Disruption of the Striated Muscle Glycogen Targeting Subunit PPP1R3A of Protein Phosphatase 1 Leads to Increased Weight Gain, Fat Deposition, and Development of Insulin Resistance
Disruption of the PPP1R3A gene encoding the glycogen targeting subunit (GM/RGL) of protein phosphatase 1 (PP1) causes substantial lowering of the glycogen synthase activity and a 10-fold decrease in the glycogen levels in skeletal muscle. Homozygous GM−/− mice show increased weight gain after 3 months of age and become obese, weighing ∼20% more than their wild-type (WT) littermates after 12 months of age. Glucose tolerance is impaired in 11-month-old GM−/− mice, and their skeletal muscle is insulin-resistant at ≥12 months of age. The massive abdominal and other fat depositions observed at this age are likely to be a consequence of impaired blood glucose utilization in skeletal muscle. PP1-GM activity, assayed after specific immunoadsorption, was absent from GM−/− mice and stimulated in the hind limb muscles of WT mice by intravenous infusion of insulin. PP1-R5/PTG, another glycogen targeted form of PP1, was not significantly stimulated by insulin in the skeletal muscle of WT mice but showed compensatory stimulation by insulin in GM−/− mice. Our results suggest that dysfunction of PP1-GM may contribute to the pathophysiology of human type 2 diabetes.
Insulin stimulates glycogen synthesis in skeletal muscle through the stimulation of glucose transport and the activation of glycogen synthase (GS), which catalyzes the final step in this pathway (1–3). One of the routes involved in the activation of glycogen synthesis is the phosphatidylinositol-3-kinase/protein kinase B (PKB) pathway, which leads to the inhibition of glycogen synthase kinase-3 (GSK-3) and thus to net dephosphorylation of GS with concomitant activation (4–7). The serine residues that are phosphorylated by GSK-3 are dephosphorylated by glycogen bound PP1 (8), raising the question of whether insulin not only inhibits GSK-3 but also activates PP1.
PP1 interacts with a wide variety of regulatory subunits and is targeted to glycogen by several glycogen-targeting subunits (9). The most abundant glycogen-targeting subunit in rodent skeletal muscle is GM/RGL (124–126 kDa, encoded by the PPP1R3A gene), which is also highly expressed in other striated muscles (10–12). Stimulation with epinephrine leads to phosphorylation of Ser 67 in rabbit GM. This dissociates PP1 from GM, causing its release from glycogen and thereby inhibiting the dephosphorylation of GS and glycogen phosphorylase with a resultant increase in glycogenolysis (13). However, the participation of the PP1-GM complex in the action of insulin on glycogen metabolism is questionable. Recent studies argue against insulin acutely activating PP1-GM by phosphorylation of GM at Ser 48 (14). Analysis of a GM “knockout” mouse strain indicates that PP1-GM is not required for the insulin-activation of GS in skeletal muscle, but rather another insulin-activated form of PP1 seems to be involved (15). This form was considered unlikely to be either of the other two glycogen-targeting subunits (R5/PTG and R6) of PP1 that are known to be expressed in rodent skeletal muscle, because they are not restricted to insulin-responsive tissues. R5/PTG (36 kDa, the product of the PPP1R3C gene) is expressed in a variety of tissues with the highest mRNA levels being in liver, skeletal muscle, and cardiac muscle (16–19). The expression of hepatic R5/PTG is downregulated in streptozotocin-induced diabetes and restored by insulin treatment (20), but muscle R5/PTG is not altered by these treatments (21). In addition, R5/PTG is not known to be acutely regulated by insulin or epinephrine (22). R6 (33 kDa, the product of the PPP1R3D gene) is present in a wide variety of tissues, and its expression was not altered in streptozotocin-induced diabetes (18,20). To analyze the action of insulin on glycogen-targeted forms of PP1 and to examine further the physiological role of GM, we have disrupted the GM gene in mice. The GM−/− animals produced become obese, glucose-intolerant, and insulin-resistant in later life, and assays of different glycogen-targeted forms of PP1 support the concept that, in GM−/− mice, stimulation of muscle PP1-R5/PTG by insulin occurs as a compensatory response to the absence of insulin stimulation of PP1-GM.
