Disruption of the Striated Muscle Targeting Subunit PPP1R3A of Leads to Increased Weight Gain, Fat Deposition, and Development of Resistance Mirela Delibegovic,1 Christopher G. Armstrong,1 Lorraine Dobbie,2 Peter W. Watt,1 Andrew J.H. Smith,2 and Patricia T.W. Cohen1

Disruption of the PPP1R3A encoding the glycogen targeting subunit (GM/RGL) of protein phosphatase 1 (PP1) causes substantial lowering of the glycogen syn- nsulin stimulates glycogen synthesis in skeletal mus- thase activity and a 10-fold decrease in the glycogen cle through the stimulation of glucose transport and ؊/؊ levels in skeletal muscle. Homozygous GM mice show the activation of (GS), which increased weight gain after 3 months of age and become Icatalyzes the final step in this pathway (1–3). One of obese, weighing ϳ20% more than their wild-type (WT) the routes involved in the activation of glycogen synthesis littermates after 12 months of age. Glucose tolerance is is the phosphatidylinositol-3-kinase/protein kinase B ؊/؊ impaired in 11-month-old GM mice, and their skeletal (PKB) pathway, which leads to the inhibition of glycogen muscle is insulin-resistant at >12 months of age. The synthase kinase-3 (GSK-3) and thus to net dephosphoryla- massive abdominal and other fat depositions observed tion of GS with concomitant activation (4–7). The serine at this age are likely to be a consequence of impaired residues that are phosphorylated by GSK-3 are dephos- blood glucose utilization in skeletal muscle. PP1-G M phorylated by glycogen bound PP1 (8), raising the ques- activity, assayed after specific immunoadsorption, was ؊/؊ tion of whether insulin not only inhibits GSK-3 but also absent from GM mice and stimulated in the hind limb muscles of WT mice by intravenous infusion of insulin. activates PP1. PP1-R5/PTG, another glycogen targeted form of PP1, PP1 interacts with a wide variety of regulatory subunits was not significantly stimulated by insulin in the skele- and is targeted to glycogen by several glycogen-targeting tal muscle of WT mice but showed compensatory stim- subunits (9). The most abundant glycogen-targeting sub- ؊/؊ ulation by insulin in GM mice. Our results suggest unit in rodent skeletal muscle is GM/RGL (124–126 kDa, that dysfunction of PP1-GM may contribute to the patho- encoded by the PPP1R3A gene), which is also highly physiology of human type 2 diabetes. Diabetes 52: expressed in other striated muscles (10–12). Stimulation 596–604, 2003 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 dephos- phorylation 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 From the 1Medical Research Council Protein Phosphorylation Unit, School of phosphorylation of G at Ser 48 (14). Analysis of a G Life Sciences, University of Dundee, Dundee, Scotland, United Kingdom; and M M 2GeneTargeting Laboratory, Centre for Genome Research, University of “knockout” mouse strain indicates that PP1-GM is not Edinburgh, Edinburgh, Scotland, United Kingdom. required for the insulin-activation of GS in skeletal muscle, Address correspondence and reprint requests to Professor Patricia T.W. but rather another insulin-activated form of PP1 seems to Cohen, MRC Protein Phosphorylation Unit, School of Life Sciences, MSI/WTB Complex, University of Dundee, Dow Street, Dundee DD1 5EH, Scotland, U.K. be involved (15). This form was considered unlikely to be E-mail: [email protected]. either of the other two glycogen-targeting subunits (R5/ Received for publication 4 November 2002 and accepted in revised form 21 November 2002. PTG and R6) of PP1 that are known to be expressed in P.T.W.C. receives salary and research grants from the UK Medical Research rodent skeletal muscle, because they are not restricted to Council. Some research support funds are derived from a consortium of five insulin-responsive tissues. R5/PTG (36 kDa, the product of pharmaceutical companies: AstraZeneca, Boehringer Ingelheim, GlaxoSmith- Kline, Novo Nordisk, and Pfizer. C.G.A. received salary during the course of the PPP1R3C gene) is expressed in a variety of tissues this work from Diabetes UK. Since January 2002, he has been employed by with the highest mRNA levels being in liver, skeletal Upstate Ltd (Dundee Discovery Services Division). C.G.A.’s current affiliation is Upstate Ltd, Dundee Technology Park, Dundee, muscle, and cardiac muscle (16–19). The expression of Scotland, United Kingdom. P.W.W.’s current affiliation is Sport & Exercise hepatic R5/PTG is downregulated in streptozotocin- Science, University of Brighton, Eastbourne, England, United Kingdom. induced diabetes and restored by insulin treatment (20), 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, but muscle R5/PTG is not altered by these treatments (21). wild type. In addition, R5/PTG is not known to be acutely regulated

