Bartholomew A. Pederson

Exercise Capacity of Mice Genetically Lacking Muscle Glycogen Synthase IN MICE, MUSCLE GLYCOGEN IS NOT ESSENTIAL FOR EXERCISE

The glucose storage polymer glycogen is generally considered to be an important source of energy for skeletal muscle contraction and a factor in exercise endurance. A genetically modified mouse model lacking muscle glycogen was used to examine whether the absence of the polysaccharide affects the ability of mice to run on a treadmill. The MGSKO mouse has the GYS1 gene, encoding the muscle isoform of glycogen synthase, disrupted so that skeletal muscle totally lacks glycogen. The morphology of the soleus and quadriceps muscles from MGSKO mice appeared normal. MGSKO-null mice, along with wild type littermates, were exercised to exhaustion. There were no significant differences in the work performed by MGSKO mice as compared with their wild type littermates. The amount of liver glycogen consumed during exercise was similar for MGSKO and wild type animals. Fasting reduced exercise endurance, and after overnight fasting, there was a trend to reduced exercise endurance for the MGSKO mice. These studies provide genetic evidence that in mice muscle glycogen is not essential for strenuous exercise and has relatively little effect on endurance.

Abnormal Cardiac Development in the Absence of Heart Glycogen

ABSTRACT Glycogen serves as a repository of glucose in many mammalian tissues. Mice lacking this glucose reserve in muscle, heart, and several other tissues were generated by disruption of the GYS1 gene, which encodes an isoform of glycogen synthase. Crossing mice heterozygous for the GYS1 disruption resulted in a significant underrepresentation of GYS1-null mice in the offspring. Timed matings established that Mendelian inheritance was followed for up to 18.5 days postcoitum (dpc) and that ∼90% of GYS1-null animals died soon after birth due to impaired cardiac function. Defects in cardiac development began between 11.5 and 14.5 dpc. At 18.5 dpc, the hearts were significantly smaller, with reduced ventricular chamber size and enlarged atria. Consistent with impaired cardiac function, edema, pooling of blood, and hemorrhagic liver were seen. Glycogen synthase and glycogen were undetectable in cardiac muscle and skeletal muscle from the surviving null mice, and the hearts showed normal morphology and function. Congenital heart disease is one of the most common birth defects in humans, at up to 1 in 50 live births. The results provide the first direct evidence that the ability to synthesize glycogen in cardiac muscle is critical for normal heart development and hence that its impairment could be a significant contributor to congenital heart defects.

Brain glycogen supercompensation in the mouse after recovery from insulin-induced hypoglycemia

Brain glycogen is proposed to function under both physiological and pathological conditions. Pharmacological elevation of this glucose polymer in brain is hypothesized to protect neurons against hypoglycemia‐induced cell death. Elevation of brain glycogen levels due to prior hypoglycemia is postulated to contribute to the development of hypoglycemia‐associated autonomic failure (HAAF) in insulin‐treated diabetic patients. This latter mode of elevating glycogen levels is termed “supercompensation.” We tested whether brain glycogen supercompensation occurs in healthy, conscious mice after recovery from insulin‐induced acute or recurrent hypoglycemia. Blood glucose levels were lowered to less than 2.2 mmol/liter for 90 min by administration of insulin. Brain glucose levels decreased at least 80% and brain glycogen levels decreased approximately 50% after episodes of either acute or recurrent hypoglycemia. After these hypoglycemic episodes, mice were allowed access to food for 6 or 27 hr. After 6 hr, blood and brain glucose levels were restored but brain glycogen levels were elevated by 25% in mice that had been subjected to either acute or recurrent hypoglycemia compared with saline‐treated controls. After a 27‐hr recovery period, the concentration of brain glycogen had returned to baseline levels in mice previously subjected to either acute or recurrent hypoglycemia. We conclude that brain glycogen supercompensation occurs in healthy mice, but its functional significance remains to be established.