RESEARCH DESIGN AND METHODS
Generation of GM-targeting construct for homologous recombination.
Recombinant phage containing genomic DNA of the GM locus were isolated from a 129/Ola mouse library using a cDNA probe encoding the NH2-terminal 691 amino acids of GM. The GM targeting vector was constructed by subcloning an 8.2-kb EcoRI/XbaI fragment with the XbaI cut end filled in using Klenow enzyme, into the EcoRI/XhoI (filled in) sites of a modified Bluescript pKS+ in which the XbaI site was destroyed. The construct was then digested with StyI to excise a 2.6-kb region spanning the entire exon 1 of GM (encoding amino acids 1–262) and including 0.168 kb of the promoter region and 1.638 kb of the first intron. Subsequently, the StyI cut plasmid DNA was modified by the ligation of complementary oligonucleotides to create an XbaI site at this position. A loxP-flanked HSVtk/neo cassette containing a herpes simplex virus thymidine kinase gene (conferring gancyclovir sensitivity) and a neomycin phosphotransferase gene (conferring resistance to the antibiotic G418) under the control of a phosphoglycerate kinase promoter was inserted into the newly generated plasmid at the position of the XbaI site. The resultant GM targeting vector comprised a 2.5-kb 5′ homology arm and a 3.2-kb 3′ homology arm separated by the HSVtk/neo cassette. Before electroporation, the targeting vector was linearized at the unique NotI site.
Generation of GM-deficient (GM−/−) mice.
E14-TG2a-IV ES cells (107; 129/Ola strain), cultured according to standard protocol (23), were electroporated with linearized targeting vector and selected after 48 h in G418. G418-resistant ES cell clones were obtained after 10 days and screened by Southern blotting of XbaI and PstI digests of clone DNAs using hybridization probes flanking and external to the vector homology arm sequences (see Fig. 1). Several G418-resistant clones were identified containing the targeted disruption, and one of these was chosen for an additional round of transfection to remove the HSVtk/neo cassette from the targeted allele by Cre-mediated recombination between its directly repeated flanking loxP sites. This involved electroporation with a Cre expression construct pBS185 (24) and application of gancyclovir selection after 6 days. Gancyclovir-resistant clones were isolated after 12 days, and excision of the cassette was confirmed by Southern blot analysis as described above and in Fig. 1. ES cells from two gancyclovir-resistant clones were then used to generate GM−/− mice, by standard protocol, using C57BL/6J blastocysts and pseudopregnant female mice as foster mothers (25). Chimeric male mice were test-crossed to C57BL/6 female mice and agouti offspring screened by Southern blotting for the presence of the disrupted GM allele. Mice heterozygous for the disrupted GM allele were back-crossed to C57BL/6J for several generations. Second- and third-generation back-cross heterozygotes were intercrossed to produce mice homozygous for the disrupted allele, heterozygous and wild type (WT), which were used in the analyses described herein. All mice were maintained in temperature- and humidity-controlled conditions with a 12-h light/12-h dark cycle and allowed food and water ad libitum. Mice were genotyped by Southern blotting of tail DNA digested with XbaI (Fig. 1B) or PstI as described for ES clone DNAs above.