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FIG. 1. Targeted disruption of the GM gene. A: Restriction maps of the mouse GM gene locus, the targeting vector, and the disrupted GM gene locus before and after excision of the selection cassette by Cre/loxP recombination. The initial selection of the targeted allele was by resistance to G418, and the subsequent selection of the disrupted allele, with the selection cassette deleted, was by gancyclovir resistance. Thick bars below the map (marked as P1 and P2) indicate the flanking probes used for the Southern blotting analysis. P1 (a 1-kb SacI fragment) hybridizes to a 9-kb XbaI fragment in WT ES cell genomic DNA digests and a 3-kb band upon correct targeting. Probe P2 (a 0.8-kb XbaI/NdeI fragment) hybridizes to an 18-kb PstI fragment in the WT ES cells and a 6.5-kb band in the recombinants. B: Genotyping by Southern blot analysis of tail ؊/؉ DNA digested with XbaI. Examples of DNAs from mice heterozygous for the disrupted GM allele (GM ), homozygous for the disrupted GM allele ؊/؊ ؊/؉ (GM ), and WT. A 9-kb band is indicative of the WT allele, and a 3-kb band is indicative of the disrupted GM. C: Immunoblotting of WT, GM , ␤ ␣ ␣ ؊/؊ and GM mice using the anti-GM antibody, anti-PP1 antibody to detect PP1 as a control to assess equal loading of the samples, and anti-PP1 ␮ ؊/؊ antibody. Each lane was loaded with 20 g of protein from a skeletal muscle lysate. No GM protein was detected in the pellet fractions of the GM ␮ homogenates (data not shown). D: PP1c complex with GM was immunoadsorbed from 100 l of 1 mg/ml mouse muscle lysates, and the immune pellets were assayed for phosphorylase phosphatase activity in the presence of 4 nmol/l okadaic acid. The immunoadsorbed phosphorylase phosphatase activity is expressed in mU/mg protein in the mouse skeletal muscle lysate. The results are the mean ؎ SE for four mice of each ؊/؉ ؊/؊ genotype. E: Phosphorylase phosphatase activity in skeletal muscle lysates of WT, GM , and GM mice, measured in the presence of 4 nmol/l .okadaic acid. The results are the mean ؎ SE for four mice of each genotype

by insulin or epinephrine (22). R6 (33 kDa, the product of RESEARCH DESIGN AND METHODS

the PPP1R3D gene) is present in a wide variety of tissues, Generation of GM-targeting construct for homologous recombina- tion. Recombinant phage containing genomic DNA of the G locus were and its expression was not altered in streptozotocin- M induced diabetes (18,20). To analyze the action of insulin 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 on glycogen-targeted forms of PP1 and to examine further by subcloning an 8.2-kb EcoRI/XbaI fragment with the XbaI cut end filled in the physiological role of GM, we have disrupted the GM using Klenow , into the EcoRI/XhoI(filled in) sites of a modified Ϫ Ϫ ϩ / Bluescript pKS in which the XbaI site was destroyed. The construct was then gene in mice. The GM animals produced become obese, digested with StyI to excise a 2.6-kb region spanning the entire exon 1 of G glucose-intolerant, and insulin-resistant in later life, and M assays of different glycogen-targeted forms of PP1 support (encoding amino acids 1–262) and including 0.168 kb of the promoter region Ϫ/Ϫ and 1.638 kb of the first intron. Subsequently, the StyI cut plasmid DNA was the concept that, in GM mice, stimulation of muscle modified by the ligation of complementary oligonucleotides to create an XbaI PP1-R5/PTG by insulin occurs as a compensatory response site at this position. A loxP-flanked HSVtk/neo cassette containing a herpes to the absence of insulin stimulation of PP1-GM. simplex virus thymidine kinase gene (conferring gancyclovir sensitivity) and a