Inhibiting glycogen synthesis prevents lafora disease in a mouse model

Lafora disease (LD) is a fatal progressive myoclonus epilepsy characterized neuropathologically by aggregates of abnormally structured glycogen and proteins (Lafora bodies [LBs]), and neurodegeneration. Whether LBs could be prevented by inhibiting glycogen synthesis and whether they are pathogenic remain uncertain. We genetically eliminated brain glycogen synthesis in LD mice. This resulted in long‐term prevention of LB formation, neurodegeneration, and seizure susceptibility. This study establishes that glycogen synthesis is requisite for LB formation and that LBs are pathogenic. It opens a therapeutic window for potential treatments in LD with known and future small molecule inhibitors of glycogen synthesis. Ann Neurol 2013;74:297–300

Overexpression of glycogen synthase in mouse muscle results in less branched glycogen

Glycogen, a branched polymer of glucose, serves as an energy reserve in many organisms. The degree of branching likely reflects the balance between the activities of glycogen synthase and branching enzyme. Mice overexpressing constitutively active glycogen synthase in skeletal muscle (GSL30) have elevated muscle glycogen. To test whether excess glycogen synthase activity affected glycogen branching, we examined the glycogen from skeletal muscle of GSL30 mice. The absorption spectrum of muscle glycogen determined in the presence of iodine was shifted to higher wavelengths in the GSL30 animals, consistent with a decrease in the degree of branching. As judged by Western blotting, the levels of glycogenin and the branching enzyme were also elevated. Branching enzyme activity also increased approximately threefold. However, this compared with an increase in glycogen synthase of some 50-fold, so that the increase in branching enzyme in response to overexpression of glycogen synthase was insufficient to synthesize normally branched glycogen.

Correction: A prevalent variant in PPP1R3A impairs glycogen synthesis and reduces muscle glycogen content in humans and mice

Background Stored 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 Findings In 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. Conclusions Thus, 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.

Gene expression profiling of mice with genetically modified muscle glycogen content

Glycogen, a branched polymer of glucose, forms an energy re-serve in numerous organisms. In mammals, the two largest glyco-gen stores are in skeletal muscle and liver, which express tissue-specific glycogen synthase isoforms. MGSKO mice, in which mGys1 (mouse glycogen synthase) is disrupted, are devoid of muscle glycogen [Pederson, Chen, Schroeder, Shou, DePaoli-Roach and Roach (2004) Mol. Cell. Biol. 24, 7179-7187]. The GSL30 mouse line hyper-accumulates glycogen in muscle [Manchester, Skurat, Roach, Hauschka and Lawrence (1996) Proc. Natl. Acad. Sci. U.S.A. 93, 10707-10711]. We performed a microarray analysis of mRNA from the anterior tibialis, medial gastrocnemius and liver of MGSKO mice, and from the gastroc-nemius of GSL30 mice. In MGSKO mice, transcripts of 79 genes varied in their expression in the same direction in both the anterior tibialis and gastrocnemius. These included several genes encoding proteins proximally involved in glycogen metabolism. The Ppp1r1a [protein phosphatase 1 regulatory (inhibitor) sub-unit 1A] gene underwent the greatest amount of downregulation. In muscle, the downregulation of Pfkfb1 and Pfkfb3, encoding isoforms of 6-phosphofructo-2-kinase/fructose-2,6-bisphospha-tase, is consistent with decreased glycolysis. Pathways for branched-chain amino acid, and ketone body utilization appear to be downregulated, as is the capacity to form the gluconeogenic precursors alanine, lactate and glutamine. Expression changes among several members of the Wnt signalling pathway were identified, suggesting an as yet unexplained role in glycogen meta-bolism. In liver, the upregulation of Pfkfb1 and Pfkfb3 expression is consistent with increased glycolysis, perhaps as an adaptation to altered muscle metabolism. By comparing changes in muscle expression between MGSKO and GSL30 mice, we found a subset of 44 genes, the expression of which varied as a function of muscle glycogen content. These genes are candidates for regulation by glycogen levels. Particularly interesting is the observation that 11 of these genes encode cardiac or slow-twitch isoforms of muscle contractile proteins, and are upregulated in muscle that has a greater oxidative capacity in MGSKO mice.