The animals were studied after a 16-h overnight fast and killed by suffocation in CO2, followed by cervical dislocation, unless otherwise stated. Tissues were freeze-clamped and stored at −80°C. After freeze fracturing, skeletal muscle was homogenized at 4°C in 6 vol of 50 mmol/l Tris-HCl (pH 7.5), 150 mmol/l NaCl, 2 mmol/l EGTA, 2 mmol/l EDTA, 5% glycerol, 0.1% Triton X-100, 0.1% (vol/vol) 2-mercaptoethanol, “Complete” protease inhibitor cocktail from Roche Diagnostics Ltd (Lewes, East Sussex), and okadaic acid (Calbiochem, Nottingham, U.K.) or microcystin (Life Technologies, Paisley, U.K.) where stated. The supernatant obtained after centrifugation at 16,000g for 10 min was snap-frozen in liquid nitrogen and stored at −80°C. Glucose levels were measured in blood taken rapidly from the tail veins of mice fasted for 12 h using a Glucotrend 2 meter (Roche Diagnostics). Glucose tolerance tests were performed on mice after a 16-h overnight fast. Mice were injected with 2 mg of d-glucose/g i.p., and blood glucose levels were determined immediately before and at 15, 30, 60, and 120 min after injection. Insulin tolerance was assessed by measuring blood glucose levels after mice had received an intraperitoneal injection of 0.75 mU/g insulin (Human Actrapid, 100 iU/ml; Novo Nordisk Pharmaceuticals Ltd, Crawley, Sussex, U.K.) after a 6-h fast. In vivo tissue glucose transport was determined after intraperitoneal injection of 2-deoxy-d-[1,2-3H]-glucose mixed with 20% dextrose (2 g/kg body wt; 10 μCi/mouse) according to Zisman et al. (26). For determining the effects of insulin on enzymes in skeletal muscle, mice that were anesthetized with 6 mg of Sagatal/100 g body wt (Rhone Merieux Ltd, Harlow, U.K.) were infused with 2 mU of insulin into the femoral vein over a period of 10 min. Animals were killed by cervical dislocation, and skeletal muscle was freeze-clamped and stored in liquid nitrogen. Responsiveness of the animals to insulin was confirmed by determination of blood glucose levels before and after insulin administration.
Antibodies and immunological methods.
Antibodies to human glutathione-S-transferase‐GM (1–243) were affinity-purified against maltose-binding protein fused to GM. Antibodies to human PP1α (301-KNKGKYGQFSGLNPGG-316) and human PP1β (316-TPPRTANPPKKR-327) were affinity-purified against their respective peptides by Dr. Jane Leitch. The peptides were synthesized by Dr. G. Bloomberg (University of Bristol, U.K.), and antibodies were raised in sheep by Diagnostics Scotland (Penicuik, Midlothian, U.K.). Affinity-purified antibodies to glutathione-S-transferase‐human R5 protein and mouse R5 peptide (residues 36–49) have been described previously (20). Phospho-GSK-3 α/β (Ser 21/9) antibodies were purchased from New England Biolabs (Hitchin, Herts, U.K.). Immunoblotting was performed as described by Browne et al. (20).
Enzyme assays and glycogen content.
PP1 activities were determined by release of [32P]phosphate from phosphorylase a (10 μmol/l) or GS (3 μmol/l, phosphorylated with GSK-3β) in the presence of 4 nmol/l okadaic acid for 10 min at 30°C. For immunoadsorption of PP1-GM and PP1-R5 with anti-GM and anti-R5 peptide antibodies, respectively, lysates were prepared in the presence of 100 nmol/l okadaic acid. The immune pellets were washed five times in the presence of 4 nmol/l okadaic acid, and PP1 activity in the immune pellets were assayed as described above either before (“spontaneous” activity) or after (“total” activity) preincubation with 0.1 mg/ml “dissociating” peptide that causes the release of free PP1c from the glycogen-targeting subunit (20). One unit of activity is the amount of enzyme that catalyzes the release of 1 μmol of 32P-phosphate per minute. For GS and phosphorylase assays, skeletal muscle was homogenized in 3 vol of 25 mmol/l Tris (pH 7.5), 50 mmol/l NaF, 2 mmol/l EDTA, 2 mmol/l EGTA, 1 mmol/l sodium orthovanadate, 1 μmol/l microcystin, 0.1% 2-mercaptoethanol, and “Complete” protease inhibitor cocktail (Roche) and centrifuged at 13,000g for 5 min. GS activity in the supernatant was determined by measuring incorporation of [14C]glucose from UDP-[14C] glucose into glycogen in the absence or presence of 7.2 mmol/l glucose-6-phosphate (G-6P) (27). Glycogen phosphorylase activity was determined by the incorporation of [14C]glucose-1-phosphate (Amersham Biosciences, Little Chalfont, Bucks, U.K.) into glycogen in the absence or presence of the allosteric activator AMP (2 mmol/l) (28). Glycogen was measured with anthrone reagent after extraction from skeletal muscle with 1 mol/l NaOH at 100°C for 60 min (29).