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FIG. 2. Skeletal muscle glycogen and blood glucose levels. A: The glycogen content in skeletal muscle of ؊/؊ WT and GM mice fasted for 16 h. Glycogen concen- tration is expressed in micromoles of glycosyl units per gram of muscle (wet weight). Statistical signifi- cance was determined by Student’s t test (P < 0.01), and the error bars represent the SE. Number of mice is indicated inside the bars. B: Blood glucose levels in ؊/؊ 11-month-old WT and GM mice fasted for 12 h. Six mice of each genotype were examined. The differ- ences are not statistically significant. C: Glucose tolerance tests were performed on 11-month-old male mice of the indicated genotype. The differences be- ؊/؊ tween GM and WT mice are statistically significant for all values subsequent to the bolus of glucose (P < 0.01 for 15, 60, 120 min, P < 0.02 for 30 min). Results are the mean values ؎ SE for seven male mice of each ؊/؊ genotype. Two female GM mice examined at 11 months of age also showed glucose intolerance. Nei- ther male nor female mice at 6–8 months of age exhibited glucose intolerance. D: Insulin tolerance tests were performed on male mice of the indicated genotype that were >12 months old. Results are the mean values ؎ SE for seven male mice of each ؊/؊ genotype. The differences between GM and WT mice are statistically significant subsequent to the bolus of insulin (P < 0.05 for 15 min and P < 0.02 for 60 min). Although there was a tendency toward insu- ؊/؊ lin resistance in 11-month-old GM mice, there were no statistically significant differences in insulin tol- erance tests performed on mice up to 11 months of age (data not shown). neomycin phosphotransferase gene (conferring resistance to the antibiotic and okadaic acid (Calbiochem, Nottingham, U.K.) or microcystin (Life Tech- G418) under the control of a phosphoglycerate kinase promoter was inserted nologies, Paisley, U.K.) where stated. The supernatant obtained after centrif- into the newly generated plasmid at the position of the XbaI site. The resultant ugation at 16,000g for 10 min was snap-frozen in liquid nitrogen and stored at Ј Ј Ϫ GM targeting vector comprised a 2.5-kb 5 homology arm and a 3.2-kb 3 80°C. Glucose levels were measured in blood taken rapidly from the tail homology arm separated by the HSVtk/neo cassette. Before electroporation, veins of mice fasted for 12 h using a Glucotrend 2 meter (Roche Diagnostics). the targeting vector was linearized at the unique NotI site. Glucose tolerance tests were performed on mice after a 16-h overnight fast. ؊/؊ 7 Generation of GM-deficient (GM ) mice. E14-TG2a-IV ES cells (10 ; Mice were injected with 2 mg of D-glucose/g i.p., and blood glucose levels were 129/Ola strain), cultured according to standard protocol (23), were electropo- determined immediately before and at 15, 30, 60, and 120 min after injection. rated with linearized targeting vector and selected after 48 h in G418. Insulin tolerance was assessed by measuring blood glucose levels after mice G418-resistant ES cell clones were obtained after 10 days and screened by had received an intraperitoneal injection of 0.75 mU/g insulin (Human Southern blotting of XbaI and PstI digests of clone DNAs using hybridization Actrapid, 100 iU/ml; Novo Nordisk Pharmaceuticals Ltd, Crawley, Sussex, probes flanking and external to the vector homology arm sequences (see Fig. U.K.) after a 6-h fast. In vivo tissue glucose transport was determined after 1). Several G418-resistant clones were identified containing the targeted intraperitoneal injection of 2-deoxy-D-[1,2-3H]-glucose mixed with 20% dex- disruption, and one of these was chosen for an additional round of transfec- trose (2 g/kg body wt; 10 ␮Ci/mouse) according to Zisman et al. (26). For tion to remove the HSVtk/neo cassette from the targeted allele by Cre- determining the effects of insulin on in skeletal muscle, mice that mediated recombination between its directly repeated flanking loxP sites. were anesthetized with 6 mg of Sagatal/100 g body wt (Rhone Merieux Ltd, This involved electroporation with a Cre expression construct pBS185 (24) Harlow, U.K.) were infused with 2 mU of insulin into the femoral vein over a and application of gancyclovir selection after 6 days. Gancyclovir-resistant period of 10 min. Animals were killed by cervical dislocation, and skeletal clones were isolated after 12 days, and excision of the cassette was confirmed muscle was freeze-clamped and stored in liquid nitrogen. Responsiveness of by Southern blot analysis as described above and in Fig. 1. ES cells from two the animals to insulin was confirmed by determination of blood glucose levels Ϫ/Ϫ gancyclovir-resistant clones were then used to generate GM mice, by before and after insulin administration. standard protocol, using C57BL/6J blastocysts and pseudopregnant female Antibodies and immunological methods. Antibodies to human glutathione- mice as foster mothers (25). Chimeric male mice were test-crossed to C57BL/6 S-transferase–GM (1–243) were affinity-purified against maltose-binding pro- ␣ female mice and agouti offspring screened by Southern blotting for the tein fused to GM. Antibodies to human PP1 (301-KNKGKYGQFSGLNPGG- ␤ presence of the disrupted GM allele. Mice heterozygous for the disrupted GM 316) and human PP1 (316-TPPRTANPPKKR-327) were affinity-purified allele were back-crossed to C57BL/6J for several generations. Second- and against their respective peptides by Dr. Jane Leitch. The peptides were third-generation back-cross heterozygotes were intercrossed to produce mice synthesized by Dr. G. Bloomberg (University of Bristol, U.K.), and antibodies homozygous for the disrupted allele, heterozygous and wild type (WT), which were raised in sheep by Diagnostics Scotland (Penicuik, Midlothian, U.K.). were used in the analyses described herein. All mice were maintained in Affinity-purified antibodies to glutathione-S-transferase–human R5 protein temperature- and humidity-controlled conditions with a 12-h light/12-h dark and mouse R5 peptide (residues 36–49) have been described previously (20). cycle and allowed food and water ad libitum. Mice were genotyped by Phospho-GSK-3 ␣/␤ (Ser 21/9) antibodies were purchased from New England Southern blotting of tail DNA digested with XbaI (Fig. 1B)orPstI as described Biolabs (Hitchin, Herts, U.K.). Immunoblotting was performed as described by for ES clone DNAs above. Browne et al. (20). Metabolic studies. The animals were studied after a 16-h overnight fast and Enzyme assays and glycogen content. PP1 activities were determined by 32 ␮ ␮ killed by suffocation in CO2, followed by cervical dislocation, unless otherwise release of [ P]phosphate from phosphorylase a (10 mol/l) or GS (3 mol/l, stated. Tissues were freeze-clamped and stored at Ϫ80°C. After freeze phosphorylated with GSK-3␤) in the presence of 4 nmol/l okadaic acid for 10 fracturing, skeletal muscle was homogenized at 4°C in 6 vol of 50 mmol/l min at 30°C. For immunoadsorption of PP1-GM and PP1-R5 with anti-GM and Tris-HCl (pH 7.5), 150 mmol/l NaCl, 2 mmol/l EGTA, 2 mmol/l EDTA, 5% anti-R5 peptide antibodies, respectively, lysates were prepared in the presence glycerol, 0.1% Triton X-100, 0.1% (vol/vol) 2-mercaptoethanol, “Complete” of 100 nmol/l okadaic acid. The immune pellets were washed five times in the protease inhibitor cocktail from Roche Diagnostics Ltd (Lewes, East Sussex), presence of 4 nmol/l okadaic acid, and PP1 activity in the immune pellets were

