mobilization by extracellular stimuli (18, 19). Cumulatively, these data from PTG overexpression and reduction studies suggest that glycogen phosphorylase is the principal enzymatic target of the PTG-PP1 complex.

Studies in skeletal muscle indicate that the cell can adapt its metabolic responses to maintain glycogen levels at a certain set point (21). In rodents or humans, glycogen depletion following exercise induces an increase in insulin sensitivity and responsiveness, resulting in enhanced glucose uptake and glycogen synthase activation. Conversely, glycogen supercompensation following exercise and a high-carbohydrate meal induces a transient insulin resistance in muscle, which persists until glycogen levels are reduced to the physiological set point (25, 26). The molecular mechanisms by which skeletal muscle determines a set point for glycogen stores, "senses" intracellular glycogen levels, and adjusts the hormonal regulation of glucose uptake, metabolism, and storage are uncertain. In 3T3-L1 adipocytes, the reduction of glycogen stores achieved in this study correlated with several responses geared toward increasing glucose storage as glycogen, suggesting that 3T3-L1 adipocytes can also adapt to changes in intracellular glycogen stores (24). The marked drop in cellular glycogen levels following delivery of the PTG siRNA construct resulted in increased GLUT1 expression, enhanced basal and insulin-stimulated glucose uptake, and acute glycogen synthetic rates. Disruption of the PTG-PP1 complex also corresponded to a signification reduction in glycogen phosphorylase levels. The effects on GLUT1 and glycogen phosphorylase appeared to be specific, as there was no corresponding change in GLUT4 or glycogen synthase protein amounts. It is tempting to speculate that the physiological expression of PTG and subsequent targeting of PP1 to glycogen in 3T3-L1 adipocytes establish a basal tone of glucose flux into the cell and storage as glycogen. This physiological glycogen set point is somehow monitored by the cell, resulting in appropriate expression of proteins involved in glucose uptake, metabolism, and storage. Disruption of PTG expression in 3T3-L1 adipocytes resulted in a significant decrease in glycogen levels, which the cell attempted to counteract through several compensatory mechanisms that increased acute insulin metabolic action. Ultimately, however, these changes were unable to overcome the loss of PTG, resulting in inappropriate glycogen phosphorylase phosphorylation and dysregulation of glycogenolysis. Further study will be required to explore and define the molecular links between altered glycogen metabolism and these potential changes in gene transcription and increased insulin responsiveness in 3T3-L1 adipocytes.


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ACKNOWLEDGMENTS
 
This work was supported by a Career Development Award from the American Diabetes Association and grant DK064772 from the National Institutes of Health.


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FOOTNOTES
 
* Corresponding author. Mailing address: Department of Medicine, University of Chicago, MC1027, 5841 S. Maryland Ave., Chicago, IL 60637-1470. Phone: (773) 702-2346. Fax: (773) 834-0486. E-mail: mbrady{at}medicine.bsd.uchicago.edu. Back


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Molecular and Cellular Biology, January 2006, p. 334-342, Vol. 26, No. 1
0270-7306/06/$08.00+0     doi:10.1128/MCB.26.1.334-342.2006
Copyright © 2006, American Society for Microbiology. All Rights Reserved.



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