GM−/− mice exhibit weight gain, fat deposition, glucose intolerance, and insulin resistance.
To examine the physiological role of GM, we disrupted the murine GM gene by homologous recombination, deleting all of exon 1, which encodes the translation initiation codon as well as the PP1 and glycogen-binding domains (Fig. 1A). The GM−/− mice created had no obvious developmental or morphological defects in the first 3 months of life and gave the expected ratio of male/female offspring for an autosomal gene. Analysis of skeletal muscle from the GM−/− mice showed no GM protein on immunoblotting and decreased levels of PP1β, whereas the level of PP1α was unchanged (Fig. 1C), suggesting that the PP1β isoform interacts with GM and that its expression is decreased in the absence of the targeting subunit. Assay of immunoadsorbed PP1-GM using glycogen phosphorylase as substrate confirmed the absence of PP1-GM activity (Fig. 1D). Consistent with these results, PP1 activity in skeletal muscle lysates of GM−/− and GM−/+ mice was decreased by 49 and 22%, respectively, compared with WT levels, indicating that GM binds approximately half of the PP1 in skeletal muscle (Fig. 1E).
A major metabolic difference between GM−/− and WT mice was that the glycogen levels in skeletal muscle of GM−/− mice were drastically decreased to ∼10% of their WT littermates (Fig. 2A). This result is consistent with the tenet that PP1-GM dephosphorylates and activates GS, leading to an increase in glycogen synthesis. Although fasting blood glucose levels of GM−/− and WT mice up to 11 months of age showed no statistically significant differences (Fig. 2B), 11-month-old mice exhibited glucose intolerance (Fig. 2C). After intraperitoneal administration of a bolus of glucose, blood glucose levels in GM−/− male mice rose to ∼40% higher levels than those observed in WT mice, and blood glucose was much slower to return to basal levels (Fig. 2C). In addition, mice older than 11 months developed insulin resistance (Fig. 2D). The weights of GM−/− and WT mice were similar at 3 months of age, but thereafter, both male and female GM−/− mice gained weight more rapidly, weighing ∼20% more than WT littermates when older than 1 year (Fig. 3A and C). Deposition of fat was readily apparent in GM−/− mice with increased fatty tissue around the heart and massive increases in abdominal fat (Fig. 3B and D). GM−/− mice older than 1 year were not only obese but also showed a small increase in body length compared with their WT littermates, suggesting that alteration of metabolism influences the growth of mice, which is known to continue throughout adult life.