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FIG. 3. Alteration in body weight and fat deposits of ؊/؊ mice. A: Female GM and WT mice. Numbers of animals at 3, 6, 8, 11, and >12 months of age were 3, ؊/؊ 13, 4, 2, and 10 for WT and 6, 15, 6, 5, and 11 for GM ؊/؊ mice. B: Male GM and WT mice. Numbers of ani- mals at 3, 6, 8, 11, and >12 months of age were 9, 6, ؊/؊ 20, 7, and 8 for WT and 8, 9, 8, 6, and 11 for GM mice. Statistical significance for the weight differ- ences, assessed by Student’s t test, was P < 0.01 for female and male mice at 6, 11, and >12 months of age. ؊/؉ The growth curves for heterozygous (GM ) female and male mice showed no significant differences from ؊/؊ those for WT mice. Female (C) and male (D)GM and WT littermates older than 12 months dissected to show the fat deposits around the heart and the large ؊/؊ deposits of abdominal fat in GM mice. assayed as described above either before (“spontaneous” activity) or after creased levels of PP1␤, whereas the level of PP1␣ was (“total” activity) preincubation with 0.1 mg/ml “dissociating” peptide that unchanged (Fig. 1C), suggesting that the PP1␤ isoform 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 ␮mol interacts with GM and that its expression is decreased in of 32P-phosphate per minute. For GS and phosphorylase assays, skeletal the absence of the targeting subunit. Assay of immunoad- muscle was homogenized in 3 vol of 25 mmol/l Tris (pH 7.5), 50 mmol/l NaF, sorbed PP1-GM using glycogen phosphorylase as substrate 2 mmol/l EDTA, 2 mmol/l EGTA, 1 mmol/l sodium orthovanadate, 1 ␮mol/l confirmed the absence of PP1-G activity (Fig. 1D). Con- microcystin, 0.1% 2-mercaptoethanol, and “Complete” protease inhibitor cock- M sistent with these results, PP1 activity in skeletal muscle tail (Roche) and centrifuged at 13,000g for 5 min. GS activity in the superna- Ϫ Ϫ Ϫ ϩ 14 / / tant was determined by measuring incorporation of [ C]glucose from UDP- lysates of GM and GM mice was decreased by 49 and [14C] glucose into glycogen in the absence or presence of 7.2 mmol/l 22%, respectively, compared with WT levels, indicating glucose-6-phosphate (G-6P) (27). Glycogen phosphorylase activity was deter- 14 that GM binds approximately half of the PP1 in skeletal mined by the incorporation of [ C]glucose-1-phosphate (Amersham Bio- muscle (Fig. 1E). sciences, Little Chalfont, Bucks, U.K.) into glycogen in the absence or Ϫ/Ϫ presence of the allosteric activator AMP (2 mmol/l) (28). Glycogen was A major metabolic difference between GM and WT measured with anthrone reagent after extraction from skeletal muscle with 1 mice was that the glycogen levels in skeletal muscle of mol/l NaOH at 100°C for 60 min (29). Ϫ/Ϫ ϳ GM mice were drastically decreased to 10% of their WT littermates (Fig. 2A). This result is consistent with the RESULTS tenet that PP1-GM dephosphorylates and activates GS, ؊/؊ GM mice exhibit weight gain, fat deposition, glu- leading to an increase in glycogen synthesis. Although Ϫ/Ϫ cose intolerance, and insulin resistance. To examine fasting blood glucose levels of GM and WT mice up to the physiological role of GM, we disrupted the murine GM 11 months of age showed no statistically significant differ- gene by homologous recombination, deleting all of exon 1, ences (Fig. 2B), 11-month-old mice exhibited glucose which encodes the translation initiation codon as well as intolerance (Fig. 2C). After intraperitoneal administration Ϫ/Ϫ the PP1 and glycogen-binding domains (Fig. 1A). The of a bolus of glucose, blood glucose levels in GM male Ϫ/Ϫ ϳ GM mice created had no obvious developmental or mice rose to 40% higher levels than those observed in morphological defects in the first 3 months of life and gave WT mice, and blood glucose was much slower to return to the expected ratio of male/female offspring for an auto- basal levels (Fig. 2C). In addition, mice older than 11 Ϫ/Ϫ somal gene. Analysis of skeletal muscle from the GM months developed insulin resistance (Fig. 2D). The Ϫ/Ϫ mice showed no GM protein on immunoblotting and de- weights of GM and WT mice were similar at 3 months of

