Disruption of the Striated Muscle Glycogen Targeting Subunit
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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 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 gene 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 glycogen synthase (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 596 DIABETES, VOL. 52, MARCH 2003 M. DELIBEGOVIC AND ASSOCIATES 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 enzyme, 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 DIABETES, VOL. 52, MARCH 2003 597 MUSCLE PP1 GLYCOGEN-TARGETING SUBUNIT 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.