In mice older than 12 months, basal glucose transport, estimated by glucose tolerance tests incorporating the tracer 2-deoxy-d-[1,2-3H]-glucose (2-DOG) (26), was decreased in skeletal muscle by 69% in GM−/− mice compared with WT (Fig. 4A), indicating that this tissue is insulin-resistant in GM−/− mice. In contrast, glucose transport into adipose tissue (Fig. 4B), glycogen levels, and GS activity in adipose tissue (Table 1) were similar in GM−/− and WT mice. Incorporation of 2-DOG into glycogen, through the direct pathway (30), was not significantly elevated in the adipose tissue (WT 0.030 ± 0.01, GM−/− 0.069 ± 0.01 μmol · min−1 · mg−1 glycogen, P < 0.2) or liver (WT 0.107 ± 0.02, GM−/− 0.101 ± 0.02 μmol · min−1 · mg−1 glycogen; P < 0.5) of GM−/− mice. However, low levels of 2-DOG incorporation and low glycogen content make precise ascertainment of these values difficult for adipose tissue. Overall, it seems likely to be largely the presence of more adipose tissue in older GM−/− mice (abdominal adipose tissue is increased approximately fourfold compared with WT) that accounts for the uptake of glucose that is not utilized by skeletal muscle.
Insulin infusion into skeletal muscle stimulates PP1-GM activity in WT mice and PP1-R5/PTG activity in GM−/− mice.
To assess whether basal or insulin-stimulated GS activity was impaired in skeletal muscle by disruption of the GM gene, we infused, intravenously, saline or doses of insulin to achieve levels only slightly higher than physiological concentrations and took samples of muscles from the hind limbs of GM−/− mice and their WT littermates. The basal GS activity was only 0.1 in the skeletal muscle of GM−/− mice compared with 0.3 for WT littermates (Fig. 5A). Consequently, although insulin increased the GS activity by 1.5- to 2-fold in GM−/− and WT mice, the activity in GM−/− mice did not reach even the basal level observed in WT mice, rising only to 0.2 after stimulation with insulin. Consistent with the effects of insulin on GS activity, there was significant phosphorylation of GSK-3α at Ser21 and GSK-3β at Ser9 (Fig. 5B). The low basal and insulin-stimulated GS activities are in accordance with the very low glycogen level observed in GM−/− mice, but, interestingly, differ from the usual situation in which the activity of GS is regulated by the glycogen content, a low glycogen level being associated with a high activity state of GS (31). Our results demonstrate that loss of GM overrides the normal effects of glycogen on GS activity. Loss of GM also leads to an activation of phosphorylase (Fig. 5C), indicating that the lower glycogen content of GM−/− skeletal muscle may arise from the combined effects of inactivation of GS and activation of phosphorylase. However, the total levels of both phosphorylase and GS are decreased by 40–50% in GM−/− mice compared with WT, probably because the enzymes are more easily degraded when their glycogen-binding sites are limited by the low glycogen levels.
To ascertain whether PP1-GM was being stimulated by insulin in WT mice, we assayed its activity in the absence and presence of a peptide that dissociates PP1 from GM after immunoadsorption of PP1-GM from the muscle extracts (20). These assays showed that in WT mice, PP1-GM activity was stimulated slightly by insulin and that the total PP1 activity associated with GM was increased (Fig. 6A). As expected, PP1-GM activity was absent from GM−/− mice, indicating that the assays were specific for this particular glycogen-targeted form of PP1. Immunoblotting with an anti-PP1β antibody raised against a COOH-terminal peptide revealed an increase in PP1β immunostaining after insulin stimulation (Fig. 6B).
Although the level of insulin-stimulated GS activity was low in GM−/− mice, insulin stimulated basal GS activity 1.5- to 2-fold in GM−/− mice (Fig. 5A), raising the question of which phosphatase was dephosphorylating GS under these conditions. We therefore tested whether PP1-R5/PTG, which is expressed at appreciable levels in skeletal muscle (18), responds acutely to insulin in WT and GM−/− mice. After infusion with saline, assays carried out after immunoadsorption of PP1-R5/PTG revealed a decrease in the basal activity in the skeletal muscle of GM−/− mice, probably as a consequence of the low glycogen levels (Fig. 6C). It is interesting that although PP1-R5/PTG activity showed little or no stimulation in WT mice after infusion with insulin, the activity of PP1-R5/PTG doubled in GM−/− mice upon insulin stimulation and now approached the level of activity found in WT mice. In addition, as found for the GM complex, the immunostaining of the PP1β present in the PP1-R5/PTG immunopellets increased (Fig. 6D). The low levels of PP1-R6 activity in murine skeletal muscle precluded ascertainment of whether this glycogen-targeted form of PP1 was acutely regulated by insulin in WT or GM−/− mice.