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estimated by glucose tolerance tests incorporating the 3 tracer 2-deoxy-D-[1,2- H]-glucose (2-DOG) (26), was de- Ϫ/Ϫ creased in skeletal muscle by 69% in GM mice com- pared with WT (Fig. 4A), indicating that this tissue is Ϫ/Ϫ insulin-resistant in GM mice. In contrast, glucose trans- port into adipose tissue (Fig. 4B), glycogen levels, and GS Ϫ/Ϫ 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 Ϯ Ϫ/Ϫ elevated in the adipose tissue (WT 0.030 0.01, GM 0.069 Ϯ 0.01 ␮mol ⅐ minϪ1 ⅐ mgϪ1 glycogen, P Ͻ 0.2) or Ϯ Ϫ/Ϫ Ϯ ␮ ⅐ Ϫ1 ⅐ liver (WT 0.107 0.02, GM 0.101 0.02 mol min Ϫ1 Ͻ Ϫ/Ϫ mg 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 Ϫ/Ϫ 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 sam- Ϫ/Ϫ ples of muscles from the hind limbs of GM mice and their WT littermates. The basal GS activity was only 0.1 in Ϫ/Ϫ FIG. 4. Comparison of glucose uptake into skeletal muscle, adipose the skeletal muscle of GM mice compared with 0.3 for tissue, and liver in G ؊/؊ and WT mice in vivo. 2-DOG uptake into M WT littermates (Fig. 5A). Consequently, although insulin skeletal muscle (A) and abdominal adipose tissue (B) during a glucose Ϫ/Ϫ tolerance test. Unlabeled glucose and 2-DOG tracer were injected increased the GS activity by 1.5- to 2-fold in GM and WT ؊/؊ > Ϫ/Ϫ simultaneously into six WT and seven GM mice that were 12 mice, the activity in G mice did not reach even the months old. Data represent ␮mol ⅐ min؊1 ⅐ g؊1 tissue (mean values ؎ M SE). Statistical significance, determined by Student’s t test, was P < basal level observed in WT mice, rising only to 0.2 after 0.01 for skeletal muscle and P < 0.5 for adipose tissue. stimulation with insulin. Consistent with the effects of insulin on GS activity, there was significant phosphoryla- Ϫ/Ϫ ␣ ␤ age, but thereafter, both male and female GM mice tion of GSK-3 at Ser21 and GSK-3 at Ser9 (Fig. 5B). The gained weight more rapidly, weighing ϳ20% more than WT low basal and insulin-stimulated GS activities are in accor- Ϫ/Ϫ littermates when older than 1 year (Fig. 3A and C). dance with the very low glycogen level observed in GM Ϫ/Ϫ Deposition of fat was readily apparent in GM mice with mice, but, interestingly, differ from the usual situation in increased fatty tissue around the heart and massive in- which the activity of GS is regulated by the glycogen Ϫ/Ϫ creases in abdominal fat (Fig. 3B and D). GM mice older content, a low glycogen level being associated with a high than 1 year were not only obese but also showed a small activity state of GS (31). Our results demonstrate that loss increase in body length compared with their WT litter- of GM overrides the normal effects of glycogen on GS mates, suggesting that alteration of metabolism influences activity. Loss of GM also leads to an activation of phos- the growth of mice, which is known to continue through- phorylase (Fig. 5C), indicating that the lower glycogen Ϫ/Ϫ out adult life. content of GM skeletal muscle may arise from the In mice older than 12 months, basal glucose transport, combined effects of inactivation of GS and activation of

