Date Published: January 29, 2008
Publisher: Public Library of Science
Author(s): David B Savage, Lanmin Zhai, Balasubramanian Ravikumar, Cheol Soo Choi, Johanna E Snaar, Amanda C McGuire, Sung-Eun Wou, Gemma Medina-Gomez, Sheene Kim, Cheryl B Bock, Dyann M Segvich, Antonio Vidal-Puig, Nicholas J Wareham, Gerald I Shulman, Fredrik Karpe, Roy Taylor, Bartholomew A Pederson, Peter J Roach, Stephen O’Rahilly, Anna A DePaoli-Roach, Leif C Groop
Abstract: BackgroundStored glycogen is an important source of energy for skeletal muscle. Human genetic disorders primarily affecting skeletal muscle glycogen turnover are well-recognised, but rare. We previously reported that a frameshift/premature stop mutation in PPP1R3A, the gene encoding RGL, a key regulator of muscle glycogen metabolism, was present in 1.36% of participants from a population of white individuals in the UK. However, the functional implications of the mutation were not known. The objective of this study was to characterise the molecular and physiological consequences of this genetic variant.Methods and FindingsIn this study we found a similar prevalence of the variant in an independent UK white population of 744 participants (1.46%) and, using in vivo 13C magnetic resonance spectroscopy studies, demonstrate that human carriers (n = 6) of the variant have low basal (65% lower, p = 0.002) and postprandial muscle glycogen levels. Mice engineered to express the equivalent mutation had similarly decreased muscle glycogen levels (40% lower in heterozygous knock-in mice, p < 0.05). In muscle tissue from these mice, failure of the truncated mutant to bind glycogen and colocalize with glycogen synthase (GS) decreased GS and increased glycogen phosphorylase activity states, which account for the decreased glycogen content.ConclusionsThus, PPP1R3A C1984ΔAG (stop codon 668) is, to our knowledge, the first prevalent mutation described that directly impairs glycogen synthesis and decreases glycogen levels in human skeletal muscle. The fact that it is present in ∼1 in 70 UK whites increases the potential biomedical relevance of these observations.
Partial Text: The dissection of the genetic basis for interindividual variation in human metabolism is a major goal of contemporary metabolic research. Recently we identified a novel frameshift (FS) premature stop mutation in PPP1R3A (C1984ΔAG; stop codon 668; referred to subsequently as PPP1R3A FS ), a gene encoding the muscle-specific glycogen-targeting subunit RGL (also called GM) of protein phosphatase 1 (PP1) [2,3]. The RGL polypeptide contains an extended C-terminal tail with a short hydrophobic segment responsible for association with the sarcoplasmic reticulum  as well as carbohydrate- (glycogen)  and PP1-binding domains  in the N-terminal 240 residues. The latter facilitate localization of the catalytic subunit of the phosphatase (PP1c) to glycogen where it dephosphorylates glycogen synthase (GS) and glycogen phosphorylase (GP), and thereby promotes glycogen synthesis [7–9]. We have previously shown that Ppp1r3a-disrupted mice exhibit a 90% reduction in muscle glycogen , whereas RGL-overexpressing mice accumulate excess glycogen in muscle . The PPP1R3A FS mutation, which was initially described in a large white kindred, results in a mutant protein lacking the long C-terminal tail including the hydrophobic segment that tethers it to the sarcoplasmic reticulum . In that pedigree, severe insulin resistance was restricted to individuals who were doubly heterozygous for the PPP1R3A FS variant and an unlinked loss-of-function mutation in PPARG (AAA553T; stop codon 186), which encodes a key transcriptional regulator of adipocyte differentiation . Whilst the PPARG variant was uniquely present in that kindred, the allelic frequency of the PPP1R3A FS variant was 1.36% in a population of UK whites. Although the truncated RGL was shown to be mistargeted within the cell, its functional impact on glycogen synthesis was not determined. Here we sought to characterise the molecular and in vivo biological consequences of the PPP1R3A FS variant.
All human studies were approved by the relevant Local Research Ethics Committees (Cambridge, Oxford, and Nottingham), and all participants provided written informed consent. The RGL kin mice were generated by Cheryl Bock at the Comprehensive Cancer Center Transgenic Facility, Duke University, Durham, North Carolina, United States. All animals were maintained on a 12:12 h light–dark cycle in a temperature- and humidity-controlled facility with free access to food and water. All mouse studies were conducted in accordance with federal guidelines and were approved by the Institutional Animal Use and Care Committees of Indiana, Duke, and Yale Universities.
Muscle glycogen is one of two major energy sources for muscle contraction, the other being fatty acids. The fuel utilized by muscle depends on factors such as the type, intensity and duration of exercise, glycogen being primarily used during short bursts of high-intensity exercise . Glycogen turnover is tightly regulated by two enzymes, GS and GP. PP1 catalyzes the dephosphorylation of GS and GP, thereby activating GS, inactivating GP, and promoting net glycogen synthesis [7,8]. Its activity is regulated by a large family of targeting subunits, of which RGL is the major glycogen targeting subunit in muscle . Several human genetic disorders of glycogen metabolism have been described affecting muscle alone or muscle, liver, and other tissues. Until very recently, all of those affecting muscle alone impaired glycogen breakdown and caused either episodic exercise intolerance or fixed, progressive muscle weakness . Kollberg et al.  described a consanguineous kindred in which three individuals were homozygous for premature stop mutations in GYS1. Affected family members presented in childhood with hypertrophic cardiomyopathy (which appeared to cause sudden death in one case) and exercise intolerance. Muscle histology revealed severe glycogen depletion and a marked increase in mitochondria-rich type 1 fibres. Glucose tolerance appeared to be normal in the single individual in whom it was assessed. This human phenotype is similar to that of the GYS1 knockout mice [16,23]. A number of mutations in PPP1R3A have been identified in humans but, to date, none have been convincingly linked to in vivo alterations in glycogen metabolism [9,24–27]. We genotyped 744 nondiabetic adults from the Oxford Biobank in order to (1) assess prevalence rates of the PPP1R3A FS variant in an unselected population and (2) identify carriers of the variant whom we might approach for phenotyping. Prevalence figures of 1.46% are consistent with our original observation of 1.36% prevalence in a Cambridgeshire-based study . Fasting and postprandial muscle glycogen levels were significantly decreased in nondiabetic PPP1R3A FS carriers, making this the second genetic condition known to specifically reduce muscle glycogen accumulation.