The very low glycogen levels in skeletal muscle of GM−/− mice result from a decreased −/+G-6P GS activity ratio and an increased −/+AMP phosphorylase activity ratio. They are in accordance with GM being the most abundant glycogen-targeting subunit of PP1 in rodent skeletal muscle, dephosphorylating both GS (with activation) and phosphorylase (causing inactivation). The phenotype demonstrates that PP1-GM is an essential enzyme for the maintenance of the normal skeletal muscle glycogen levels. In addition, our data show that GM specifically interacts with PP1β and not other isoforms of PP1 (Fig. 6B), consistent with previous studies (32,33).
The increased weight gain of GM−/− mice from 3 months of age and massive increases in adipose tissue present at 12 months of age are consistent with the concept that when blood glucose cannot be converted into glycogen in skeletal muscle, much of this glucose is taken up by adipocytes and converted into fat deposits, which increase gradually with the age and lead to obesity. Glucose intolerance, which is clearly evident in GM−/− mice at 11 months of age, and the subsequent development of insulin resistance may arise as a consequence of the increased fatty acids present in GM−/− mice of this age (34,35). Because glucose intolerance and insulin resistance usually precede overt diabetes, our results suggest that disruption of GM may predispose to insulin resistance and diabetes. In accordance with this data, association of PPP1R3/GM mutations with insulin resistance have been noted in some human populations (36–38) but not in others (39–41). For example, a mutation-lowering expression of PPP1R3 (GM) was associated with diabetes in the Pima Indians (37) and a Japanese population (38). In addition, interaction of a truncated GM with a peroxisome proliferator‐activated receptor γ mutant was found to give rise to a rare severe insulin resistance phenotype (42).
Suzuki et al. (15) recently reported a GM/RGL knockout model in which the mice exhibited very low skeletal muscle glycogen levels. However, in contrast to our GM−/− mice, those in the study of Suzuki et al. did not show weight gain, fat deposition, glucose intolerance, or insulin resistance when examined up to 12 months of age. The routes for disposal of glucose that is not utilized for glycogen synthesis in the skeletal muscles of the Suzuki et al. GM knockout mice are unclear. Both GM knockout models have been produced in very similar genetic backgrounds of 129 crossed to C57BL/6J, although the 129 substrains differ (129/Ola compared with 129/Sv). The precise nature of the genetic modification that abrogates the PP1 and glycogen-binding domains of GM in the two knockout models is slightly different; the one described herein deletes a larger region of both promoter and intron 1 sequence. Moreover, in this model, the selection markers have been removed by Cre/loxP recombination to avoid any potential complicating effects as a result of their interference with the expression of neighboring genes, whereas the selection marker is retained in the model of Suzuki et al. Nevertheless, the difference between the two GM−/− models is of considerable interest because it may provide an avenue to uncover the factors that lead to obesity, glucose intolerance, and insulin resistance when blood glucose conversion into muscle glycogen is inadequate for the postprandial removal of blood glucose.
In the GM−/− mice reported here, both the basal −/+G-6P GS activity ratio and −/+G-6P GS activity ratio measured after stimulation by near physiological doses of insulin were observed to be low. Disruption of the GM-targeting subunit of PP1 therefore causes a major decrease in the GS activity ratio that cannot be overcome by insulin treatment under the conditions that we have studied. The data indicate that PP1-GM may contribute to action of insulin on GS activity in skeletal muscle in these conditions. Although Suzuki et al. found that upon insulin stimulation GS activity in the skeletal muscle of GM−/− mice rose well above the basal levels in WT mice, they used higher doses of insulin (2 units/kg i.v.) to obtain this stimulation.