TABLE 1 Comparison of the glycogen contents (␮mol of glycosyl units per g wet weight of tissue), GS activity ratios, and the total GS activities ⅐ Ϫ1 ⅐ Ϫ1 Ϫ/Ϫ in the presence of G-6P (nmol min mg protein) in the skeletal muscle, liver, and adipose tissue of GM and WT mice Glycogen synthase activity ratio Glycogen synthase activity Glycogen content (␮mol/g tissue) (Ϫ/ϩ G-6P) (nmol ⅐ minϪ1 ⅐ mgϪ1) Ϫ/Ϫ Ϫ/Ϫ Ϫ/Ϫ Tissue WT (n)GM (n) WT (n)GM (n) WT (n)GM (n) Muscle 5.84 Ϯ 0.46 (10) 0.56 Ϯ 0.13 (9) 0.3 Ϯ 0.09 (5) 0.1 Ϯ 0.06 (5) 40 Ϯ 1.2 (5) 24 Ϯ 0.75 (5) Liver 101.56 Ϯ 10.44 (6) 86.7 Ϯ 10.89 (6) 0.36 Ϯ 0.09 (6) 0.39 Ϯ 0.08 (6) 145.5 Ϯ 23.14 (6) 147.98 Ϯ 23.1 (6) Adipose 0.61 Ϯ 0.09 (9) 0.50 Ϯ 0.06 (10) 0.15 Ϯ 0.07 (6) 0.18 Ϯ 0.09 (6) 2.96 Ϯ 0.59 (6) 3.1 Ϯ 0.6 (6) The number of animals (n) examined is given in brackets. The differences in the values for glycogen content, GS activity ratio, and GS activity Ϫ/Ϫ Ͻ in GM and WT skeletal muscle were statistically significant using Student’s t test (P 0.001). There were no statistically significant Ϫ/Ϫ differences for liver and adipose tissue values in GM and WT mice.

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phosphorylase. However, the total levels of both phos- Ϫ/Ϫ phorylase 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 ex- tracts (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). Ϫ/Ϫ 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-PP1␤ antibody raised against a COOH-termi- nal peptide revealed an increase in PP1␤ immunostaining after insulin stimulation (Fig. 6B). Although the level of insulin-stimulated GS activity was Ϫ/Ϫ low in GM mice, insulin stimulated basal GS activity 1.5- Ϫ/Ϫ 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 Ϫ/Ϫ 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 Ϫ/Ϫ 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 Ϫ/Ϫ 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 PP1 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 Ϫ/Ϫ WT or GM mice.

DISCUSSION Ϫ/Ϫ The very low glycogen levels in skeletal muscle of GM mice result from a decreased Ϫ/ϩG-6P GS activity ratio and an increased Ϫ/ϩAMP phosphorylase activity ratio. They are in accordance with GM being the most abundant glycogen-targeting subunit of PP1 in rodent skeletal mus- cle, dephosphorylating both GS (with activation) and ؊/؊ FIG. 5. Effect of insulin on GS activity in the skeletal muscle of GM phosphorylase (causing inactivation). The phenotype dem- and WT mice. A: GS activity was assayed in the presence or absence of G-6P in supernatant of skeletal muscle from either saline- or insulin- onstrates that PP1-GM is an essential enzyme for the ؊/؊ treated GM and WT mice. GS activity is presented as a ratio of the maintenance of the normal skeletal muscle glycogen lev- activity in the absence of G-6P divided by the total activity in the els. In addition, our data show that G specifically inter- presence of G-6P. Error bars indicate ؎SE for duplicate assays on the M number of mice analyzed (shown inside the bars). Removal of G-6P acts with PP1␤ and not other isoforms of PP1 (Fig. 6B), from muscle extracts by gel filtration did not significantly alter the consistent with previous studies (32,33). results obtained. B: Muscle extracts were immunoblotted with anti– Ϫ/Ϫ phospho-GSK-3 antibodies. Equal loading of the lanes was assessed The increased weight gain of GM mice from 3 months using the control anti-GAPDH antibody. C: Phosphorylase activity was of age and massive increases in adipose tissue present at assayed in the presence or absence of AMP in supernatant of skeletal ؊/؊ 12 months of age are consistent with the concept that muscle from either saline- or insulin-treated GM and WT mice. Error bars indicate ؎SE for duplicate assays on the number of mice analyzed when blood glucose cannot be converted into glycogen in ؊/؊ (shown inside the bars). The differences between GM and WT mice skeletal muscle, much of this glucose is taken up by are statistically significant (P < 0.01 for saline infusion, P < 0.02 for adipocytes and converted into fat deposits, which increase insulin infusion). Total phosphorylase activity in the skeletal muscle of -G ؊/؊ mice (1.3 ؎ 0.05 ␮mol ⅐ min؊1 ⅐ mg؊1) was lower than in WT gradually with the age and lead to obesity. Glucose intol M Ϫ Ϫ / .(␮mol ⅐ min؊1 ⅐ mg؊1 0.1 ؎ 2.54) erance, which is clearly evident in GM mice at 11

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FIG. 6. Effect of insulin treatment on the expression

of GM and R5/PTG and their associated phosphatase activities. The PP1c complex with GM was immuno- adsorbed from 100 ␮l of 1 mg/ml mouse muscle lysate (A and B). The PP1c complex with R5 was immuno- adsorbed from 100 ␮l of 1 mg/ml mouse muscle lysate (C and D). A and C: The immune pellets were assayed for spontaneous phosphatase activity (speckled bars) and in the presence of dissociating peptide (hatched bars) for determination of the total phos- phatase activity using either phosphorylase or GS as substrate. The immunoadsorbed phosphatase activity is expressed in mU/mg total protein in the mouse muscle lysate. The mice hind limbs were infused with saline or insulin for 10 min. The number of mice analyzed is shown inside the bars. Error bars indicate the SE for triplicate immunoadsorption assays, re- peated at least four times. Statistical significance for

the stimulation of the PP1-GM total phosphorylase phosphatase activity and total GS phosphatase activ- ity in WT mice, assessed by Student’s t test, was P < 0.05. Statistical significance for the insulin stimula- tion of the PP1-R5/PTG spontaneous and total phos- ؊/؊ phorylase phosphatase activity in the GM mice was P < 0.001. B: The immunopellets were blotted with anti-PP1␤ antibodies. No PP1␣ or PP1␥ was found in

the PP1-GM immunopellets. The same membrane was then blotted with anti-GM as a control. Two represen- tative independent samples per treatment are shown, and the blot is one of several that show the same result. D: The immunopellets were blotted with an- ti-R5 and anti-PP1␤ antibodies. The lower band was identified as R5 by depletion of this band from the lysates with anti-R5. Two representative indepen- dent samples per treatment are shown, and the blot is one of several that show the same result.