To examine the activities of different glycogen-targeted forms of PP1 separately, we developed a coupled glycogen-targeting subunit immunoadsorption and PP1 assay system (20). In contrast, previous assays have assessed the effect of hormones on total PP1 activity or a mixture of different glycogen-targeted forms of PP1 (15,43–46). Our PP1 assay is also performed in the presence of a peptide that dissociates the glycogen-targeting subunit from PP1 (measuring of total PP1 activity bound to the targeting subunit) or in the absence of the peptide (measuring the spontaneous activity of the glycogen-targeted PP1 complex). The activity of the free catalytic subunit of PP1 is unaffected by the dissociating peptide. Using this assay, we observed an increase in the total PP1 activity bound to GM after insulin treatment in the skeletal muscle of WT mice. An increase in GM-associated PP1β immunostaining after insulin stimulation was also observed and may reflect an real increase in the level of PP1β or alternatively the COOH-terminus of PP1β may undergo a posttranslational modification that allows it to be recognized more efficiently by the antibody. Because this region contains two threonine residues, it is possible that the activity of PP1β bound to GM may be regulated by insulin via reversible phosphorylation.
In contrast to PP1-GM, PP1-R5/PTG activity was not appreciably stimulated by insulin in the skeletal muscle of WT mice, but both the spontaneous and total PP1 activity associated with R5/PTG was stimulated by insulin in GM−/− mice with a parallel increase in PP1-β immunostaining. Thus, the absence of PP1-GM leads to a compensatory effect of insulin on PP1-R5/PTG. In summary, our results are consistent with the following model. In WT mice, insulin-activates GS not only by inhibiting GSK-3 but also by activating PP1-GM. The activation of PP1-GM is caused by an increase in the activity or amount of PP1β bound to the glycogen-targeting subunit. In GM−/− mice, this modification of PP1β elevates the activity of the PP1-R5/PTG complex, but the activity of PP1-R5/PTG in the presence or absence of insulin is insufficient to restore GS activity to the levels observed in WT mice.
The work was supported by the Medical Research Council, U.K. and Diabetes U.K. The CGR Gene Targeting Laboratory was supported by the Biotechnology and Biological Sciences Research Council, U.K. M.D. is a recipient of a postgraduate studentship from the Royal Society and holds the Pat and Muriel McPherson Studentship of the Medical Research Council Protein Phosphorylation Unit. C.G.A. was supported by a postdoctoral research assistantship from Diabetes U.K.
We thank Gareth Browne and Ann Burchell for helpful advice and Derek Black for technical assistance.
Address correspondence and reprint requests to Professor Patricia T.W. Cohen, MRC Protein Phosphorylation Unit, School of Life Sciences, MSI/WTB Complex, University of Dundee, Dow Street, Dundee DD1 5EH, Scotland, U.K. E-mail:.
Received for publication 4 November 2002 and accepted in revised form 21 November 2002.
P.T.W.C. receives salary and research grants from the UK Medical Research Council. Some research support funds are derived from a consortium of five pharmaceutical companies: AstraZeneca, Boehringer Ingelheim, GlaxoSmithKline, Novo Nordisk, and Pfizer. C.G.A. received salary during the course of this work from Diabetes UK. Since January 2002, he has been employed by Upstate Ltd (Dundee Discovery Services Division).
C.G.A.’s current affiliation is Upstate Ltd, Dundee Technology Park, Dundee, Scotland, United Kingdom. P.W.W.’s current affiliation is Sport & Exercise Science, University of Brighton, Eastbourne, England, United Kingdom.
2DOG, 2-deoxy-d-[1,2-3H]-glucose; G-6P, glucose-6-phosphate; GS, glycogen synthase; GSK-3, glycogen synthase kinase-3; PP1, protein phosphatase 1; WT, wild type.