602 DIABETES, VOL. 52, MARCH 2003 M. DELIBEGOVIC AND ASSOCIATES months of age, and the subsequent development of insulin PP1 assay is also performed in the presence of a peptide resistance may arise as a consequence of the increased that dissociates the glycogen-targeting subunit from PP1 Ϫ/Ϫ fatty acids present in GM mice of this age (34,35). (measuring of total PP1 activity bound to the targeting Because glucose intolerance and insulin resistance usually subunit) or in the absence of the peptide (measuring the precede overt diabetes, our results suggest that disruption spontaneous activity of the glycogen-targeted PP1 com- of GM may predispose to insulin resistance and diabetes. plex). The activity of the free catalytic subunit of PP1 is In accordance with this data, association of PPP1R3/GM unaffected by the dissociating peptide. Using this assay, mutations with insulin resistance have been noted in some we observed an increase in the total PP1 activity bound to human populations (36–38) but not in others (39–41). For GM after insulin treatment in the skeletal muscle of WT mice. An increase in G -associated PP1␤ immunostaining example, a mutation-lowering expression of PPP1R3 (GM) M was associated with diabetes in the Pima Indians (37) and after insulin stimulation was also observed and may reflect a Japanese population (38). In addition, interaction of a an real increase in the level of PP1␤ or alternatively the COOH-terminus of PP1␤ may undergo a posttranslational truncated GM with a peroxisome proliferator–activated receptor ␥ mutant was found to give rise to a rare severe modification that allows it to be recognized more effi- insulin resistance phenotype (42). ciently by the antibody. Because this region contains two threonine residues, it is possible that the activity of PP1␤ Suzuki et al. (15) recently reported a GM/RGL knockout model in which the mice exhibited very low skeletal bound to GM may be regulated by insulin via reversible Ϫ/Ϫ phosphorylation. muscle glycogen levels. However, in contrast to our GM mice, those in the study of Suzuki et al. did not show In contrast to PP1-GM, PP1-R5/PTG activity was not weight gain, fat deposition, glucose intolerance, or insulin appreciably stimulated by insulin in the skeletal muscle of WT mice, but both the spontaneous and total PP1 activity resistance when examined up to 12 months of age. The associated with R5/PTG was stimulated by insulin in routes for disposal of glucose that is not utilized for Ϫ Ϫ G / mice with a parallel increase in PP1-␤ immuno- glycogen synthesis in the skeletal muscles of the Suzuki et M staining. Thus, the absence of PP1-G leads to a compen- al. G knockout mice are unclear. Both G knockout M M M satory effect of insulin on PP1-R5/PTG. In summary, our models have been produced in very similar genetic back- results are consistent with the following model. In WT grounds of 129 crossed to C57BL/6J, although the 129 mice, insulin-activates GS not only by inhibiting GSK-3 but substrains differ (129/Ola compared with 129/Sv). The also by activating PP1-GM. The activation of PP1-GM is precise nature of the genetic modification that abrogates caused by an increase in the activity or amount of PP1␤ Ϫ Ϫ the PP1 and glycogen-binding domains of GM in the two / bound to the glycogen-targeting subunit. In GM mice, knockout models is slightly different; the one described this modification of PP1␤ elevates the activity of the herein deletes a larger region of both promoter and intron PP1-R5/PTG complex, but the activity of PP1-R5/PTG in 1 sequence. Moreover, in this model, the selection markers the presence or absence of insulin is insufficient to restore have been removed by Cre/loxP recombination to avoid GS activity to the levels observed in WT mice. any potential complicating effects as a result of their interference with the expression of neighboring , ACKNOWLEDGMENTS whereas the selection marker is retained in the model of Suzuki et al. Nevertheless, the difference between the two The work was supported by the Medical Research Council, Ϫ/Ϫ U.K. and Diabetes U.K. The CGR Gene Targeting Labora- GM models is of considerable interest because it may provide an avenue to uncover the factors that lead to tory was supported by the Biotechnology and Biological Sciences Research Council, U.K. M.D. is a recipient of a obesity, glucose intolerance, and insulin resistance when postgraduate studentship from the Royal Society and blood glucose conversion into muscle glycogen is inade- holds the Pat and Muriel McPherson Studentship of the quate for the postprandial removal of blood glucose. Ϫ Ϫ Medical Research Council Protein Phosphorylation Unit. In the G / mice reported here, both the basal M C.G.A. was supported by a postdoctoral research assis- Ϫ/ϩG-6P GS activity ratio and Ϫ/ϩG-6P GS activity ratio tantship from Diabetes U.K. measured after stimulation by near physiological doses of We thank Gareth Browne and Ann Burchell for helpful insulin were observed to be low. Disruption of the G - M advice and Derek Black for technical assistance. 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 REFERENCES data indicate that PP1-G may contribute to action of 1. Craig JW, Larner J: Influence of epinephrine and insulin on uridine M diphosphate glucose-␣-glucan transferase and phosphorylase in muscle. insulin on GS activity in skeletal muscle in these condi- Nature 202:971–973, 1964 tions. Although Suzuki et al. found that upon insulin 2. 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